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https://openalex.org/W3124607895	https://europepmc.org/articles/pmc7864193?pdf=render	English	null	Alteration of Pituitary Tumor Transforming Gene 1 by MicroRNA-186 and 655 Regulates Invasion Ability of Human Oral Squamous Cell Carcinoma	International journal of molecular sciences	2,021	cc-by	10,105	Citation: Lee, S.S.; Choi, J.H.; Lim, S.M.; Kim, G.J.; Lee, S.K.; Jeon, Y.K. Alteration of Pituitary Tumor Transforming Gene 1 by MicroRNA- 186 and 655 Regulates Invasion Ability of Human Oral Squamous Cell Carcinoma. Int. J. Mol. Sci. 2021, 22, 1021. https://doi.org/ 10.3390/ijms22031021 Keywords: pituitary tumor-transforming gene 1; oral squamous cell carcinoma; invasion; microRNA Alteration of Pituitary Tumor Transforming Gene 1 by MicroRNA-186 and 655 Regulates Invasion Ability of Human Oral Squamous Cell Carcinoma Sang Shin Lee 1,, Jong Ho Choi 1 , Seung Mook Lim 2, Gi Jin Kim 2 , Suk Keun Lee 1 and Yoon Kyung Jeon 3, 1 Department of Oral Pathology, College of Dentistry, Gangneung-Wonju National University, Gangneung 25457, Korea; jhchoi@gwnu.ac.kr (J.H.C.); sukkeunlee@hanmail.net (S.K.L.) 2 Department of Biomedical Science, CHA University, Seoul 13488, Korea; lsmook17@naver.com (S.M.L.); gjkim@cha.ac.kr (G.J.K.) 3 Department of Pathology, Seoul National University College of Medicine, Seoul 03080, Korea * Correspondence: sangshin@gwnu.ac.kr (S.S.L.); ykjeon@snu.ac.kr (Y.K.J.) 1 Department of Oral Pathology, College of Dentistry, Gangneung-Wonju National University, Gangneung 25457, Korea; jhchoi@gwnu.ac.kr (J.H.C.); sukkeunlee@hanmail.net (S.K.L.) 2 Department of Biomedical Science, CHA University, Seoul 13488, Korea; lsmook17@naver.com (S.M.L.); gjkim@cha.ac.kr (G.J.K.) 3 Department of Pathology, Seoul National University College of Medicine, Seoul 03080, Korea * Correspondence: sangshin@gwnu.ac.kr (S.S.L.); ykjeon@snu.ac.kr (Y.K.J.) 1 Department of Oral Pathology, College of Dentistry, Gangneung-Wonju National University, Gangneung 25457, Korea; jhchoi@gwnu.ac.kr (J.H.C.); sukkeunlee@hanmail.net (S.K.L.) 2 Department of Biomedical Science, CHA University, Seoul 13488, Korea; lsmook17@naver.com (S.M.L.); gjkim@cha.ac.kr (G.J.K.) g g , ; j g (J ); ( ) 2 Department of Biomedical Science, CHA University, Seoul 13488, Korea; lsmook17@naver.c gjkim@cha.ac.kr (G.J.K.) gj ( ) 3 Department of Pathology, Seoul National University College of Medicine, Seoul 03080, Korea * Correspondence: sangshin@gwnu.ac.kr (S.S.L.); ykjeon@snu.ac.kr (Y.K.J.) Abstract: Background: Pituitary tumor-transforming gene 1 (PTTG1) was recently shown to be involved in the progression as well as the metastasis of cancers. However, their expression and function in the invasion of oral squamous cell carcinoma (SCC) remain unclear. Methods: The expressions of PTTG1 and PTTG1-targeted miRNA in oral SCC cell lines and their invasion capability depended on PTTG1 expression were analyzed by quantitative RT-PCR, Western blots, the transwell insert system and Zymography. Results: Invasion abilities were decreased in oral SCC cells treated with siRNA-PTTG1. When PTTG1 were downregulated in oral SCC cells treated with microRNA-186 and -655 inhibited their invasion abilities via MMP-9 activity. Conclusions: These results indicate that alteration of expression of PTTG1 in oral SCC cells by newly identiﬁed microRNA-186 and -655 can regulate invasion activity. Therefore, these data offer new insights into further understanding PTTG1 function in oral SCC and should provide new strategies for diagnostic markers for oral SCC. International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Oral squamous cell carcinoma (SCC) is one of the most common malignant epithelial oral neoplasms. While the major risk factors for oral SCC are smoking and alcohol, other risk factors include viral infections, mutations, and immune defects [1]. Although oral SCC is based on clinical detection, most cases of oral SCC are not detected until advanced stages because earlier detection is difﬁcult [2]. According to the last GLOBOCAN, worldwide new case and deaths per year of oral cancer, was about 350,000/18,100,000 and 180,000/9,600,027, respectively [3]. Also, the prognosis is very poor and the ﬁve-year survival rate of oral SCC is still less than 50%, despite signiﬁcant improvements in clinical surgery, radiotherapy, and chemotherapy [4]. It is necessary to conﬁrm whether oral SCC is conﬁned locally or has invaded and metastasized to determine appropriate treatments, such as surgery or irradiation [5,6]. Thus, a reliable diagnostic marker for oral SCC isneeded and it is necessary to elucidate the molecular mechanisms involved in oral SCC metastasis. Recently, reports have suggested mining and evaluating molecular diagnostic markers for invasion and metastasis-speciﬁc microRNAs of oral SCC [7]. Received: 25 November 2020 Accepted: 18 January 2021 Published: 20 January 2021 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/). MicroRNAs (miRNAs) are a family of small non-coding (~22-nt long), endogenous, single RNA molecules that control gene expression by binding to the 3′-UTRs of the target mRNAs, regulating cleavage or translational inhibition [8]. Generally, it is well known that one miRNA targets several genes in stage and time-specific manners [9]. These observations suggest that miRNAs play vital roles in numerous biologic processes, including development, https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1021. https://doi.org/10.3390/ijms22031021 Int. J. Mol. Sci. 2021, 22, 1021 2 of 16 tumorigenesis, and metastasis [10]. Thus, studies on target genes associated with oncogenes or tumor suppressors using miRNAs have recently been reported in human cancers [11]. More recently, Tavazoie et al., showed that miRNA-126 and miRNA-335 functioned as metastasis suppressors in human breast cancer, and miRNA-331-3p had suppressive effects on non-small- cell lung carcinoma (NSCLC) invasion and metastasisin vitroandin vivo [12,13]. Recently, Feng et al. 1. Introduction reported that miRNA-22 increased by a lentiviral gene delivery system suppressed the cell proliferation, migration, and invasion of oral SCCs through targeting NLRP3, which is an NLR family pyrin domain containing three inflammasomes, in oral SCC tissues and cell lines [14]. In addition, many researchers have reported that several kinds of miRNAs, such as miR-1-3p, miR-9, and miR-30a-5p, controlled the proliferation, invasion, and metastasis of oral SCCs via exchange the expression of various target genes [15–18]. Interestingly, it was reported that miRNA-186 had a role as a tumor suppressor in oral SCCs and its expression was decreased by inhibition of the signaling activity of ERK and AKT, which are downstream of protein tyrosine phosphatase SHP2 [16,19]. However, whether miRNA-186 regulates oral SCC cell invasion or modulates pituitary tumor-transforming gene (PTTG)1 in oral SCC cells invasion remains poorly understood. p y PTTG is an oncogene that was ﬁrst isolated and identiﬁed in a rat pituitary tumor GH4 cell line but not in normal pituitary [20]. PTTGs are classiﬁed into PTTG1, PTTG2 and PTTG3, according to the order in which they were identiﬁed [21]. Speciﬁcally, PTTG1 is a multifunctional protein involved and overexpressed in various endocrine-related cancers, including pituitary, thyroid, breast, ovarian, and uterine tumors [22–24]. Previous evidence has shown that it is an important oncoprotein implicated in the progression of several cancer cell types through the metaphase-anaphase transition of the cell cycle [25,26]. However, PTTG1 is highly correlated with tumor invasiveness and is known as a critical gene associated with tumor metastasis, although its expression in normal human tissue is rare [22,27]. Moreover, the downregulation of PTTG1 by small interfering RNA (siRNA) inhibited the migration ability of non-small cell lung cancer (NSCLC) cells through matrix metalloproteinase (MMP) [28]. Speciﬁcally, PTTG1 is directly associated with cancer cell migration through MMP-2 [29]. In our previous reports, the regulation of PTTG1 by miRNA-186 correlated with the proliferation and metastasis of trophoblasts, which demonstrate invasion activity during implantation and placental development through the integrin/Rho-family signaling pathway [30]. Nonetheless, the expression and function of PTTG1 in oral SCC, as well as its molecular mechanisms in the metastasis of oral SCC, are unclear and require further investigation. Therefore, the objectives of the present study were to analyze the expression of PTTG1 in oral SCC cell lines (YD-10B and YD-15) and determine whether their effect on invasion was dependent upon PTTG1 expression using siRNA. 1. Introduction In addition, we investigated whether PTTG1-targeted microRNA could regulate the expression and invasion ability of PTTG1 in oral SCC cells. 2. Results 2.1. Effect of PTTG1 on Oncogenesis of Oral SCC Cell Lines First, we conﬁrmed the expression of PTTG1 in both oral SCC cell lines YD-10B and YD-15 by qRT-PCR and Western blotting. The expression of PTTG1 mRNA and protein was increased more in the YD-10B cells than in the YD-15 cells (Figure 1A,B). Generally, PTTG1 is involved in oncogenesis [31]. To conﬁrm the effect of PTTG1 on the oral SCC cell lines, we conﬁrmed the viability of the oral SCC cell lines regardless of the PTTG1 expression using PTTG1-speciﬁc siRNA treatment (siPTTG1) (Figure 1C). As shown in Figure 1D, the expression of PTTG1 in the YD-10B and YD-15 cell lines was dramatically decreased after siPTTG1 treatment. No expression of p53 was detected in the YD-10B cell line because they have a mutant type of p53, whereas p53 was highly expressed in the YD-15 cells, which have wild-type p53 (Figure 1D). Also, the expression of p53 was decreased in the YD-15 cells by siPTTG1 treatment, but there was no difference in the expression of CK18 between the two cell types (Figure 1D). Furthermore, we conﬁrmed the Int. J. Mol. Sci. 2021, 22, 1021 3 of 16 3 of 16 expression of vimentin because it is overexpressed in most epithelial cancer and correlates with tumor growth and poor prognosis [32]. The expression of vimentin was increased in the YD-10B cells, whereas vimentin expression was decreased in the YD-15 cells compared to the mock control (Figure 1D). However, the morphological characteristics of both cell types were not changed by siPTTG1 treatment. These ﬁndings suggest that the expression of PTTG1 affected oncogenesis in the YD-10B and YD-15 cells. expression of vimentin because it is overexpressed in most epithelial cancer and correlates with tumor growth and poor prognosis [32]. The expression of vimentin was increased in the YD-10B cells, whereas vimentin expression was decreased in the YD-15 cells compared to the mock control (Figure 1D). However, the morphological characteristics of both cell types were not changed by siPTTG1 treatment. These ﬁndings suggest that the expression of PTTG1 affected oncogenesis in the YD-10B and YD-15 cells. Figure 1. Expression of PTTG1 in oral SCCcell lines. (A) The mRNA and (B) protein expres- sion of PTTG1 in YD-10B and YD-15 cell lines were analyzed by qRT-PCR and Western blots. (C)Morphologies of the YD-10B and YD-15 cell lines after 200 nM siPTTG1 treatment for 24 or 48 h. 2. Results Magniﬁcation, ×400; scale bar = 50 µm. (D) The expression of PTTG1, p53, CK18, and vimentin in YD-10B and YD-15 cell lines analyzed by Western blots after 200 nM siPTTG1 treatment for 24 h. GAPDH was used as an internal control. CK18, cytokeratin 18. Figure 1. Expression of PTTG1 in oral SCCcell lines. (A) The mRNA and (B) protein expres Figure 1. Expression of PTTG1 in oral SCCcell lines. (A) The mRNA and (B) protein expres- sion of PTTG1 in YD-10B and YD-15 cell lines were analyzed by qRT-PCR and Western blots. (C)Morphologies of the YD-10B and YD-15 cell lines after 200 nM siPTTG1 treatment for 24 or 48 h. Magniﬁcation, ×400; scale bar = 50 µm. (D) The expression of PTTG1, p53, CK18, and vimentin in YD-10B and YD-15 cell lines analyzed by Western blots after 200 nM siPTTG1 treatment for 24 h. GAPDH was used as an internal control. CK18, cytokeratin 18. sion of PTTG1 in YD-10B and YD-15 cell lines were analyzed by qRT-PCR and Western blots. (C)Morphologies of the YD-10B and YD-15 cell lines after 200 nM siPTTG1 treatment for 24 or 48 h. Magniﬁcation, ×400; scale bar = 50 µm. (D) The expression of PTTG1, p53, CK18, and vimentin in YD-10B and YD-15 cell lines analyzed by Western blots after 200 nM siPTTG1 treatment for 24 h. GAPDH was used as an internal control. CK18, cytokeratin 18. 2.2. Effect of PTTG1 on Proliferation of YD-10B and YD-15 Cells 2.2. Effect of PTTG1 on Proliferation of YD-10B and YD-15 Cells Generally, PTTG1 controls cell proliferation by regulating chromatid separation during cell mitosis [26]. Thus, we conﬁrmed the effect of PTTG1 on the proliferation of YD-10B and YD-15 cells by Western blotting and FACS analysis following siPTTG1 treatment. We determined the proportion of cells in S phase in the YD-10B (7.55%) and YD-15 (12.85%) cell lines compared to the mock control group. After siPTTG1 treatment, the proportion of YD-10B cell in S phase was increased (9.18%), whereas it was decreased in YD-15 cells (4%) (Figure 2A). The activation of cyclin D1 is mainly responsible for regulating the G1-S phase transition and useful as prognostic biomarkers in oral SCC [33,34]. These results are consistent with the marked increase in the expression of cyclin D1 protein in YD-10B cells transfected with siPTTG1 compared to the control. In contrast to YD-10B, the expression of cyclin D1 was remarkably decreased in YD-15 cells transfected with siPTTG1 compared to the control (Figure 2B). To analyze cellular proliferation and death, the expression of phosphorylated Akt (p-Akt), which used as a marker for cell proliferation, was increased in both cell lines by treatment with siPTTG1 compared to the control, although no sig- niﬁcant difference was detected in cyclin E1, phosphorylated Erk (p-Erk), Bcl-2 or Bak (Figure 2B,C). Interestingly, the expression of phosphorylated mTOR, which used as a marker for autophagy, in the YD-10B cell line was dramatically decreased, but increased in the YD-15 cells, after siPTTG1 treatment. Additionally, the expression of gp130 was increased in the YD-10B cells after siPTTG1 treatment, but decreased in the YD-15 cells, Int. J. Mol. Sci. 2021, 22, 1021 4 of 16 although the expression of STAT3 and phosphorylated STAT3 was decreased (Figure 2D). These results indicate that the knockdown of PTTG1 decreased the proliferation of YD-10B cells and increased YD-15 cell proliferation by upregulating or downregulating the cell cycle, respectively, in a p53 expression-dependent manner. Figure 2. Effect of PTTG1 on cell cycle, apoptosis, and growth in oral SCC celllines. (A) Cell cycle analysis by FACScan in YD-10B and YD-15 cell lines after treatment with siPTTG1. The expression of proteins related to (B) cell cycle and (C) apoptosis, and (D) cell growth was analyzed in cells treated with 200 nM siPTTG1 by Western blots. GAPDH was used as an internal control. p-ERK, phosphorylated ERK; p-Akt, phosphorylated Akt; p-mTOR, phosphorylated mTOR; p-STAT3, phosphorylated STAT3. 2.2. Effect of PTTG1 on Proliferation of YD-10B and YD-15 Cells 2 3 Effect of PTTG1 on Invasion Ability of YD-10B and YD-15 Cells Figure 2. Effect of PTTG1 on cell cycle, apoptosis, and growth in oral SCC celllines. (A) Cell cycle analysis by FACScan in YD-10B and YD-15 cell lines after treatment with siPTTG1. The expression of proteins related to (B) cell cycle and (C) apoptosis, and (D) cell growth was analyzed in cells treated with 200 nM siPTTG1 by Western blots. GAPDH was used as an internal control. p-ERK, phosphorylated ERK; p-Akt, phosphorylated Akt; p-mTOR, phosphorylated mTOR; p-STAT3, phosphorylated STAT3. Figure 2. Effect of PTTG1 on cell cycle, apoptosis, and growth in oral SCC celllines. (A) Cell cycle analysis by FACScan in YD-10B and YD-15 cell lines after treatment with siPTTG1. The expression of proteins related to (B) cell cycle and (C) apoptosis, and (D) cell growth was analyzed in cells treated with 200 nM siPTTG1 by Western blots. GAPDH was used as an internal control. p-ERK, phosphorylated ERK; p-Akt, phosphorylated Akt; p-mTOR, phosphorylated mTOR; p-STAT3, phosphorylated STAT3. 2.3. Effect of PTTG1 on Invasion Ability of YD-10B and YD-15 Cells 2.3. Effect of PTTG1 on Invasion Ability of YD-10B and YD-15 Cells Since several groups reported that PTTG1 promoted the invasion ability in different types of cancer cells [28,35], we investigated whether PTTG1 regulated the invasion ability of YD-10B and YD-15 cells using a Transwell chamber assay. The number of invaded YD-10B cells treated with siPTTG1 were signiﬁcantly decreased compared to the mock control (p < 0.05, Figure 3A). Although the invasion ability of the YD-15 cells was lower than that of the YD-10B cells, the number of invaded cells was signiﬁcantly decreased after siPTTG1 treatment compared to the mock control (p < 0.05, Figure 3A). Furthermore, the expression of MMP-2 and MMP-9 released from the YD-10B cells treated with siPTTG1 was markedly decreased compared to the mock control (p < 0.05, Figure 3B). In the YD-15B cells, the expression of MMP-9 was signiﬁcantly decreased but there was no difference in MMP-2 expression following siPTTG1 treatment (p < 0.05, Figure 3B). These results suggest Int. J. Mol. Sci. 2021, 22, 1021 5 of 16 that the downregulation of PTTG1 by siPTTG1 treatment suppressed the invasion ability of the YD-10B and YD-15 cells by decreasing the expression of MMPs. Figure 3. Invasion abilities of oral SCC cell linesdepend on PTTG1 expression. (A)Image (left) and numbers (right) of invaded YD-10B and YD-15 cells after siPTTG1 treatment. Magniﬁcation, ×100; scale bar = 100 µm. (B) Activities of MMP-9 and -2 in YD-10B and YD-15 cells after siPTTG1 treatment analyzed by zymography. The values are means ± standard errors. * indicates signiﬁcant differences between control and siPTTG1 treatment (p < 0.05). All experiments were performed in triplicate. Figure 3. Invasion abilities of oral SCC cell linesdepend on PTTG1 expression. (A)Image (left) and numbers (right) of invaded YD-10B and YD-15 cells after siPTTG1 treatment. Magniﬁcation, ×100; scale bar = 100 µm. (B) Activities of MMP-9 and -2 in YD-10B and YD-15 cells after siPTTG1 treatment analyzed by zymography. The values are means ± standard errors. * indicates signiﬁcant differences between control and siPTTG1 treatment (p < 0.05). All experiments were performed in triplicate. 2.4. Effect of miR-186 and -655 on Expression of PTTG1 in YD-10B and YD-15 Cells 2.4. Effect of miR-186 and -655 on Expression of PTTG1 in YD-10B and YD-15 Cells 2.4. Effect of miR-186 and -655 on Expression of PTTG1 in YD-10B and YD-15 Cells To associate miRNAs with the regulation of PTTG1 expression, a bioinformatics search was performed in three databases, Microcosm, MicroRNA, and TargetScan, for predicted miRNA-targeting PTTG1 mRNAs. The databases predicted miR-186 and miR-655 as po- tential miRNAs targeting PTTG1 (Figure 4A). Furthermore, these tools indicated that the mRNA 3′ UTR of PTTG1 matched with miR-186 and miR-655 (Figure 4B). Synthesized miRNA mimics, inhibitor or scrambled control were transfected into the YD-10B and YD-15 cells. In the qRT-PCR analysis, the expression of miR-186 and miR-655 were effec- tively decreased in YD-10B and YD-15 cells treated with the mimic sequence (p < 0.05, Figure 4C,D, respectively), resulting in signiﬁcantly decreased mRNA and protein ex- pression of PTTG1 compared to the control (p < 0.05, miR-186, Figure 4E,G; miR-655, Figure 4F,H). In contrast, YD-10B and YD-15 cells treated with the miR-186 inhibitor se- quence showed decreased expression levels, although the decreases were not statically signiﬁcant (Figure 5A), resulting in increased mRNA and protein expression of PTTG1 compared to the control (p < 0.05, Figure 5C,E). As shown in Figure 6B, the expression level of miR-655 decreased in cells treated with the miR-655 inhibitor sequence, resulting in an increased mRNA and protein expression of PTTG1 (Figure 5D,F). However, the miR-655 inhibitor had no impact on PTTG1 protein levels (Figure 5F). These results suggest that the expression of PTTG1 was modulated by miR-186 and miR-655 in the YD-10B and YD-15 cells. 6 of 16 Int. J. Mol. Sci. 2021, 22, 1021 Figure 4. Effect of up-regulated of miR-186 and -655 on PTTG1 expression in oral SCC celllines. (A) Venn diagram showin overlapping miRNAs related to PTTG1 mRNA by Microcosm, Micro RNA, and Target Scan. (B) miRNA sequence an arget of the PTTG1 gene in the 3′-UTR site. Expression of (C) miR-186 and (D) miR-655 determined by qRT-PCR in YD-10 and YD-15 cells treated with mimic sequences. mRNA expression of PTTG1 determined by qRT-PCR in YD-10B and YD-1 ells treated with (E) miR-186 and (F) miR-655 mimic sequence. * indicates signiﬁcant differences between Negative contr and mimic treatment (p < 0.05). All experiments were performed in triplicate. Protein expression of PTTG1 determined b Western blots in YD-10B and YD-15 cells treated with (G) miR-186 and (H) miR-655 mimic sequence. GAPDH was used an internal control. Figure 4. Effect of up-regulated of miR-186 and -655 on PTTG1 expression in oral SCC celllines. 2.4. Effect of miR-186 and -655 on Expression of PTTG1 in YD-10B and YD-15 Cells (A) Venn diagram showing overlapping miRNAs related to PTTG1 mRNA by Microcosm, Micro RNA, and Target Scan. (B) miRNA sequence and target of the PTTG1 gene in the 3′-UTR site. Expression of (C) miR-186 and (D) miR-655 determined by qRT-PCR in YD-10B and YD-15 cells treated with mimic sequences. mRNA expression of PTTG1 determined by qRT-PCR in YD-10B and YD-15 cells treated with (E) miR-186 and (F) miR-655 mimic sequence. * indicates signiﬁcant differences between Negative control and mimic treatment (p < 0.05). All experiments were performed in triplicate. Protein expression of PTTG1 determined by Western blots in YD-10B and YD-15 cells treated with (G) miR-186 and (H) miR-655 mimic sequence. GAPDH was used as an internal control. 7 of 16 Int. J. Mol. Sci. 2021, 22, 1021 Figure 5. Effect of downregulation of miR-186 and -655 on PTTG1 expression in oral SCC cell lines. Expression of (A) miR-186 and (B) miR-655 determined by qRT-PCR in YD-10B and YD-15 cells treated with inhibitory sequences. mRNA expression of PTTG1 determined by qRT-PCR in YD-10B and YD-15 cells treated with (C) miR-186 and (D) miR-655 inhibitory sequences. * indicates signiﬁcant differences between Negative control and inhibitor treatment (p < 0.05). All experiments were performed in triplicate. Protein expression of PTTG1 determined by Western blots in YD-10B and YD-15 cells treated with (E) miR-186 and (F) miR-655 inhibitory sequence. GAPDH was used as an internal control. Figure 5. Effect of downregulation of miR-186 and -655 on PTTG1 expression in oral SCC cell lines. Expression of (A) miR-186 and (B) miR-655 determined by qRT-PCR in YD-10B and YD-15 cells treated with inhibitory sequences. mRNA expression of PTTG1 determined by qRT-PCR in YD-10B and YD-15 cells treated with (C) miR-186 and (D) miR-655 inhibitory sequences. * indicates signiﬁcant differences between Negative control and inhibitor treatment (p < 0.05). All experiments were performed in triplicate. Protein expression of PTTG1 determined by Western blots in YD-10B and YD-15 cells treated with (E) miR-186 and (F) miR-655 inhibitory sequence. GAPDH was used as an internal control. 2.5. The Expression of PTTG1 Modulated by miR-186 and miR-655 Regulated Invasion Ability of YD-10B and YD-15 Cells 2.5. The Expression of PTTG1 Modulated by miR-186 and miR-655 Regulated Invasion Ability of YD-10B and YD-15 Cells To identify the role of miR-186 and miR-655 in the invasion ability of YD-10B and YD-15 cells, Transwell chamber assays were performed in both cells treated with mimic or inhibitor sequences. The number of YD-10B and YD-15 cells treated with the miR-186 mimic sequence that invaded through the chamber was considerably less compared to the control cells. Quantitative analysis of the cell numbers showed that the YD-10B and YD-15 cells treated with the miR-186 mimic sequence had invasion rates 1.7 or 3.2 times less, respectively, than that of the control cells (p < 0.05, Figure 6A). Also, the number of invaded YD-10B and YD-15 cells was 2.6 or 2 times less, respectively, than that of the control cells after treatment with the miR-655 mimic sequence (p < 0.05, Figure 6B). Our previous studies showed that MMP-2 and MMP-9 activities can promote oral SCC cell invasion via PTTG1 expression [36]. Considering that, we performed zymography to analyze the activities of MMP-2 and MMP-9 in YD-10B and YD-15 cells treated with or without the miR-186 and miR-655 mimic sequence. The activity of MMP-9 was considerably decreased in YD-10B and YD-15 cells treated with both mimic sequences compared to the control cells. The activity of MMP-2 was decreased in the YD-10B cells treated with the miR-186 mimic Int. J. Mol. Sci. 2021, 22, 1021 8 of 16 sequence. However, that of the YD-15 cells treated with the miR-186 mimic sequence were increased compared to the control cells (Figure 6C). As shown in Figure 6D, the activity of MMP-9 was effectively decreased in the YD-10B and YD-15 cells treated with miR-655 mimic sequence compared to the control cells, although the MMP-2 activities were not signiﬁcantly different. sequence. However, that of the YD-15 cells treated with the miR-186 mimic sequence were increased compared to the control cells (Figure 6C). As shown in Figure 6D, the activity of MMP-9 was effectively decreased in the YD-10B and YD-15 cells treated with miR-655 mimic sequence compared to the control cells, although the MMP-2 activities were not signiﬁcantly different. mimic sequence compared to the control cells, although the MMP-2 activities were no signiﬁcantly different. Figure 6. Effect of upregulated miR-186 and -655 on invasion abilities of oral SCCcell lines. 2.5. The Expression of PTTG1 Modulated by miR-186 and miR-655 Regulated Invasion Ability of YD-10B and YD-15 Cells Image (left) and numbers (right) of invaded YD-10B and YD-15 cells treated with (A) miR-186 and (B) miR-655 mimic sequences. Magniﬁcation, ×100; scale bar = 100 µm. The values are means ± standard errors.* indicates signiﬁcant differences between negative control and mimic treatment (p < 0.05). All experiments were performed in triplicate. Activity of MMP-9 and -2 analyzed by zymography in YD-10B and YD-15 cells treated with (C) miR-186 and (D) miR-655 mimic sequences. In contrast to the mimic sequence results, the number of invaded YD-10B and YD-1 cells treated with the miR-186 or miR-655 inhibitor sequences was higher than that of th control cells. The number of invaded YD-10B and YD-15 cells treated with the miR-18 gure 6. Effect of upregulated miR-186 and -655 on invasion abilities of oral SCCcell lines. Image (left) and numbers ight) of invaded YD-10B and YD-15 cells treated with (A) miR-186 and (B) miR-655 mimic sequences. Magniﬁcation, 100; scale bar = 100 µm. The values are means ± standard errors.* indicates signiﬁcant differences between negative ontrol and mimic treatment (p < 0.05). All experiments were performed in triplicate. Activity of MMP-9 and -2 analyzed y zymography in YD-10B and YD-15 cells treated with (C) miR-186 and (D) miR-655 mimic sequences. In contrast to the mimic sequence results, the number of invaded YD-10B and YD-15 cells treated with the miR-186 or miR-655 inhibitor sequences was higher than that of the l ll h b f d d Y 1 d Y 1 ll d h h 1 Figure 6. Effect of upregulated miR-186 and -655 on invasion abilities of oral SCCcell lines. Image (left) and numbers (right) of invaded YD-10B and YD-15 cells treated with (A) miR-186 and (B) miR-655 mimic sequences. Magniﬁcation, ×100; scale bar = 100 µm. The values are means ± standard errors.* indicates signiﬁcant differences between negative control and mimic treatment (p < 0.05). All experiments were performed in triplicate. Activity of MMP-9 and -2 analyzed by zymography in YD-10B and YD-15 cells treated with (C) miR-186 and (D) miR-655 mimic sequences. In contrast to the mimic sequence results, the number of invaded YD-10B and YD-15 cells treated with the miR-186 or miR-655 inhibitor sequences was higher than that of the control cells. 2.5. The Expression of PTTG1 Modulated by miR-186 and miR-655 Regulated Invasion Ability of YD-10B and YD-15 Cells The number of invaded YD-10B and YD-15 cells treated with the miR-186 inhibitor sequence was 1.3 or 1.6 times higher, respectively, than that of the control cells (p < 0.05, Figure 7A). In addition, the number of invaded YD-10B and YD-15 cells treated In contrast to the mimic sequence results, the number of invaded YD-10B and YD-15 cells treated with the miR-186 or miR-655 inhibitor sequences was higher than that of the control cells. The number of invaded YD-10B and YD-15 cells treated with the miR-186 inhibitor sequence was 1.3 or 1.6 times higher, respectively, than that of the control cells (p < 0.05, Figure 7A). In addition, the number of invaded YD-10B and YD-15 cells treated Int. J. Mol. Sci. 2021, 22, 1021 9 of 16 9 of 16 with the miR-655 inhibitor sequence was 1.2 times higher than the control cells (p < 0.05, Figure 7B). Furthermore, the activities of MMP-9 were increased in YD-10B and YD-15B cells treated with the inhibitor sequence, regardless of the miRNAs, compared to control cells. However, the MMP-2 activities were not signiﬁcantly different, regardless of the miRNA treatment (Figure 7C,D). These results strongly suggest that miR-186 and miR- 655 were capable of directly targeting the 3′-UTRs of the PTTG1 genes, resulting in the alteration of PTTG1 expression associated with the invasion ability of oral SCC cells via MMP-9 activity. alteration of PTTG1 expression associated with the invasion ability of oral SCC cells v MMP-9 activity. re 7. Effect of downregulated miR-186 and -655 on invasion abilities of oral SCC cell lines. Image (left) and numbers t) of invaded YD-10B and YD-15 cells treated with (A) miR-186 and (B) miR-655 inhibitory sequences. Magniﬁcation, 0; scale bar = 100 µm. The values are means ± standard errors. * indicates signiﬁcant differences between negative rol and inhibitor treatment. All experiments were performed in triplicate. Activity of MMP-9 and -2 analyzed by ography in YD-10B and YD-15 cells treated with (C) miR-186 and (D) miR-655 mimic sequences. gure 7. Effect of downregulated miR-186 and -655 on invasion abilities of oral SCC cell lines. Image (left) and numbers ght) of invaded YD-10B and YD-15 cells treated with (A) miR-186 and (B) miR-655 inhibitory sequences. Magniﬁcation, 00; scale bar = 100 µm. The values are means ± standard errors. * indicates signiﬁcant differences between negative ntrol and inhibitor treatment. All experiments were performed in triplicate. 3. Discussion In the present study, we demonstrated that PTTG1 was expressed in oral SCC cells in a p53-dependent manner that regulated the proliferation of oral SCC cell lines via cell-cycle regulation. In addition, the inhibition of PTTG1 reduced the invasion ability of oral SCC cell lines and their invasion ability was regulated through MMP activity via miR-186 and miR-655. PTTG1 is a recently identiﬁed oncogene in pituitary tumors and high expression levels of PTTG1 were reported in several different cancers [25,37,38]. In the oral SCC ﬁeld, several groups recently reported the overexpression of PTTG1 in patients with oral SCC and that the expression of PTTG1 was a potential predictor of oral SCC progression. [39,40]. However, the studies did not investigate the mechanisms related to tumorigenesis through PTTG1 and did not conﬁrm a correlation between PTTG1 expression and oral SCC cells in in vitro experiments. In our study, two types of SCC cells originating from oral SCC, YD- 10B and YD-15 cells, were used to verify and investigate the mechanism of PTTG1 in oral cancer tumorigenesis.There are differences in the pathologic diagnosis and p53 expression in the two cell lines. The YD-10B cells are oral SCC, which has a frameshift mutation in p53, resulting in no expression of the p53 protein, whereas YD-15 cells strongly express p53 protein [41], which is known as a transcription factor with pro-apoptotic function. Therefore, its expression is associated with tumor suppression by promoting cell cycle arrest and apoptosis [42]. Interestingly, the PTTG1 expression and p53 expression were negative correlation in these cell lines in the present study. These ﬁndings are correlated with reports that high expression of PTTG1 was found to be associated with mutant p53 protein stability [43] Additionally, Tong et al. showed that p53 binds to PTTG1 and their interaction was shown to regulate cell cycle regulation via cyclin D using chromatin immunoprecipitation-of-chip studies [44]. Furthermore, several studies reported that p53-mediated PTTG1 expression induced cell death by regulating DNA-damage and/or apoptotic factors [38,45], In our preliminary data, we conﬁrmed the expression patterns of PTTG1 and their localization were observed in oral SCC tissues of patients. Their expression was localized in nuclei of tumor cells in well and moderate-differentiated oral SCC tissues (data not shown). 2.5. The Expression of PTTG1 Modulated by miR-186 and miR-655 Regulated Invasion Ability of YD-10B and YD-15 Cells Activity of MMP-9 and -2 analyzed by mography in YD-10B and YD-15 cells treated with (C) miR-186 and (D) miR-655 mimic sequences. Figure 7. Effect of downregulated miR-186 and -655 on invasion abilities of oral SCC cell lines. Image (left) and numbers (right) of invaded YD-10B and YD-15 cells treated with (A) miR-186 and (B) miR-655 inhibitory sequences. Magniﬁcation, ×100; scale bar = 100 µm. The values are means ± standard errors. * indicates signiﬁcant differences between negative control and inhibitor treatment. All experiments were performed in triplicate. Activity of MMP-9 and -2 analyzed by zymography in YD-10B and YD-15 cells treated with (C) miR-186 and (D) miR-655 mimic sequences. Int. J. Mol. Sci. 2021, 22, 1021 10 of 16 10 of 16 3. Discussion In this study, we showed that the altered expression of PTTG1 regulated the cell cycle of oral SCC cells and their function was involved with p53-dependent cell cycle arrest, although no differences in apoptotic and anti-apoptotic changes were seen. However, these different mechanisms mediated by p53 should be studied in the future. Recent studies suggested that miRNAs are involved in post-transcriptional gene expression and play critical roles in biological and cellular processes, such as metabolism, tumorigenesis, and metastasis. In cancer studies, miRNA acts as a metastasis regulator, as well as an oncogene and tumor suppressor, depending on the cancer type. MetastamiRs regulate metastasis through diverse mechanisms, including migration, invasion, and epithelial-mesenchymal transition (EMT) [10,46]. To further study the mechanisms of PTTG1 on the invasion ability of oral SCC, we used bioinformatics algorithms and methods, including microRNA [47], TargetScan human [48], and Microcosm Targets [49]. Based on the predictions, we found that miR-186 and -655 might be regulators that interact with PTTG1. Previous studies reported that low plasma levels of miR-655 were associated with lymphatic invasion and lymph node metastasis in patients with esophageal squamous cell carcinoma (ESCC) [50]. The overexpression of miR-655 inhibited the invasion ability of ESCC cells by targeting PTTG1 [51]. Li et al. demonstrated that PTTG1 modulated by miR- 186 regulated the invasion and metastasis of NSCLC [28]. Wang et al. showed that PTTG1 was overexpressed in patients with neuroendocrine tumors (CNET), which was regulated by miR-186. However, all previous reports on PTTG1 expression by targeting miR-186 and miR-655 were insufﬁcient to clearly explain the mechanisms of cancer invasion and metastasis. The relationship between miR, including miR-186 and miR-655, and PTTG1, which is involved with the invasion ability of oral SCC, remains unknown although PTTG1- targeting miR-655 inhibit pituitary tumor cell tumorigenesis via p53/PTTG1 regulation Int. J. Mol. Sci. 2021, 22, 1021 11 of 16 11 of 16 feedback loop [52]. In our previous study, we showed that miR-186 directly targeted PTTG1, which controls human trophoblast invasion via integrin and the Rho-family [30]. In the current study, we ﬁrst demonstrated that miR-186 and miR-655 were direct targets of PTTG1 in oral SCC cell lines. Using Transwell assays, we found that the altered expression of miR- 186 and -655 controlled the invasion ability of oral SCC cell lines. 3. Discussion These observations may explain roles for miR-186 and -655 in the regulation of oral SCC invasion and metastasis, regardless of cell type. However, the further study on the correlation between microRNAs (e.g., miR-186 and -655) and p53 expression on migration ability of oral SCC should be need in the future. MMPs are a highly regulated family of zinc-dependent endopeptidases that are asso- ciated with tumor progression and metastasis [53,54]. The role of MMPs in cancer invasion is primarily overexpression in invasive malignant tumors [55,56]. In oral SCCs, MMP- 1, MMP-8, MMP-10, MMP-12, and MMP-13 are highly expressed in patients with oral SCC [57]. MMP expression may determine the level of metastasis in oral SCC. Makinen et al. demonstrated that the nuclear expression of MMP-13 or the cytoplasmic expression of MMP-2, -8, and -9 was associated with invasion depth and tumor size in oral tongue squamous cell carcinoma [58]. MMP-7 and -9 and MT1-MMP were shown to be closely correlated to invasion depth and progression [59]. In gelatin zymography, we found that the upregulation of miR-186 and miR-655 reduced the activity of MMP-9, whereas down- regulated miR-186 and miR-655 induced the activity of MMP-9 in YD-10B and YD-15 cells. These ﬁndings suggest that miR-186 and miR-655 negatively controlled the expression of PTTG1 and MMP-9 in the oral SCC cell lines. These data are consistent with previous reports on the PTTG1 control of oral SCC cell lines through the regulation of MMP-2 and MMP-9 activity. 4. Materials and Methods 4.1. Cell Culture Two oral SCC cell lines, YD-10B cell line (p53 frameshift mutation, Tongue) and YD- 15 cell line (p53 wild type, Tongue) were used [41]. YD-10B and YD-15 were obtained from Dr. Jin Kim (Yonsei University, Seoul, Korea). Both cell lines were cultivated in a deﬁned medium [3:1 mixture ofDulbecco’s modiﬁed Eagle’s medium (11965, Gibco, Rockville, MD, USA) and Ham’s F12 nutrient Mixture (11765-054, Gibco) supplemented with 10% fetal bovine serum (16000-044, Gibco) and 100 U/mL penicillin and streptomycin (15140-122, Gibco), and 2 mM L-glutamine (25030, Gibco) at 37 ◦C in a humidiﬁed atmo- sphere of 5% CO2. Synthesized miRNA mimics, inhibitor, and scrambled control were synthesized by Genolution (Genolution Inc., Seoul, Korea). All transient transfections with siRNA (Invitrogen, Waltham, MA, USA) and miRNA (Genolution, Seoul, Korea) in YD-10B and YD-15 cells were performed using Lipofectamine 2000 Transfection Reagent (Invitrogen), according to the manufacturer’s protocol. Brieﬂy, 1 × 106 cells were seeded onto 100 mm culture dishes with serum-free OPTI-MEM (Gibco). Next, the cells were transfected with 200 nM siRNA-PTTG1 (siPTTG1) or 30 nM miRNAs (-186, -655, or scram- bled negative control) for 24–48 h. The sequences used in the present study are shown as below. hsa-miR-186-5p; F: 5′- CAA AGA ATT CTC CTT TGG GCT-3′; hsa-miR-655-5p; F: 5′-AGAGGUUAUCCGUGUUAUGUUC-3′.All transfection experiments were optimized for high efﬁciency and minimal cell damage. 4.2. Fluorescence-Activated Cell Sorting Analysis 4.3. Quantitative Real-Time Polymerase Chain Reaction Analysis (qRT-PCR) 4.3. Quantitative Real-Time Polymerase Chain Reaction Analysis (qRT-PCR) Total RNA was extracted using Trizol reagent (Invitrogen). cDNA (500 ng) was synthesized using Superscript III reverse transcriptase (Invitrogen) or a miR-X miRNA First-Strand Synthesis Kit (Clontech Laboratories, Mountain View, CA, USA) according to the manufacturer’s protocol. qRT-PCR was performed on an Exicycler 96 (Bioneer, Deajeon, Korea) using SYBR Green Master Mix (Roche, Basal, Switzerland). The PCR cycling conditions for the mRNAs were as follows: 95 ◦C for 5 min, followed by 40 cycles at 95 ◦C for 5 s and 60 ◦C for 30 s. The microRNA ampliﬁcation conditions were 95 ◦C for 10 s, followed by 40 cycles at 95 ◦C for 5 s and 60 ◦C for 20 s, with a ﬁnal step at 95 ◦C for 60 s, 55 ◦C for 30 s, and 95 ◦C for 30 s. All normalizations were done using β-actin or U6 levels and the relative expression levels were calculated by the ∆∆CT method. Also, Reverse transcriptase PCR (RT-PCR) was used to detect mRNA expression of the PTTG1. Total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was carried out using 500ng of total RNA and Superscript III reverse transcriptase (Invitrogen), the synthesized cDNAs were ampliﬁed by PCR. The ampliﬁcation conditions were as follows: 5 min 95◦C, followed by 35 cycles of 94 ◦C for 30 s, 52~65 ◦C for 1 min, and 72◦C for 1 min. The PCR products were visualized by electrophoresis on a 1~2% (w/v) agarose gel (Cambrex, ME, USA) containing 0.5µg/mLethidium bromide (Promega, WI, USA).The primer sequences used in the present study are shown as the below. PTTG1; F:5′-AAG GAA AAT GGA GAA CA GGC-3′, R: 5′- GCT TGG CTG TTT TTG TTT GAG G-3′; GAPDH; F:5′-GATTCCACCCATGGCAAATTC, R:5′-GTCATGAGTCCTTCCACGATAC. y GCT TGG CTG TTT TTG TTT GAG G-3′; GAPDH; F:5′-GATTCCACCCATGGCAAATTC, R:5′-GTCATGAGTCCTTCCACGATAC. 4.4. Western Blots The cells were extracted on ice in lysis buffer [RIPA buffer (Sigma), complete protease inhibitor cocktail (Mini Tablet; Roche), phosphatase inhibitor cocktail II (A.G. Scientiﬁc, San Diego, CA, USA)], and cleared by centrifugation at 13,000 rpm for 20 min at 4 ◦C. The protein concentration of the lysates was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equal concentrations of total lysates were mixed with 5× loading dye [50mM-Tris HCL(pH 6.8), 2% sodium dodecyl sulfate (SDS), 1% 2-mercaptoethanol, 12.5mM ethylenediaminetetraacetic acid, and 0.02% bromophenol blue], and separated by 8–15% gradient SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the proteins were transferred to polyvinylidene diﬂuoride membranes (Bio-Rad Laboratories, Berkeley, CA, USA). The membranes were incubated in 8% skim milk or 5% bovine serum albumin (BSA, AMRESCO, Cincinnati, OH, USA) for 1 h at room temperature and then incubated overnight with primary antibodies at 4 ◦C. The antibodies are shown in Supplementary Table S1. The following day, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; 1:20,000), anti- mouse IgG (1:20,000) or anti-goat IgG (1:20,000) secondary antibodies for 1 h at room temperature. The speciﬁc protein bands were visualized with an Electrochemiluminescence Advance Western Blot Detection Kit (Amersham Biosciences, Uppsala, Sweden). 4.2. Fluorescence-Activated Cell Sorting Analysis YD-10B and YD-15 cells were seeded onto 100 mm culture dishes at a density of 1 × 106 cells/dish and were transfected with 50–200nM siPTTG1 for 24 h. The adherent and ﬂoating cells were both collected and ﬁxed in ice-cold 70% ethanol at room temperature (RT) for 10 min. The ﬁxed cells were rehydrated in phosphate-buffered saline (PBS) and stained with 50 µg/mLpropidium iodide (Sigma-Aldrich, St. Louis, MO, USA) and 10µg/mLRNase A (Sigma-Aldrich) at room temperature for 30 min. The samples were analyzed by a FACSCalibur ﬂow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) Int. J. Mol. Sci. 2021, 22, 1021 12 of 16 and the cell cycle phase distribution was quantiﬁed with CellQuest Pro software (BD Biosciences, Heidelberg, Germany). All experiments were performed in two separate experiments in duplicate. 4.5. Transwell Chamber Assay The invasion ability of oral SCC cells was determined using a 24-well cell culture insert system pre-coated matrigel (8 µm pore size; BD Biosciences) and zymography. For the transwell chamber assay, a total of 3.5–4.5 × 104 cells were collected and added to the upper chamber with serum-free OPTI-MEM media (Gibco). Culture media containing 10% FBS was put into the lower chamber as a chemo-attractant, and the cells were transfected with siRNA or miRNA for 24 h. After 24 h, the membranes were ﬁxed with ice-cold methanol for 20 min and stained with Mayer’s hematoxylin (Dako, Santa Clara, CA USA) at 37 ◦C Int. J. Mol. Sci. 2021, 22, 1021 13 of 16 for 10 min. The non-invading cells remaining in the upper chamber were scraped out using a cotton swab. The numbers of invaded cells were counted in at least ﬁve randomly selected ﬁelds under a light microscope. All experiments were performed in two separate experiments in duplicate. 4.7. Statistical Analysis The results are presented as means ± standard errors. The data were analyzed using Student’s t-test, with p < 0.05 considered to indicate statistical signiﬁcance. 4.6. Zymography After the invasion assay, the supernatants from the invaded cells in the lower chamber- containing secreted MMP-2 and MMP-9 were collected. Equal volumes of the supernatants were electrophoresed on 10% SDS-PAGE gel with 1mg/mLgelatin. The gel was renatu- rated in renaturation buffer (Bio-Rad Laboratories) at 37 ◦C for 30 min and incubated in developing buffer (Bio-Rad Laboratories) at 37 ◦C for 24 h. After incubation, the gel was stained for 2 h with staining solution [10% acetic acid/40% methanol supplemented with 0.5% Coomassie brilliant blue R-250 (Sigma)] and destained for 1 h in destaining solution [50% methanol/10% acetic acid/40%water]. 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Author Contributions: S.S.L.: analysis of data, data interpretation, manuscript drafting, ﬁnancial support, ﬁnal approval of manuscript; J.H.C.: data interpretation and manuscript drafting; S.M.L.: analysis of data; G.J.K.: critical discussion and manuscript drafting; S.K.L.: critical discussion; Y.K.J.: critical discussion, ﬁnal approval of manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0014098, 2019R1I1A3A01057886). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: We special thank Jin Kim (Yonsei University, Seoul, South Korea) for donation Oral SCC cell lines (YD-10B and YD-15). Conﬂicts of Interest: The authors declare that they have no competing interests. Conﬂicts of Interest: The authors declare that they have no competing interests. Conﬂicts of Interest: The authors declare that they have no competing interests. 14 of 16 Int. J. Mol. Sci. 2021, 22, 1021 Abbreviations SCC Squamous cell carcinoma PTTG1 Pituitary tuor-transforming gene 1 miRNA MicroRNA p-Akt Phosphorylated Akt siRNA Small interfering RNA MMP Matrix metalloproteinase References [CrossRef] g g [ ] 10. Kim, J.; Yao, F.; Xiao, Z.; Sun, Y.; Ma, L. MicroRNAs and metastasis: Small RNAs play big roles. Cancer Metastasis Rev. 2018, 37, 5–15. [CrossRef] 11. Wang, D.; Qiu, C.; Zhang, H.; Wang, J.; Cui, Q.; Yin, Y. Human microRNA oncogenes and tumor suppressors show signiﬁcantly different biological patterns: From functions to targets. PLoS ONE 2010, 5, e13067. [CrossRef] [PubMed] 12. Li, X.; Zhu, J.; Liu, Y.; Duan, C.; Chang, R.; Zhang, C. 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https://openalex.org/W4287864848	https://zenodo.org/records/5716388/files/CESpsicologia.pdf	English	null	The role of emotional intelligence for teachers efficacy and classroom management	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	7,911	PSICOLOGÍA PSICOLOGÍA The role of the teacher’s emotional intelligence for efficacy and classroom management El papel de la inteligencia emocional del profesor para la eficacia y la gestión del aula Sabina Valente1 ORCID, Abílio Afonso Lourenço2 ORCID, Paulo Alves3 ORCID, Sergio Dominguez-Lara4 ORCID 1 Center for Research in Education and Psychology of the University of Évora, Portugal 2 University of Minho, Braga, Portugal 3 Research in Education and Community Intervention; Piaget Institute of Viseu, Portugal 4 Universidad de San Martín de Porres, Lima, Perú El papel de la inteligencia emocional del profesor para la eficacia y la gestión del aula El papel de la inteligencia emocional del profesor para la eficacia y la gestión del aula Sabina Valente1 ORCID, Abílio Afonso Lourenço2 ORCID, Paulo Alves3 ORCID, Sergio Dominguez-Lara4 ORCID 1 Center for Research in Education and Psychology of the University of Évora, Portugal 2 University of Minho, Braga, Portugal 3 Research in Education and Community Intervention; Piaget Institute of Viseu, Portugal 4 Universidad de San Martín de Porres, Lima, Perú Sabina Valente1 ORCID, Abílio Afonso Lourenço2 ORCID, Paulo Alves3 ORCID, Sergio Dominguez-Lara4 ORCID 1 Center for Research in Education and Psychology of the University of Évora, Portugal 2 University of Minho, Braga, Portugal 3 Research in Education and Community Intervention; Piaget Institute of Viseu, Portugal 4 Universidad de San Martín de Porres, Lima, Perú Abstract Fecha correspondencia: Recibido: julio 31 de 2019. Aceptado: febrero 24 de 2020. Forma de citar: Valente, S., Lourenço, A.A., Alves, P., & Dominguez-Lara, S. (2020). The role of emotional intelligence capacity for teacher’s efficacy and classroom management efficacy. Rev. CES Psico, 13(2), 18-31. In recent years, several studies have revealed multiple benefits of teachers’ emotional intelligence (EI) concerning their professional performance, re- garding teaching and learning process, students’ school performance, job satisfaction, reduction of stress and burnout, and the importance of inter- personal relationships at school. However, few studies have explored how teachers’ EI capacity affect their effectiveness in teaching and classroom management. In this sense, the aim of this study was twofold: to examine the relationship between teacher’s EI capacities, teacher efficacy, and class- room management effectiveness, and to analyze the relationship between teacher’s characteristics (gender) and professional background (service time and academic formation) and teacher’s EI capacities. 634 Portuguese teachers filled out a form with personal and professional data and answered an adaptation of the Emotional Skills and Competence Questionnaire, and the Teacher Efficacy in Classroom Management and Discipline Scale. Struc- tured Equation Model analyses were computed and showed that teachers who tended to have higher levels of capacity to perceive, express and manage emotions reported higher levels of teaching effectiveness and classroom management effectiveness. Therefore, is important for Portuguese teachers developing their emotional skills during academic formation, to a more effec- tive future professional activity. Fecha correspondencia: Recibido: julio 31 de 2019. Aceptado: febrero 24 de 2020. Keywords: Teacher’s, Emotional Intelligence, Teacher Efficacy, Classroom Management Efficacy. Sobre los autores: 1. Ph.D. Education. Researcher at the Center for Research in Education and Psychology, University of Évora (CIEP-UE), and International Association for Development of Emotional Education (PAIDEIA). 2. Ph.D. Education. Researcher at the Psychology and Education Research Center of School Group Alexandre Herculano - Porto. Palabras clave: Maestros, Inteligencia Emocional, Eficacia del Maestro, Eficacia de la Gestión del Aula. 3. Ph.D. Psychology. Professor at Piaget Institute Viseu. Researcher at the Research in Education and Community Intervention (RECI). 3. Ph.D. Psychology. Professor at Piaget Institute Viseu. Researcher at the Research in Education and Community Intervention (RECI). Resumen En los últimos años, diversos estudios revelaron múltiples beneficios de la inteligencia emocional (IE) de los maestros con respecto a su desempeño profesional, el proceso de enseñanza y aprendizaje, el rendimiento escolar de los estudiantes, su satisfacción laboral, la reducción del estrés y el ago- tamiento, y la importancia de las relaciones interpersonales en la escuela. PSICOLOGÍA PSICOLOGÍA Valente, S., Lourenço, A.A., Alves, P., & Domínguez-Lara, S. Pág 19 Pág 19 Sin embargo, pocos estudios han investigado cómo las capacidades de IE de los maestros afectan su efectividad en la enseñanza y el manejo del aula. En este sentido, el objetivo de este estudio fue doble: examinar la relación entre las capacidades de IE del maestro, su eficacia en la enseñanza y su eficacia en la gestión del aula; y analizar la relación entre las características del maestro (género), su formación profesional (tiempo de servicio y formación académica) y sus capacidades de IE. 634 maestros portugueses completaron un formulario con datos personales y profesio- nales, y respondieron una adaptación del Cuestionario de Habilidades Emocionales y Competencia, y la Escala de Eficacia del Maestro en el Manejo del Aula y la Disciplina. Los análisis del modelo de ecuaciones estructuradas mostraron que los maestros que tienden a tener niveles más altos de capacidad para percibir, expresar y manejar las emociones mostraron niveles más altos de eficacia docente y niveles más altos de eficacia en el manejo del aula. Por lo tanto, es importante que los maestros Portu- gueses desarrollen sus habilidades emocionales durante la formación académica, con el fin de lograr una futura actividad profesional más efectiva. Sobre los autores: 1. Ph.D. Education. Researcher at the Center for Research in Education and Psychology, University of Évora (CIEP-UE), and International Association for Development of Emotional Education (PAIDEIA). 2. Ph.D. Education. Researcher at the Psychology and Education Research Center of School Group Alexandre Herculano - Porto. 3. Ph.D. Psychology. Professor at Piaget Institute Viseu. Researcher at the Research in Education and Community Intervention (RECI). 4. Ph.D. Psychology. Professor at University of San Martín de Porres. Researcher at the Psychology Research Institute. Introduction Teaching requires great emotional labor and teachers EI is recognized as the basis of their http://dx.doi.org/10.21615/cesp.13.2.2 The role of the teacher’s emotional intelligence for efficacy and classroom management The role of the teacher’s emotional intelligence for efficacy and classroom management The role of the teacher’s emotional intelligence for efficacy and classroom management PSICOLOGÍA Pág 20 Pág 20 attitudes toward their students and to provide a steady and wholesome classroom environment (Hen & Sharabi-Nov, 2014). Also, teachers recognized the importance of EI in classrooms and how it becomes a crucial constituent of the teaching-learning process resulting in holistic learning and development of students (Allen et al., 2014; Becker et al., 2014; Maamari & Majdalani, 2019; Pugazhenthi & Srinivasan, 2018). Be- sides, different studies showed that EI is positively correlated with teacher’s efficacy (Hassan et al., 2015; Koçoğlu, 2011; Wenn et al., 2018), and with classroom management efficacy (Valente et al., 2019; Wahyuddin, 2016). EI theory concerns the perception of emotions, the use of emotions to facilitate thinking, the understanding of emotions and their management (Mayer & Salovey, 1997). In this sense, teachers with a great capacity for emotional perception are those who perceive and are aware of student’s state of mind and know when and how to inter- vene (Fernandéz-Berrocal & Extremera, 2005). One of the most difficult capacities to master is the management of our emotional states, one of the most significant dimensions of EI construct and with greater predictive capacity (Bracket et al., 2006). When the teacher regulates his emotions, he often manages to modify his own and others’ feelings (Fernandéz-Berrocal & Extremera, 2005), providing coping strategies (Lazarus, 2000) that focus on emotion change or solving the problem. Effectiveness in teaching and quality learning are the two most significant factors for high standards in the field of education (Pugazhenthi & Srinivasan, 2018). In the classroom where individuals with different characteristics, feelings, and personalities interact, it is expected that several problems arise. The- se problems require skills to successfully manage them, across great effectiveness capacities for teaching and classroom management. On teaching work, considered one of the professions with the greatest emotional exhaustion (Fernandéz-Berrocal & Extremera, 2005), apply correct emotional manage- ment is essential and recommended for teaching efficacy, and classroom management. Introduction For teachers, it is not enough to have only academic knowledge, it is also essential having emotional knowledge. Studies on teacher’s emotional intelligence (EI) have al- ready provided evidence that EI is the foundation for positives relationships, and for a good functioning in a school environment (Hargraves, 2017; Maamari & Majdalani, 2019), namely concerning the benefits regarding professional performance, (Cejudo & López-Delgado, 2017), teaching and learning process (Allen et al., 2014), students’ school achievement (Becker et al., 2014), job satisfaction (Cejudo & López-Delgado, 2017), reducing stress and burnout (Subalakshmi et al., 2019), and the significance for interpersonal relationships in an educational context (Yin et al., 2013). 4. Ph.D. Psychology. Professor at University of San Martín de Porres. Researcher at the Psychology Research Institute. Emotions influence teacher-student interactions and shape the atmosphere of the classroom (Meyer & Turner, 2007). In addition, Iskandar, Majzub and Mahmud (2009) points that EI plays a significant role in teaching efficacy, and in increasing performan- ce at teacher’s work. Moreover, Perry and Ball (2007) refer that there is a need to cla- rify the relation between teachers’ EI differences and classroom behavior. However, few studies have examined the relationships between school teacher’s EI capacities, teacher’s efficacy and classroom management efficacy. In this context, the present study analyzes the relationships between Portuguese school teacher’s EI, teacher’s efficacy, and classroom management efficacy. It also analyzes the relationships between teacher’s EI and teacher’s characteristics (gender) and professional background (service time and academic formation). Teacher’s emotional intelligence capacities Teacher’s emotional intelligence capacities School teachers currently work in a society full of instability, of many natures, and at schools which in the middle of successive reforms are slow to find a solution that meets the needs of the whole educational community. Increasingly, the significance of teacher’s EI development is perceptible, so that they have tools to work in a rewarding and effective way the teaching and learning process. During the last years, EI has received more attention in the educational field. Introduction It is not just about whether teachers can ‘handle’ the inclusive classroom, but also about their confidence in choosing the strategies that promote student’s success (Lancaster, 2014; Kiel et al., 2019). In this sense, tea- cher efficacy is strained from Bandura (1997) conceptualization of self-efficacy and alludes to teacher’s perceptions of their skill in achieving the roles prescribed for them to realize a set of educational objectives, such as learning easing and student development. Studies suggest that teachers who perceive themselves as more effective show higher professional performance and personal well-being at work (Holzberger et al., 2013; Klassen & Chiu, 2010), low levels of stress and burnout (Dicke et al., 2014) and use appropriate classroom management strategies (Woolfolk & Hoy, 1990). It has been found that teacher efficacy affects teacher levels of effort, planning, orga- nization, persistence, and reliance on others. Specifically, teachers “with a high sense of efficacy employ more behaviors that have the potential to enhance student learning and motivation” (Lancaster, 2014, p. 240). The study of Ahsan et al. (2012) suggests that the teacher’s efficacy affects classroom management skills. Indeed, Martin, Linfoot and Stephenson (1999) proposed that teacher’s responses in the classroom misbehavior may be mediated by their confidence with their ability to deal with student behavior. Studies have showed that classroom management efficacy is an essential pre- requisite for cognitive lear- ning, and if the teacher fails to solve problems arising from unruly and conflicting student behavior, the entire teaching and learning pro- cess will be affected (Taxer et al., 2018; Valente, 2019). Classroom management is analyzed by several authors as a set of actions carried out by the teacher to develop a favorable and stimulating environment for school students. For Djigić and Stojiljkovic (2012), classroom management includes the ma- nagement of space, time and activities, as well as management of student behavior, articulating the characteristics of the teacher and his capacities. Teacher’s capacity to establishing and maintain a productive learning environment through effective classroom management is considered by experienced teachers as an essential competency (O’Neill & Stephensen, 2011). Studies have showed that classroom management efficacy is an essential prerequisite for cognitive learning, and if the teacher fails to solve problems arising from unruly and conflicting student behavior, the entire teaching and learning process will be affected (Taxer et al., 2018; Valente, 2019). Introduction The interactions in classroom context are instituted and sustained through emotions expressed by the teacher during the work he develops with students, being significant for education quality, since emotions help on discovery and understanding of others, playing an essential role in teachers-students relationships. Studies analyze the role of personal and professional variables (e.g., gender, service time and academic formation) whit school teacher’s EI. Some of these studies reveal high ove- rall EI scores for women compared to men (Gill & Sankulkar, 2017; Valente, 2019). About the influence of service time on teacher’s EI, some studies with Portuguese teachers indicate that teachers with more service time tend to have lower levels of EI (Sousa, 2011; Valente, 2019). Teachers who have taught for less than six years have greater emotional perception and can regulate their negative emotional states and prolong the positive ones, contrary to their colleagues with more than six years of service time (Sousa, 2011). Regarding academic training, studies showed that teachers with more academic formation (e.g., Ph.D.) are the ones who commit most attention to their emotions and tend to have higher levels of EI (Fernandes, 2015; Valente, 2019). Efficacy for teaching and for classroom management Efficacy for teaching and for classroom management Effectiveness in teaching and quality learning are the two most significant factors for high standards in the field of education (Pugazhenthi & Srinivasan, 2018). In the classroom where individuals with different characteristics, feelings, and personali- ties interact, it is expected that several problems arise. These problems require skills to successfully manage them, across great effectiveness capacities for teaching and classroom management. Personal teaching efficacy is defined as the teacher’s belief in their capacity to bring about modification in students, it refers to teacher’s judgments of their teaching abi- lities, and consists of teacher beliefs that they know suitable teaching techniques and http://dx.doi.org/10.21615/cesp.13.2.2 PSICOLOGÍA Valente, S., Lourenço, A.A., Alves, P., & Domínguez-Lara, S. PSICOLOGÍA Pág 21 Pág 21 can help students learn, achieve more, do better than usual, and increase retention, among other skills (Gibson & Dembo, 1984). For Lopes and Oliveira (2017) efficacy to teach is defined as the method of making student’s learning possible, promoting engagement and discussion, concerning and respecting students, and maximizing student’s academic performance. Introduction Given the above, in the present study, the following hypotheses were presented: hypothesis 1) A statistically positive correlation is expected between gender and EI capacities; hypothesis 2) A statistically negative correlation is expected between service time and EI capacities; hypothesis 3) A statistically positive correlation is ex- pected between academic formation and EI capacities; hypothesis 4) A statistically positive correlation is expected between the capacity for perceive and understand emotions and the capacity to manage and regulate emotions; hypothesis 5) A statis- tically positive correlation is expected between the capacity to express and classify emotions and the capacity for manage and regulate emotions; hypothesis 6) A statis- tically positive correlation is expected between EI capacities and “personal teaching efficacy”; hypothesis 7) A statistically positive correlation is expected between EI ca- pacities and “efficacy for classroom management and discipline”; and hypothesis 8) http://dx.doi.org/10.21615/cesp.13.2.2 PSICOLOGÍA The role of the teacher’s emotional intelligence for efficacy and classroom management The role of the teacher’s emotional intelligence for efficacy and classroom management Pág 22 Pág 22 A statistically positive correlation is expected between “personal teaching efficacy” and efficacy for “classroom management and discipline”. Measures A personal and professional information form was developed by the researchers to collect information from participates (gender, service time, and academic formation). Emotional Intelligence was assessed through the Emotional Skills and Competence Questionnaire for Teachers (ESCQ-T; Valente & Lourenço, peer review), adaptation of Emotional Skills and Competence Questionnaire (ESCQ; Takšić, 2000). The answers were evaluated on a 6-point Likert scale, ranging from 1 (never) to 6 (always). ESCQ-T includes 45-item distributed in three dimensions: (a) Perceive and understand emo- tions, 15 items (α = 0.91) (e.g., When I see how a student feels, I usually know what has happened to him/her); (b) Express and classify emotions, 14 items (α = 0.85) (e.g., I can express my emotions well); and (c) Manage and regulate emotions, 16 items (α = 0.87) (e.g., I can stay in a good mood even if something unpleasant happens in the classroom). Cronbach’s alpha in this study was α = 0.91. Teacher efficacy was assessed through the Teacher Efficacy in Classroom Manage- ment and Discipline Scale (TECMDS; Emmer & Hickman, 1991), adapted from the scale of Gibson and Dembo (1984). The TECMDS includes 36-item distributed in three dimensions: Personal teaching efficacy (teacher’s efficacy); External influences; and Efficacy for classroom management and discipline. The answers were evaluated on a Likert scale with 5 points, ranging from 1 (strongly disagree) to 5 (strongly agree). In this study, only the “personal teaching efficacy” and the “efficacy for classroom management and discipline” dimensions were applied: (a) Personal teaching effi- cacy, 7 items (α = 0.79) (e.g., If one of my students cannot perform an activity, I am able to accurately evaluate the correct level of difficulty); (b) and Efficacy for class- room management and discipline, 15 items (α = 0.86) (e.g., If a student in my class becomes disturbing and noisy, I make sure I know techniques to correct it quickly). Cronbach’s alpha in this study was α = 0.85. Participants p The participants were 634 school teachers (5th to 12th grade), 68.8% women and 31.2% men. Service time among participants varied: 7.3% with less than 10 years, 27.1% between 10-20 years, 41.5% between 21-30 years, and 24.1% with more than 30 years of service time. Regarding academic formation varied: 2.8% had a bachelor’s degree, 75.7% undergraduate degree, 19.7% master’s degree and 1.7% had a Ph.D. The sample of the study was of convenience. All teachers had Portuguese nationality and working in public schools. Data analysis The present study used descriptive statistics, Pearson’s correlation tests, and the Structural Equation Models (SEM; Lowe, Winzar, & Ward, 2007) technique. The sta- tistical analyses were performed using SPSS/AMOS 25 (Arbuckle, 2012). No missing data were recorded since all the collaborating school teachers completed the ques- tionnaires in full. So, all the collected data were considered valid. The model fit was estimated considering the statistical indices: GFI and AGFI: values ≥ 0.90 indicate an acceptable fit, values ≥ 0.95 indicate a good fit; CFI: values ≥ 0.95 indicate a good fit of the model (Hu & Bentler, 1999); TLI: values ≥ 0.95 reveal a robust fit (Hair, Anderson, Tatham, & Black, 2005); RMSEA: values between 0.08-0.05 indicate a reasonable fit, and values < 0.05 indicate a good fit (Byrne, 2010); and CN: a value > 200 is indicative that the model adequately represents the sample data (Hoelter, 1983). It was considered as criterion that asymmetry values greater than two and kurtosis values greater than seven should not be considered (Finney & DiStefano, 2013). For Pearson’s coefficient r it is assumed that: (a) an r < 0.200 indicates a very low; (b) between 0.200-0.399 low; (c) between 0.400-0.699 moderate; (d) between 0.700-0.899 high; and (e) and between 0.900-1 very high (Pestana & Gageiro, 2014). Procedure All the procedures were conducted with permission from the General Directorate of Education (Ministry of Education), Ethics committees of the authors’ institutions, schools’ Directors, and Teachers participants. Researchers explained the purpose of the study and administered the questionnaires, in groups of 10-20 teachers, during a session lasting from 30 minutes, at school, during 2018. The conventional ethical and deontological procedures were defined, and teachers were informed about the confidentiality and anonymity of the collected data before they gave their consent to participate voluntarily in the study. In total, 700 questionnaires were delivered and http://dx.doi.org/10.21615/cesp.13.2.2 PSICOLOGÍA Valente, S., Lourenço, A.A., Alves, P., & Domínguez-Lara, S. Pág 23 634 fully filled were returned. 90.6% of teachers agreed to participate with the study, and only 9.4% were not filled in due to a lack of teacher availability. 634 fully filled were returned. 90.6% of teachers agreed to participate with the study, and only 9.4% were not filled in due to a lack of teacher availability. http://dx.doi.org/10.21615/cesp.13.2.2 http://dx.doi.org/10.21615/cesp.13.2.2 Results The overall goodness indices of proposed SEM are very robust: χ2 = 9.132; p = 0.243; χ2/d.f. = 1.305; GFI = 0.996; AGFI = 0.982; CFI = 0.987; TLI = 0.947; RMSEA = 0.022, confirming the hypothesis that projected model represents the relations between existing variables in our empirical matrix. The Hoelter index values were also adjus- ted [CN = 976 (0.05) and 1281 (0.01)]. Figure 1 specifies the hypothesized model for 634 school teachers. Figure 1. Individual standardized parameters of hypothesized specified model (n = 634) Figure 1. Individual standardized parameters of hypothesized specified model (n = 634) http://dx.doi.org/10.21615/cesp.13.2.2 The role of the teacher’s emotional intelligence for efficacy and classroom management PSICOLOGÍA Pág 24 Pág 24 Table 1 shows the descriptive data (minimum, maximum, mean, standard deviation, asymmetry and kurtosis) corresponding to variables included on SEM. In sample, no variable reveals values close to defined by Finney and DiStefano (2013), so is justi- fied to proceed with estimation of the fit model. Table 1 shows the descriptive data (minimum, maximum, mean, standard deviation, asymmetry and kurtosis) corresponding to variables included on SEM. In sample, no variable reveals values close to defined by Finney and DiStefano (2013), so is justi- fied to proceed with estimation of the fit model. Table 1. Descriptive Statistics Corresponding to the Variables Variable Min. Max. Mean SD Assymmetry Kurtosis Gender 1 2 - - -0.81 -1.34 Service time 1 4 - - -0.29 -0.67 Academic formation 1 4 - - 1.13 1.99 PUE 15 90 68.33 10.60 -0.49 2.83 ECE 14 84 60.56 17.91 -1.26 1.04 MRE 16 96 73.69 16.13 -1.52 3.41 PTE 7 35 19.62 7.77 0.18 -0.79 ECMD 15 75 57.04 9.60 -0.40 1.46 Note. SD = Standard Deviation; PUE = Perceive and Understand Emotions; ECE = Express and Classify Emotions; MRE = Manage and Regulate Emotions; PTE = Personal Teaching Efficacy; ECMD = Efficacy for Classroom Management and Discipline Table 1. Descriptive Statistics Corresponding to the Variables Note. SD = Standard Deviation; PUE = Perceive and Understand Emotions; ECE = Express and Classify Emotions; MRE = Manage and Regulate Emotions; PTE = Personal Teaching Efficacy; ECMD = Efficacy for Classroom Management and Discipline From the analysis of Table 2 and Figure 1, it can be determined that most of the hypo- theses guiding the specifications have been confirmed and are statistically significant. Results From the analysis of Table 2 and Figure 1, it can be determined that most of the hypo- theses guiding the specifications have been confirmed and are statistically significant. Results indicated that women presented higher results than men in all EI capacities, na- mely a positive relation with the capacities for perceive/understand emotions (β = 0.15; p < 0.001), for express/classify emotions (β = 0.09; p < 0.05), and to manage/regulate emotions (β = 0.08; p < 0.05). When considering service time, results indicated that teachers with more teaching expe- rience show lower EI capacities, in the three EI capacities, namely lower capacity for per- ceive/understand emotions (β = -0.11; p< 0.01), for express/classify emotions (β = -0.11; p < 0.01), and for manage/regulate emotions (β = -0.09; p< 0.05). Regarding academic formation, results suggest teachers with more academic formation have higher level of EI in all dimensions, namely, more capacity for perceiving/understanding emotions (β = 0.10; p < 0.01), for expressing/classifying emotions (β = 0.09; p < 0.05) and for managing/ regulating emotions (β = 0.15; p < 0.001). These relations are positive and statistically significant. Considering existing relations between dimensions of the two constructs, the results indicated that teachers who tend to have higher levels of capacity in perceiving/unders- tanding emotions showed higher levels of teacher efficacy (β = 0.08; p < 0.05), and higher levels of efficacy for classroom management/discipline (β = 0.08; p < 0.05). Teachers who tend to have higher levels for capacity to manage/regulate emotions showed higher levels of teacher efficacy (β = 0.08; p< 0.05), and higher levels in efficacy for classroom management/discipline (β = 0.10; p < 0.05). Teachers who tend to have higher levels in expressing/classifying emotions showed higher levels of teacher efficacy (β = 0.13; p < 0.001) and higher levels in efficacy for classroom management/discipline (β = 0.08; p < 0.05). http://dx.doi.org/10.21615/cesp.13.2.2 PSICOLOGÍA Valente, S., Lourenço, A.A., Alves, P., & Domínguez-Lara, S. Pág 25 Pág 25 Table 2. Results Covariance Structure Hypothesized for the Sample Variables EVnS SEV EE p Gender → PUE 3.53 0.15 0.89 * Gender → MRE 2.83 0.08 1.36 0.037 Gender → ECE 3.59 0.09 1.52 0.019 Service time → PUE -1.32 -0.11 0.47 0.005 Service time → MRE -1.69 -0.09 0.71 0.018 Service time → ECE -2.15 -0.11 0.81 0.008 Academic formation → PUE 2.14 0.10 0.82 0.009 Academic formation → MRE 4.86 0.15 1.24 * Academic formation → ECE 3.18 0.09 1.41 0.024 PUE → MRE 0.20 0.13 0.06 * ECE → MRE 0.08 0.09 0.04 0.026 PUE → PTE 0.06 0.08 0.03 0.035 MRE→ PTE 0.04 0.08 0.02 0.046 ECE → PTE 0.06 0.13 0.02 * PUE → ECMD 0.07 0.08 0.04 0.042 MRE→ ECMD 0.06 0.10 0.02 0.014 ECE → ECMD 0.05 0.08 0.02 0.034 PTE → ECMD 0.11 0.09 0.05 0.029 Covariances Gender ↔ Service time -0.04 -0.09 0.02 0.028 Gender ↔ Academic formation -0.02 -0.08 0.01 0.047 Academic formation ↔ Service time -0.05 -0.12 0.02 0.004 Note. PUE = Perceive and Understand Emotions; ECE = Express and Classify Emotions; MRE = Manage and Regulate Emotions ; PTE = Personal Teaching Efficacy; ECMD = Efficacy for Classroom Management and Discipline; EVnS = Estimated Values not Standardized; SEV = Standardized Estimated Values; EE = Estimated Errors; p = significance level;* = 0.000 Table 2. Covariance Structure Hypothesized for the Sample Note. PUE = Perceive and Understand Emotions; ECE = Express and Classify Emotions; MRE = Manage and Regulate Emotions ; PTE = Personal Teaching Efficacy; ECMD = Efficacy for Classroom Management and Discipline; EVnS = Estimated Values not Standardized; SEV = Standardized Estimated Values; EE = Estimated Errors; p = significance level;* = 0.000 Furthermore, teachers who tend to have higher values for the capacities to perceive/ understand emotions (β = 0.13; p < 0.001), and to express/classify emotions (β = 0.09; p < 0.05) showed more capacity for manage/regulate emotions, and both relations are statistically significant. It was also possible observed that teacher’s whit more effi- cacy for teaching showed more efficacy for classroom management/discipline (β = 0.09; p < 0.05). Furthermore, teachers who tend to have higher values for the capacities to perceive/ understand emotions (β = 0.13; p < 0.001), and to express/classify emotions (β = 0.09; p < 0.05) showed more capacity for manage/regulate emotions, and both relations are statistically significant. Results It was also possible observed that teacher’s whit more effi- cacy for teaching showed more efficacy for classroom management/discipline (β = 0.09; p < 0.05). Moreover, it can be mentioned that female gender had less service time (β = -0.9; p < 0.05), and lower academic formation (β = -0.08; p < 0.05). However, teachers who had more service time presented less academic formation (β = -0.12; p < 0.001). These relations are negative and statistically significant (cf. Table 2). Regarding the multiple square correlations, these indicate that variables such as gender, service time and academic formation explain the capacities for perceive/ understand emotions in 5 % (η2 = 0.049), express/classify emotions in 3 % (η2 = 0.030), and for manage/regulate emotions with a value close to 8 % (η2 = 0.083). Regar- ding the variables efficacy for teaching and for classroom management/discipline, they are both explained indirectly by gender, service time and academic formation; and, directly, by the variables concerning the EI capacities and both are explained by approximately 4 %, with η2 = 0.037 for the first and η2 = 0.041 for the second. http://dx.doi.org/10.21615/cesp.13.2.2 The role of the teacher’s emotional intelligence for efficacy and classroom management PSICOLOGÍA Pág 26 Pág 26 As can be seen in Table 3, concerning the Pearson r correlations between the varia- bles included in the model, it appears that the majority of the model variables are related to each other and are statistically significant, although the adjustment has very low values. Table 3. Pearson’s Correlation between the Variables G ST AF PUE ECE MRE PTE ECMD Gender (G) - Service time (ST) -0.088* - Academic formation (AF) -0.079* -0.116 - PUE 0.156 -0.135 0.102 - ECE 0.095* -0.124** 0.094* 0.121 - MRE 0.106 -0.145 0.177 0.182 0.135 - PTE 0.036 -0.080* 0.060 0.114 0.155 0.113** - ECMD 0.087* -0.082* .040 0.118 0.120 0.134 0.120 - Note. PUE = Perceive and Understand Emotions; ECE = Express and Classify Emotions; MRE = Manage and Regulate Emotions; PTE = Personal Teaching Efficacy; ECMD = Efficacy for Classroom Management and Discipline; ** p<0.01; * p<0.05 Table 3. Pearson’s Correlation between the Variables Note. Results PUE = Perceive and Understand Emotions; ECE = Express and Classify Emotions; MRE = Manage and Regulate Emotions; PTE = Personal Teaching Efficacy; ECMD = Efficacy for Classroom Management and Discipline; ** p<0.01; * p<0.05 The most obvious associations correlate between capacity to manage/regulate emotions with the capacity for perceive/understand emotions (r = 0.182; p <0.01), and the academic formation (r = 0.177; p <0.01), being positive and statistically sig- nificant. There was also a positive and statistically significant association between the capacity to perceive /understand emotions and the capacity to express/classify emotions (r = 0.121; p <0.01). Note that the three EI capacities are associated with all other variables, which showed some cohesion in the estimation of proposed model. It should also be noted that service time is the only exogenous variable associated with all other variables, and these values are negative and statistically significant. Discussion Which enables teachers have a good ability for manage/regulate emotions. Like in the study of Iskandar et al. (2009), the findings of the present investigation indi- cate that EI plays a significant role in teacher’s efficacy, and in increasing performance at work. Results have showed that EI capacities (perceive/understand emotions; ex- press/classify emotions; and manage/regulate emotions) have a statistically positive correlation with teacher’s efficacy (PUE: r = 0.114; p < 0.01; ECE: r = 0.155; p< 0.01; MRE: r = 0.113; p< 0.01), as previous study (Hassan et al., 2015; Koçoğlu, 2011; Wenn et al., 2018), confirming hypotheses 6. These results showed that a teacher with de- veloped EI capacities presents more methods to enhance the teaching and learning process. Also, Bandura (1997) indicated that teacher’ EI influence teaching compe- tence and teacher efficacy by impacting their motivation. The teacher who tends to have higher levels of EI ca- pacities, have more efficacy for teaching and for class- room management. These teachers easily perceive the students’ emotional state and adapt their behavior, altering the activity within the classroom. When they perceive that students are disinterested, they have the sensitivity to criticize a more sensitive student, arran- ges the organization of the tables in the room according to the class, and separates the students who are likely to have more friction. Concerning hypotheses 7, the results have showed a statistically positive correlation between EI capacities and efficacy for classroom management/discipline (PUE: r = 0.118; p < 0.01; ECE: r = 0.120; p < 0.01; MRE: r = 0.134; p < 0.01). Similar to other EI studies (Hen & Sharabi-Nov, 2014; Valente, 2019; Valente et al., 2019; Wahyuddin, 2016) confirming hypotheses 7. Therefore, EI capacities favor the teacher’s work in classroom management. Thus, providing better management of activities, space, time, and students’ behavior, which promotes the teaching and learning process. Aiming consideration on analysis the role of the EI capacities on teacher’s efficacy and classroom management efficacy, the results showed that the way teachers perceive their emotions, express them and internalize the capacity to manage and regulate with those emotions, influences their school praxis, essentially in their understanding of their self-efficacy, as well as their efficacy for manage all kinds of experiences that are developing in classroom space. The relevance of this study showed that teacher’s EI capacities must be understood as something to be managed in a constructive and proactive way. Discussion Similar to other studies (Gill & Sankulkar, 2017; Valente, 2019), the results have showed that gender was a statistically positive correlation with EI capacities (PUE: r = 0.156; p <0. 01; ECE: r = 0.095; p <0. 05; MRE: r = 0.106; p <0.01). Therefore, tea- chers’ women presented higher results than men in all EI capacities, confirming hypothesis 1. These results may be justified by the fact that women are more so- cially accepted to show their emotions, allowing them to develop their emotional capacities. Concerning hypotheses 2, similar with other studies, with Portuguese teachers (Sousa, 2011; Valente, 2019), results have showed that service time was a statistically negative correlation with EI capacities (PUE: r = -0.135; p <0. 01; ECE: r = -0.124; p <0.01; MRE: r = -0.145; p<0.01). These results can be interpreted by the fact that teachers, in Portugal, are an aged class, suffering many of them from stress and burnout which affects their emotional capacity. When teacher’s academic formation was considered the results have showed a statistically positive correlation with EI capacities (PUE: r = 0.102; p <0.01; ECE: r = 0.094; p<0.05; MRE: r = 0.177; p < 0.01), as in other studies on Portuguese school teachers (Fernandes, 2015; Valente et al. 2019), confirming hypothesis 3. Concerning hypotheses 4, the results have showed a statistically positive correla- tion between the capacity for perceive and understand emotions and the capacity to manage and regulate emotions (r = 0.182; p < 0.01). For example, teachers who can http://dx.doi.org/10.21615/cesp.13.2.2 PSICOLOGÍA Valente, S., Lourenço, A.A., Alves, P., & Domínguez-Lara, S. PSICOLOGÍA Pág 27 perceive the emotions students feel are better able to manage the emotions (posi- tive or negative) that occur in the classroom and change the pace of the classroom in accordance with their emotional recognition. Also found a statistically positive correlation between the capacity to express/classify emotions and the capacity for manage/regulate emotions (r = 0.135; p < 0.01), confirming hypotheses 5. In short, this capacity to distinguish differences between verbal behavior and expressive manifestations of students allows teacher to know that, even when student initially claims that nothing happened; it is not entirely true and offers to speak and listen to the same, providing support and understanding. In order to do this the teacher needs to have good capacities in the scope of emotional perception and expression. Conclusion The present study investigated the relationship between EI capacities, teacher efficacy, and classroom management efficacy, and also analyzed how school teacher’s EI capa- cities are influenced by gender, service time and academic formation. The findings of this study supported all hypotheses. This result demonstrated the benefits of teacher’s EI capacities, confirming the relationship between EI capacities and the efficacy for teaching and classroom management. Although the results of this study are encouraging, they must be analyzed in light of some limitations. First, this study used two self-report questionnaires. They measure perception, which may lead teachers to respond according to what they consider socially necessary. Second, the sample size limits the generalization of results, as it represents a small sample of the population of teachers in Portugal. Another limitation was that the teachers in the study were mostly women, limiting the variability of the sample. However, this reveals the reality of Portuguese schools, where men teachers are the minority. Therefore, the primary recommendation for future studies is complement the re- sults through triangulation of data collection such as interviews and classroom ob- servation. Second, future studies should involve larger samples. Finally, we should also examine the effect age on teacher’s EI capacities. In conclusion, the finding of this study proved the significance of EI and their influen- ce on the teachers’ work, specifically that EI capacities play an important role in effi- cacy for teaching and classroom management. In light of these findings, emotional education programs should be included in teachers' academic formation, to develop EI capacities so that they have a more effective professional activity. Discussion The teacher who tends to have higher levels of EI capacities, have more efficacy for teaching and for classroom management. These teachers easily perceive the students’ emotional state and adapt their behavior, altering the activity within the classroom. When they perceive that students are disinterested, they have the sensitivity to criticize a more sensitive student, arranges the organization of the tables in the room according to the class, and separates the students who are likely to have more friction. An intelligent perception and management of the aspects associated with a daily praxis in the school context will certainly be salutary for the personal development of students, as well as for the structuring of positive and self-regulating environ- ments in their learning. In general, it can be emphasized that results found indicate that EI capacities influence the perceptions that teachers have about their efficacy for teaching, and so for classroom management. Aware of this complexity, currently http://dx.doi.org/10.21615/cesp.13.2.2 The role of the teacher’s emotional intelligence for efficacy and classroom management The role of the teacher’s emotional intelligence for efficacy and classroom management PSICOLOGÍA Pág 28 lived in the school, it is expected that this framework of successive constructions and reconstructions will design education for a desirable renewal of commitments. The results also have shown a statistically positive correlation between teacher efficacy and efficacy for classroom management/discipline (r = 0.120; p < 0.01), confirming hypotheses 8. Therefore, teacher’s classroom management strategies are affected by their point of view on their teaching skill. References Ahsan, M. T., Sharma, U., & Deppeler, J. M. (2012). Exploring pre-service teachers perceived teaching-efficacy, attitudes and concerns about inclusive education in Bangladesh. International Journal of Whole Schooling, 8(2), 1–20. Allen, V. 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https://openalex.org/W4307808722	http://journal.waocp.org/article_90318_ae4f07e683ae4692bcb05b1e31d5422d.pdf	English	null	Association of Handgrip Strength with Quality of Life in Breast Cancer Survivors: A Systematic Review and Meta-Analysis	Asian Pacific journal of cancer prevention	2,022	cc-by	5,577	Asian Pacific Journal of Cancer Prevention, Vol 23 3237 1Federal University of Pernambuco. Graduate program of Physical Therapy. Recife-PE, Brazil. ²Clinical Epidemiology Program, Brazilian National Cancer Institute (INCA), Rio de Janeiro, Brazil. For Correspondence: diego.sdantas@ufpe.br Abstract Objective: Handgrip strength (HGS) is an indicator of general muscular strength and in cancer patients acts as a relevant marker associated with mortality and health. This study aimed to evaluate the association between peripheral muscle function and health-related quality of life (HRQoL) in breast cancer (BC) survivors. Methods: Systematic review registered on PROSPERO under number: CRD 42021225206. The searches were carried out on MEDLINE via Pubmed, PEDro, Cochrane Library, Embase, CINAHL via EBSCO and Science Direct databases. Observational studies evaluating the association between handgrip strength (HGS) and HRQoL in adult female BC survivors were included. No linguistic or time restrictions were applied. Two reviewers reviewed full texts for inclusion and performed data extraction and risk of bias using the Newcastle and Ottawa scale (NOS). Results: Five articles were included and involved 587 patients, mean age of 47 to 59 years. The percentage of decreased HGS ranged from 38.3% to 60.3%. HGS was associated with different quality of life measures. From meta-analysis including 220 patients, the correlation coefficient between HGS and HRQoL was 0.26 (95% CI: 0.07-0.35). Conclusions: Breast cancer survivors face decline of HGS. In this population HGS was correlated with HRQoL. However, more evidence are necessary. eywords: Muscle strength- hand strength- breast neoplasm- clinical evaluation- quality of life indicators Asian Pac J Cancer Prev, 23 (10), 3237-3245 Asian Pac J Cancer Prev, 23 (10), 3237-3245 al., 2018), muscle strength is a primordial component in the physical examination of these patients, and should be included as a therapeutic objective in care protocols to better direct recovery and rehabilitation strategies, thus minimizing the adverse effects of declining muscle function (Cantarero-Villanueva et al., 2012). Association of Handgrip Strength with Quality of Life in Breast Cancer Survivors: A Systematic Review and Meta-Analysis Alana Cristina Campos e Silva1, Anke Bergmann2, Camilla Medeiros Araujo1, Andrea Karla Soares Montenegro1, Angelica da Silva Tenório1, Diego Dantas1 REVIEW Editorial Process: Submission:04/26/2022 Acceptance:10/07/2022 DOI:10.31557/APJCP.2022.23.10.3237 Handgrip Strength and Quality Of Life DOI:10.31557/APJCP.2022.23.10.3237 Handgrip Strength and Quality Of Life REVIEW Introduction Breast cancer (BC) is the most common malignancy in women worldwide, and is the leading cause of cancer related-deaths in women (Bray et al., 2018). Although scientific and technological advancements have enabled the early diagnosis and timely treatment of BC, many women experience the adverse effects of the disease and complications in treatment, which eventually affect their health-related quality of life (HRQoL) negatively (Annunziata et al., 2018). Handgrip strength (HGS) is an indicator of general muscular strength that has been widely used in clinical evaluations and scientific research because of its practicality and low cost (Bohannon, 2019; Jochemet al., 2019). Previous studies have demonstrated the capability of HGS in cancer patients as a marker of clinical and nutritional status (Keaver et al., 2021), a predictor of decreased functionality and functional capacity (Duarteet al., 2020; Perez et al., 2018; Rijket al., 2016), and as a predictor of worse HRQoL and mortality in BC patients (Cantarero-Villanueva et al., 2012; Duarte et al., 2020; Paek and Choi, 2019b; Zhuang et al., 2020). However, the evidence regarding the predictive power of HGS in the HRQoL of BC survivors is not well delineated. In this context, this systematic review aimed to evaluate the association between HGS and HRQoL in women with BC. With the increase in survival and life expectancy of women with BC, the HRQoL has become an even more important therapeutic target (Koniecznyet al., 2020). This is because the QoL of cancer survivors is lower than that of individuals without cancer, thus demonstrating the need for monitoring and interventions (Schmidtet al., 2018). The various treatments for BC (surgery, chemotherapy, radiotherapy, targeted therapy, and hormone therapy) have several consequences, including a negative effect on muscle function, resulting in fatigue and sarcopenia and leading to a poorer QoL for BC survivors (Guigni et al., 2018; Klassen et al., 2017). Although BC is not typically characterized by cases of cachexia (Guigni et Alana Cristina Campos e Silva et al the quality of the articles included (Stang, 2010). The NOS is based on a star rating system, and a maximum of 9 stars can be attributed to each study (for prospective and cross-sectional studies). The quality assessment was independently verified by two authors, and any disagreements were resolved by a third reviewer. The studies that received a score equal or higher than 7 were considered of high quality (Bae, 2016). Statistical analysis R software (version 4.2.1) was used for meta-analysis of correlation. The meta-analysis method involved the inverse variance method, restricted maximum-likelihood estimator for tau^2, and Fisher’s z transformation of correlations. The studies that reported correlation coefficient (r value) were pooled in different analysis, considering the total score or quality of life questionnaire domains which are comparable with each other. The heterogeneity of studies was assessed by the Q-statistic and I2 statistics. For studies heterogeneous (I2>50%) the effect size was determined by random effects model. Otherwise, common effects model was used. Funnel plots were used to determine publication bias. The search for the articles was performed on August 18, 2021 by D.D. in MEDLINE via the Pubmed, PEDro, Cochrane Library, Embase, CINAHL EBSCO, and Science Direct databases. The search strategy involved keywords combined by boolean operators as follows: (breast cancer OR breast tumor OR breast cancer) AND (handgrip OR hand strength) AND (quality of life) AND (association OR correlation OR relationship) AND (cross- sectional study OR observational study). Studies obtained from other sources or article references, provided they met the eligibility criteria, were also included. Characteristics of the studies i The five articles included in this review (Brandini et al., 2019; Buyukakincak et al., 2014; Esteban-Simón et al., 2021; Kaya et al., 2010; Koca et al., 2020) have a cross-sectional design and involved a total of 587 women BC survivors who were mostly recruited from Turkey (Buyukakincak et al., 2014; Kaya et al., 2010; Koca et al., 2020) with a mean age >50 years. The characteristics of the studies are summarized in Table 1. Agreement between the reviewers was measured using the Kappa coefficient, and the values were interpreted as no to slight (0.01–0.20), reasonable (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80), and near perfect (0.80–1.00) agreement (McHugh, 2012). The primary endpoint of this review was the association between HGS and HRQoL, while the secondary endpoint was HGS deficit, which was calculated by the difference in HGS between the affected and non-affected sides. Of the studies included, only the study by (Buyukakincak et al., 2014) evaluated patients in the immediate postoperative period of mastectomy for BC. The other studies included patients who were in adjuvant treatment or had concluded systemic treatments for cancer. Patients with lymphedema were also included in the studies. Selection processi After identification of the citations in the databases, we proceeded with the removal of duplicate entries through Mendeley software (Elsevier Inc., USA, New York). The screening of articles was performed based on reading the title and abstract by two independent reviewers (A.C.C.S. and C.M.A.). In the next step, the reviewers evaluated the studies through a full reading, and proceeded with the application of the eligibility criteria. Disagreements were resolved by a third researcher (D.D.). Results A total of 178 studies were identified, of which 37 duplicate entries were excluded. Of the 11 studies selected for full reading, the most common reason for exclusion was not presenting association data between HGS and HRQoL. In addition, two studies were congress abstracts and the full versions were not available, one study evaluated HGS in another population, and one study was excluded because it was a systematic review. The process is detailed in Figure 1. The screening process showed substantial inter-rater agreement (k = 0.677). Material and Methods A systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (Page et al., 2021). The review protocol was registered in the Prospective Register of Systematic Reviews (PROSPERO): CRD 42021225206. For this review, transversal observational or cohort studies (prospective or retrospective) that analyzed the association between HGS and HRQoL in women BC survivors were deemed eligible. Studies where HGS was evaluated using a manual dynamometer and HRQoLHRQ through a validated questionnaire were included. Studies that did not present isolated data on the association between the outcomes HGS and HRQoL, as well as conference abstracts, were excluded. No restrictions were imposed in terms of the date or language of publication. Methodological quality and strength of evidence Methodological quality and strength of evidence In most studies, (Brandini da Silva et al., 2019; Buyukakincak et al., 2014; Kaya et al., 2010; Koca et al., 2020) the HGS was measured in both hands and the protocol involved the performance of three measurements for each side with registration of the mean value obtained. In the study by Esteban-Simón et al., (2021) two attempts were made on each hand in an alternating manner, and the best result for each was selected. In addition, in this study the authors used the HGS in absolute and relative form, which consists of the absolute value of the HGS of both hands divided by the body mass index (BMI). i The evaluation of the quality of the study using the NOS of the five studies is presented in Table 2. All studies showed high methodological quality, with their only weakness being the insufficient follow-up of participants. Data extraction The articles included were reanalyzed and the relevant data were extracted by the two reviewers using an electronic spreadsheet. The primary studies collected data on authorship, country where the study was conducted, year of publication, sample size, mean age of participants, protocol used to measure HGS, forms of QoL evaluation, type of association analysis between variables, and main results. The performance of bilateral mastectomy was an exclusion criterion for most studies (Brandini da Silva et al., 2019; Buyukakincak et al., 2014; Kaya et al., 2010; Koca et al., 2020). In the study by (Esteban-Simón et al., 2021) there was no information regarding this exclusion criterion. In all studies, participants with previous shoulder or musculoskeletal problems in the upper limbs were excluded. Methodological quality assessment The Newcastle-Ottawa scale (NOS) was used to assess Methodological quality assessment The Newcastle-Ottawa scale (NOS) was used to assess Asian Pacific Journal of Cancer Prevention, Vol 23 3238 DOI:10.31557/APJCP.2022.23.10.3237 Handgrip Strength and Quality Of Life DOI:10.31557/APJCP.2022.23.10.3237 Handgrip Strength and Quality Of Life Figure 1. Flowchart of Systematic Review Selection Figure 1. Flowchart of Systematic Review Selection instruments: Breast Cancer Treatment Outcome Scale (BCTOS), 36-Item Short Form Survey Instrument (SF- 36), European Organization for Research and Treatment of Cancer (EORTC-QLQC-30) and complementary module for BC, Functional Assessment of Cancer Therapy - Breast (FACT-B), and the World Health Organization Quality of Life (WHOQOL-bref). Assessment of HRQoL HRQoL was evaluated using different validated Asian Pacific Journal of Cancer Prevention, Vol 23 3239 HGS measures All measurements of HGS were performed with the use of a dynamometer. In two studies (Buyukakincak et al., 2014; Koca et al., 2020) the Jamar model dynamometer was used; one study (Brandini da Silva et al., 2019) used a Saehan model SH50 dynamometer; another used a digital (Esteban-Simón et al., 2021) used a digital dynamometer, model TKK 5401 Grip-D; and one study (Kaya et al., 2010) did not specify the brand. Summary of the results Only two studies reported the HGS deficit as a dichotomous variable, and the prevalence of HGS decrease ranged from 38.3% (Brandini da Silva et al., 2019) to 60.3% (Kaya et al., 2010). Regarding the association between HGS and HRQoL, the main results of the studies were summarized in table 1. The studies that performed the association between HGS and HRQoL by correlation coefficient were pooled in meta-analysis of correlation (Figure 2). The correlation coefficient between HGS x score total of HRQoL was 0.26 (95% CI: 0.07-0.35). The analysis includes 220 patients and showed heterogeneity (I2 = 53%; p= 0.12). Three studies reported data on HGS deficit of the evaluated patients. In two studies (Buyukakincak et al., 2014; Kaya et al., 2010) a value equal or greater than 10% of difference in HGS between the affected and non-affected side was considered as HGS impairment. For (Brandini da Silva et al., 2019) the cut-off point was an alteration between the limbs of 12%. Other meta-analysis of correlation were performed for quality of life questionnaire domains which comparable with each other. The correlation coefficient for HGS x Assessment of HRQoL HRQoL was evaluated using different validated Asian Pacific Journal of Cancer Prevention, Vol 23 3239 Alana Cristina Campos e Silva et al Cristina Campos e Silva et al Asian Pacific Journal of Cancer Prevention, Vol 23 Author Country/ Year Sample Size Age (mean ± SD) Measurement of outcomes Association Measure Main Results Handgrip Strength (HGS) Health-related Quality of Life (HRQoL) Brandini et al. Brazil 2019 300 58,8 ± 9,6 Equipment: Saehan Hydraulic dynamometer HGS calculated from the average of three measurements. The decrease in HGS was considered a difference of 12% of the LA compared to the LNA. Breast Cancer Treatment Outcome Scale (BCTOS) ROC Curve Analysis HGS deficit 38.3% of patients. Association HGS Deficit X BCTOS scores: Functional status (AUC=0.623; p<0.001); Breast specific pain (AUC =0.596; p=0.005); Edema (AUC=0.583; p=0.013) Büyükakincak et al. Turkey 2014 93 47,5 ± 8,8 Equipment: Jamar hydraulic dynamometer. HGS calculated from the average of three measurements in both hands of the patient. Patient with forearm in neutral position and elbow flexed 90°. 36-Item Short Form Survey Instrument (SF-36) Correlation Analysis "The HGS values of the participants were not informed. HGS X HRQoL Association: Correlation HGS in the Functional capacity domain r=0.245 p<0.05 Physical aspects domain r=0.227 p<0.05" Esteban- Simón et al. Asian Pacific Journal of Cancer Prevention, Vol 23 3241 Summary of the results Spain 2021 60 52,3± 9,0 Equipment: Digital dynamometer (TKK 5401 Grip-D) . HGS calculated as the best result of two trials on each side. It used absolute HGS and relative HGS (absolute HGS/IMC). It did not specify the measures of decrease of HGS. European Organization for Research and Treatment of Cancer (EORTC-QLQC-30 and complementary module for breast cancer. Correlation Analysis "HGS X HRQoL Association: Absolute HGS: BR23 - arm symptoms/discomfort (r=-0.346; p=0.008) Relative HGS EORTC-QLQC-30 global health status/quality of life (r=0.387; p=0.003); physical function (r=0.254; p= 0.054); fatigue (r=-0.274; p=0.038); BR23 - arm symptoms/discomfort (r=-0.332; p=0.011) " Kaya et al. Turkey 2010 67 53,7 ± 12,3 "Equipment: Dynamometer - brand and protocol not specified. HGS calculated from the average of three trials for each side. HGS decrease criterion: 10% difference of LA compared to LNA." Functional Assessment of Cancer Therapy - Breast (FACT-B) Multivariate Regression Analysis "Deficit of HGS = 60.3%. Deficit of HGS = 60.3% HGS X HRQoL Association: Physical well-being (B=-3.20; r2 =0.093 p=0.012 (Multivariate Regression). " Koca et al. Turkey 2020 67 50,4 ± 11,2 "Equipment: Jamar hydraulic dynamometer. Equipment: Jamar hydraulic dynamometer. HGS calculated as the average of three measurements obtained according to the recommendations of the American Society of Hand Therapy with a 20-second rest between measurements." World Health Organization Quality of Life (WHOQOL- bref) Correlation analysis HGS X HRQoL Association: General health score: LD (r=0.370; p=0.006)/LE (r=0.414; p=0.002); Physical health score: LD (r=0.465; p≤0.001)/LE (r=0.379; p=0.005); Social health score: LD (r= 0.352; p= 0.011)/LE (r=0.300; p=0.003); Environmental score: LD (r=0.438; p= 0.001)/LE/ r=0.459; p=0.001); Total WHOQOL score: LD/ r=0.406; p=0.003)/LE (r=0.356; p=0.01) Table 1. Characteristics of the Studies Included in the Systematic Review. Summary of the results HGS, manual grip strength; HRQoL, quality of life; LD, right side; LE, left side; SD , Standard deviation; LA, affected side; LNA, non-affected side; AUC, area under ROC curve DOI:10.31557/APJCP.2022.23.10.3237 Handgrip Strength and Quality Of Life Handgrip Strength and Quality Of Lif a) HGS x Total Score of Quality of life b) HGS x Functional Capacity domain c) HGS x Physical Function domain d)HGS x Emmotional aspects domain e)HGS x Social aspects domain f)HGS x Mental health domain a) HGS x Total Score of Quality of life a) HGS x Total Score of Quality of life b) HGS x Functional Capacity domain c) HGS x Physical Function domain d)HGS x Emmotional aspects domain d)HGS x Emmotional aspects domain e)HGS x Social aspects domain e)HGS x Social aspects domain f)HGS x Mental health domain Figure 2. Forest Plots of the Correlation between HGS and HRQoL f)HGS x Mental health domain Figure 2. Forest Plots of the Correlation between HGS and HRQoL f)HGS x Mental health domain Figure 2. Forest Plots of the Correlation between HGS and HRQoL functional capacity was 0.19 (95% CI: 0.03-0.34); and between HGS x emotional aspects was 0.18 (95% CI: 0.02-0.33). For this analysis was not verified heterogeneity (I2 = 0). All analysis and data are shown in Figure 2. functional capacity was 0.19 (95% CI: 0.03-0.34); and between HGS x emotional aspects was 0.18 (95% CI: 0.02-0.33). For this analysis was not verified heterogeneity (I2 = 0). All analysis and data are shown in Figure 2. Publication bias Publication bias functional capacity was 0.19 (95% CI: 0.03-0.34); and between HGS x emotional aspects was 0.18 (95% CI: 0.02-0.33). For this analysis was not verified heterogeneity (I2 = 0). All analysis and data are shown in Figure 2. Funnel Plot asymmetry was observed in the studies of correlation between HGS and HRQoL, total scores and domains. (Figure 3). Egger test was not performed because Funnel Plot asymmetry was observed in the studies of correlation between HGS and HRQoL, total scores and domains. (Figure 3). Discussion This systematic review including studies of high methodological quality showed that handgrip strength has a predictive value for HRQoL scores in women who survived BC. To the best of our knowledge, this is the first article that systematically reviewed evidence regarding this association. Our findings add to the body of evidence that advocates the importance of measuring HGS in clinical practice and rehabilitation protocols for BC patients. Besides that, it should also be noted that this loss of muscle strength may be related to the late effects of other treatments performed (chemotherapy, radiotherapy, targeted therapy and hormone therapy) (Guigni et al., 2018; Klassen et al., 2017) or resultant complications, such as lymphedema, adhesive capsulitis, pain, and musculoskeletal dysfunction of the shoulder (Cantarero- Villanueva et al., 2012; Esteban-Simón et al., 2021; Kaya et al., 2010; Paek and Choi, 2019a). The HGS is a measure of physical function, and plays an important general predictive role for muscle mass and strength.(Paek and Choi, 2019a) In patients with BC, HGS deficits have been associated with lower functional capacity; greater fatigue (Cantarero-Villanueva et al., 2012) shoulder deficits, and kinesiophobia (Koca et al., 2020; Lee et al., 2015); and fewer complications from cancer treatments and hospitalizations (Paek and Choi, 2019a). There is evidence that HGS in women breast cancer survivors is positively associated with various aspects of physical function, given that better HGS levels favor mobility and performance in usual and self-care activities (Paek and Choi, 2019a). This probably reflects on the quality of life, since the HGS was associated with several dimensions of quality of life, assessed through different instruments, the relationship with dimensions referring to physical function is evident. Furthermore, the association with other dimensions, such as arm symptoms/discomfort and psychosocial factors, suggests a multidimensional relationship between HGS and quality of life in this population. As a secondary outcome of this review, two papers presents to a high prevalence of women with decreased HGS on the same side to the surgical site, rates ranged 38% to 60%). This not only indicates the loss of muscle function, but also serves as a sign for several other issues that include activities and social participation, such as work and leisure (Annunziata et al., 2018; Schmidt et al., 2018). This is because muscle strength is a predictor of several clinical indicators and basic function required by these women for several daily activities. Summary of the results Egger test was not performed because Asian Pacific Journal of Cancer Prevention, Vol 23 3241 Alana Cristina Campos e Silva et al a) HGS x Total Score of Quality of life b) HGS x Functional Capacity domain c) HGS x Physical Function domain a) HGS x Total Score of Quality of life a) HGS x Total Score of Quality of life cific Journal of Cancer Prevention, Vol 23 el Plots of the Correlation between HGS and HRQoL a) HGS x Total Score of Quality of life b) HGS x Functional Capacity domain c) HGS x Physical Function domain d)HGS x Emmotional aspects domain e)HGS x Social aspects domain f)HGS x Mental health domain b) HGS x Functional Capacity domain b) HGS x Functional Capacity domain c) HGS x Physical Function domain c) HGS x Physical Function domain d)HGS x Emmotional aspects domain d)HGS x Emmotional aspects domain d)HGS x Emmotional aspects domain e)HGS x Social aspects domain e)HGS x Social aspects domain f)HGS x Mental health domain f)HGS x Mental health domain Asian Pacific Journal of Cancer Prevention, Vol 23 3242 Figure 3. Funnel Plots of the Correlation between HGS and HRQoL DOI:10.31557/APJCP.2022.23.10.3237 Handgrip Strength and Quality Of Life Study Selection Comparability Outcome Total Representativeness of the exposed cohort Selection of the non- exposed cohort Ascertainment of exposure Demonstration that outcome of interest was not present at start of study Comparability of cohorts on the basis of the design or analysis Assessment of outcome Was follow-up long enough for outcomes to occur? Adequacy of follow up of cohorts? Brandini et al. * * * * * * * * 8 Büyükakinca et al. * * * * * * - * 7 Esteban-Simón et al. * * * * * * * * 8 Kaya et al. * * * * * * - * 7 Koca et al. * * * * * * - * 7 Table 2. Quality Methodological Assessment of Included Studies by the Newcastle-Ottawa Scale *, The study met the criteria. Study Selection Comparability Outco Table 2. Quality Methodological Assessment of Included Studies by the Newcastle-Ottawa Scale the number of studies by analysis was insufficient and did not attends the requirements of the test. Summary of the results presence of initial or advanced grade lymphedema is related to lower handgrip strength, possibly due to patients avoiding using the affected arm in usual activities, and this situation usually results in muscle atrophy, weakness, and functional restrictions. The use of multimodal therapies (surgery, radiotherapy, and systemic chemotherapeutic agents) may increase the loss of HGS. Asian Pacific Journal of Cancer Prevention, Vol 23 3243 Discussion Based on cross-sectional studies it was possible to verify that HGS is predictor of HRQoL. A detailed analysis of the correlations or verification of these associations showed that the HGS has a convergence with the domains of the WHOQOL-bref questionnaire, with all the domains of the BCTOS, except for the Aesthetic Status subscale; with the scores of functional capacity and physical aspects from SF-36; with the domain of physical well-being of FACT-B; and global health status/QoL, physical function, task performance, fatigue, loss of appetite and arm symptoms/discomfort domains of EORTC-QLQC-30. Meta-analysis confirms the correlation between HGS and total score of HRQoL, functional capacity and emotional aspects. However, the effects was need interpreted with Besides that, breast cancer survivors are often overweight. These conditions are related to changes in body composition characterized by fat mass gain and lean mass loss. Consequently, it occurs reduced muscle mass and muscle strength and increased fatigue. In addition, cancer treatments such as chemotherapy contribute to structural and biochemical changes in muscle tissue (Guigni et al., 2018). The combination of these factors contributes to the reduction of HGS. Additionally, the study by Baklaci and Cols (2020) showed that the Asian Pacific Journal of Cancer Prevention, Vol 23 3243 Alana Cristina Campos e Silva et al caution because included only three studies that reported the correlation coefficient and these analyses disregard potential confounding factors. CMA: Data Analysis; Manuscript writing/editing; Approval final version of manuscript. CMA: Data Analysis; Manuscript writing/editing; Approval final version of manuscript. CMA: Data Analysis; Manuscript writing/editing; Approval final version of manuscript. AKSM: Data Analysis; Manuscript writing/editing; Approval final version of manuscript. Previously evidence points that HRQoL is a multidimensional construct directly influenced by issues related to pain (Paek and Choi, 2019a), presence of lymphedema, and associated muscle weakness (De Groef et al., 2016; Lee et al., 2015), and treatments and complications resulting in psychological (Sato et al., 2018) and cognitive issues (Baert et al., 2017). Then, these factors may act as confounding factors and need to be included in a more robust analysis. Two studies, not included in meta-analysis, adjust the association’s measures with control of confounding variables and in these studies, HGS remained a predictive variable for HRQoL scores (Brandini da Silva et al., 2019; Kaya et al., 2010). AST: Protocol/project development; Manuscript writing/editing; Approval final version of manuscript. Acknowledgements The authors thank to Universidade Federal de Pernambuco e CAPES for the financial support for translating the manuscript. Discussion DD: Protocol/project development; Data collection or management; Data Analysis; Manuscript writing/editing; Approval final version of manuscript. Conflict of Interest Statement for all authorsi The authors have no relevant financial or non-financial interests to disclose. The authors have no relevant financial or non-financial interests to disclose. Funding statementi As a fast, objective, and low-cost measurement parameter, HGS is a useful tool that can be utilized in the routine evaluation of women with BC. Adjustments in the HGS calculation have been proposed in the literature to reduce biases and increase clinical relevance. The relative HGS is a representative indicator of muscle strength, independent of the influence of body mass (Hao et al., 2020), and is taken by adjusting the value measured in HGS by the BMI (Lawman et al., 2016). In the study of (Esteban-Simón et al., 2021) which comparatively analyzed the associations of HGS and relative HGS, the adjusted calculation of relative HGS had a greater association with the HRQoL scores of women with BC, thus corroborating available evidence from other populations (Hao et al., 2020; Lawman et al., 2016). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001. – Finance Code 001. Data Availability Statement This manuscript use data previously published by other authors and all data are presented into results section. References Annunziata MA, Muzzatti B, Flaiban C, et al (2018). Long-term quality of life profile in oncology: a comparison between cancer survivors and the general population. Support Care Cancer, 26, 651–6. Limitations The limitations of this study include the diversity of questionnaires used to evaluate HRQoL and different association measures reported, which limited the generalization and power of meta-analysis performed. The poor description of the protocols to evaluate the HGS in the primary studies made it difficult to compare the procedural questions between the studies. Bae JM (2016). A suggestion for quality assessment in systematic reviews of observational studies in nutritional epidemiology. Epidemiol Health, 38. Baert IAC, Meeus M, Mahmoudian A, et al (2017). Do psychosocial factors predict muscle strength, pain, or physical performance in patients with knee osteoarthritis?. J Clin Rheumatols, 23, 308–16. Baklaci M, Eyigör S, Tanıgör G, Özgür İM, Çalışkan KS (2020). Assessment of muscle strength and volume changes in patients with breast cancer-related lymphedema. Oncol Res Treat, 43, 584–91. In conclusion, there is evidence that handgrip strength is correlated to HRQoL scores in female BC survivors. Furthermore, it is observed that cancer-related treatments are associated with decreased handgrip strength and, in turn, a decreased HRQoL in these patients. Since measuring handgrip strength has high clinical value is recommended as part of the evaluation of female cancer survivors to detect damage to muscle function and track adverse health outcomes. The HGS is a low cost measure and useful in different contexts, primary health care, offices and hospitals, its use. However, these findings need to be supported by more evidence. Bohannon RW (2019). Grip Strength: An Indispensable Biomarker For Older Adults. Clin Interv Aging, 14, 1681–91. Brandini SFC, José SJ, Sarri AJ, Paiva CE, Vieira RA (2019). Comprehensive validation study of quality-of-life questionnaire using objective clinical measures: Breast Cancer Treatment Outcome Scale (BCTOS), Brazilian Portuguese Version. 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https://openalex.org/W4364323058	https://vspu.net/sit/index.php/sit/article/download/5482/4929	Ukrainian	null	ВИКОРИСТАННЯ НАЦІОНАЛЬНОЇ ОНЛАЙН-ПЛАТФОРМИ З ЦИФРОВОЇ ГРАМОТНОСТІ ПРИ ПІДГОТОВЦІ БАКАЛАВРІВ СПЕЦІАЛЬНОСТІ «ПРАВО»	Sučasnì ìnformacìjnì tehnologìï ta ìnnovacìjnì metodiki navčannâ v pìdgotovcì fahìvcìv: metodologìâ, teorìâ, dosvìd, problemi	2,023	cc-by	4,744	Методологічні проблеми впровадження цифрових технологій та інноваційних методик навчання Методологічні проблеми впровадження цифрових технологій та інноваційних методик навчання ВИКОРИСТАННЯ НАЦІОНАЛЬНОЇ ОНЛАЙН-ПЛАТФОРМИ З ЦИФРОВОЇ ГРАМОТНОСТІ ПРИ ПІДГОТОВЦІ БАКАЛАВРІВ СПЕЦІАЛЬНОСТІ «ПРАВО» Анотація. В умовах цифровізації всіх сфер діяльності людини, юридична галузь також зазнає трансформацій. Очевидно, це має бути враховано і під час підготовки майбутніх правників. У статті обґрунтовується необхідність модернізації підготовки бакалаврів спеціальності «Право» у частині, що стосується опанування сучасними цифровими технологіями та їх подальшого використання в професійній діяльності. На основі аналізу Стандарту вищої освіти першого (бакалаврського) рівня вищої освіти галузі знань 08 Право, спеціальності 081 Право виокремлено компетентності та результати навчання, формування яких безпосередньо залежить від якісного опанування сучасних інформаційно-цифрових технологій. Виокремлено ключові питання, які варто включити до змістового наповнення дисципліни, що відповідатиме за формування цифрової грамотності. Запропоновано використовувати контент національної онлайн-платформи з цифрової грамотності веб-порталу «Дія. Цифрова освіта» під час фахової підготовки студентів спеціальності «Право». Описано структуру та особливості роботи з онлайн-платформою. Серед усього контенту виокремлено освітні серіали (курси), які доречно використовувати при підготовці студентів спеціальності «Право». Коротко охарактеризовано особливості кожного курсу та окреслено перелік питань, які розглядаються. З метою підготовки до складання національного тесту на визначення рівня цифрової грамотності, запропоновано до перегляду перелік освітніх серіалів, розміщених на онлайн-платформі. Зазначено, що освітні серіали (курси) можуть бути рекомендовані викладачем як різновид неформальної освіти. Зроблено висновки щодо векторів модернізації підготовки здобувачів освіти спеціальності «Право» та визначено необхідність подальшого вивчення методичних засад впровадження сучасних онлайн-інструментів та контенту освітніх платформ у освітній процес. Ключові слова: національна онлайн-платформа з цифрової грамотності; веб-портал «Дія. Цифрова освіта»; підготовка бакалаврів спеціальності 081 Право; освітні серіали (курси). УДК 378.147+004 DOI: 10.31652/2412-1142-2022-66-45-52 УДК 378.147+004 DOI: 10.31652/2412-1142-2022-66-45-52 © І. Ю. Слободянюк, 2022 Слободянюк Ірина Юріївна д р р ндидат педагогічних наук, викладач кафедри педагогіки, психології та фахових методик, кандидат педагогічних наук, викладач кафедри педагогіки, психології та фахових методик, Комунальний заклад вищої освіти «Барський гуманітарно-педагогічний коледж ім. М. Грушевського», м. Бар, Україна ORCID ID 0000-0002-1249-8729 кандидат педагогічних наук, викладач кафедри педагогіки, психології та фахових методик, Комунальний заклад вищої освіти «Барський гуманітарно-педагогічний коледж ім. М. Груш м. Бар, Україна д д д у , д ф др д , ф д , Комунальний заклад вищої освіти «Барський гуманітарно-педагогічний коледж ім. М. Грушевського», м. Бар, Україна ORCID ID 0000 0002 1249 8729 у Комунальний заклад вищої освіти «Барський гуманітарно-педагогічний коледж ім. М. Грушевського», м Бар Україна Комунальний заклад вищої освіти «Барський гуманітарно-педагогічний коледж ім. М. Грушев м. Бар, Україна р р ORCID ID 0000-0002-1249-8729 islobodianuk@gmail.com ORCID ID 0000-0002-1249-8729 islobodianuk@gmail.com ВИКОРИСТАННЯ НАЦІОНАЛЬНОЇ ОНЛАЙН-ПЛАТФОРМИ З ЦИФРОВОЇ ГРАМОТНОСТІ ПРИ ПІДГОТОВЦІ БАКАЛАВРІВ СПЕЦІАЛЬНОСТІ «ПРАВО» 1. ВСТУП Постановка проблеми. Розвиток інформаційних технологій та поширення цифровізації відкриває нове «вікно можливостей» як для громадян, так і для держави в цілому. Трансформаційні процеси, які відбуваються у сучасному суспільстві, обумовлюють необхідність оновлення вимог до компетенцій фахівців різних сфер діяльності. Сьогодні нашій державі особливо необхідні кваліфіковані спеціалісти, які окрім високого рівня професійних знань та умінь, здатні до самоорганізації, самоосвіти, вміють працювати активно та креативно, розуміються у сучасних технологіях та ефективно використовують їх у професійній діяльності. Розвиток цифрових технологій не оминув сферу юридичних послуг. Як зазначає В. Очеретяний, «для того, щоб мінімізувати рутинну роботу в простих юридичних питаннях чи статистичних даних правники починають звертатися до спеціалістів із цифрових технологій, або самі опановують їх» [1, с. 55]. Щодо останнього – сьогодні існує низка різноманітних вебінарів, тренінгів та курсів, які допоможуть підвищити рівень володіння сучасними технологіями та цифровими інструментами уже практикуючим спеціалістам. Однак, необхідно затрачати час на пошук відповідних курсів та їх вивчення. 10 березня 2021 року Кабінетом Міністрів України було схвалено та затверджено Положення про Єдиний державний веб-портал цифрової освіти «Дія. Цифрова освіта», у © І. Ю. Слободянюк, 2022 45 Сучасні інформаційні технології та інноваційні методики навчання в підготовці фахівців: методологія, теорія, досвід, проблеми якому задекларовано його призначення, а саме – «реалізація права кожного на якісну та доступну освіту шляхом забезпечення створення можливості дистанційного навчання через Інтернет» [2]. Таким чином контент порталу може бути використаний як здобувачами освіти, так і дипломованими спеціалістами, які бажають покращити свій фаховий рівень. Веб-портал цифрової освіти є інформаційно-телекомунікаційною системою, яка складається з національної освітньої онлайн-платформи з цифрової грамотності, веб- платформи дистанційного навчання «Всеукраїнська школа онлайн», освітніх проєктів, аудіо- та відеоматеріалів, текстового контенту, засобів перевірки отриманих знань після роботи користувача з освітніми матеріалами, особистих електронних кабінетів користувачів, карти центрів цифрової освіти, додаткових засобів комунікації, мобільного додатка, інших підсистем та програмних модулів [2]. 20 липня 2022 року Наказом Міністерства освіти і науки України №644 затверджено та введено в дію Стандарт вищої освіти першого (бакалаврського) рівня вищої освіти галузі знань 08 Право, спеціальності 081 Право [3]. У контексті нашого дослідження, звертаємо увагу на такі зазначені компетентності та результати навчання, що, на нашу думку, можуть бути повноцінно сформовані та досягнуті за умови оволодіння сучасними цифровими технологіями та їх подальшого використання (таблиця 1). Таблиця 1 Компетентності Загальні Спеціальні − здатність використовувати інформаційні та комунікаційні технології; − здатність вчитися і оволодівати сучасними знаннями. 2. РЕЗУЛЬТАТИ ДОСЛІДЖЕННЯ У сучасних умовах трансформації суспільства юридична діяльність набуває нових властивостей, які тісно пов’язані з його інформатизацією [5]. Популяризація Legal Tech змушує переглянути традиційні підходи до вирішення юридичних питань за рахунок впровадження інформаційно-цифрових технологій в сферу правових послуг, що в свою чергу обумовлює необхідність та важливість підготовки здобувачів освіти до їх вмілого використання у професійній діяльності. Контент навчальних дисциплін, що забезпечують формування вміння використовувати новітні засоби та технології, повинен бути актуальним та сучасним. На нашу думку, варто приділяти увагу розгляду таких питань: − пошук, аналіз та перевірка достовірності отриманої інформації шляхом використання можливостей сучасних інформаційних технологій для ефективного вирішення правових завдань; − робота зі спеціалізованими інформаційно-правовими системами; − використання функціоналу сучасних онлайн-сервісів, мобільних додатків та чат-ботів юридичного спрямування; − сучасні способи та інструменти візуалізації даних у професійній діяльності юриста; − робота з хмарними сервісами, які забезпечують можливість віддаленої колективної і і − сучасні способи та інструменти візуалізації даних у професійній діяльності юриста; б і і б і і сучасні способи та інструменти візуалізації даних у професійній діяльності юриста; − робота з хмарними сервісами, які забезпечують можливість віддаленої колективної співпраці; − використання сучасних засобів комунікації – сервіси відеоконференцзв’язку, месенджери тощо; б і ф ій ї б − використання сучасних засобів комунікації – сервіси відеоконференцзв’язку, месенджери тощо; − забезпечення інформаційної безпеки; − основи кібергігієни; − використання кваліфікованого електронного підпису; − сучасний стан та перспективи використання штучного інтелекту у правос − сучасний стан та перспективи використання штучного інтелекту у правосудді; − використання онлайн-платформ для самоосвіти; − вільне володіння сучасними технічними засобами, що покликані спрощувати чи автоматизувати шаблонну роботу. − вільне володіння сучасними технічними засобами, що покликані спрощувати чи автоматизувати шаблонну роботу. Розглянемо структуру та контентне наповнення національної онлайн-платформи з цифрової грамотності веб-порталу «Дія. Цифрова освіта» та можливості її використання в освітньому процесі при підготовці майбутніх правників. Перейшовши за адресою https://osvita.diia.gov.ua, користувач потрапляє на головну сторінку. Весь контент структурований за розділами: навчання, цифрограм та про проєкт. Варто зазначити, що на сайті передбачено можливість здійснення пошукового запиту. До розділу навчання входять: освітні серіали (курси), гайди та подкасти на різну тематику, інформація про експертів, які беруть участь у проєкті, рейтинг користувачів, корисні посилання та довідник із тлумаченням сучасних термінів. Щоб перевірити рівень свої цифрової грамотності необхідно скористатися розділом цифрограм. Виконавши завдання національного тесту, користувач отримає сертифікат, що засвідчує рівень цифрової грамотності та ступінь володіння визначеними компетентностями (рис. 1.). Цифрограм доступний за такими напрямами: для громадян, для вчителів, для державних службовців та медичних працівників. 1. ВСТУП 46 Методологічні проблеми впровадження цифрових технологій та інноваційних методик навчання Методологічні проблеми впровадження цифрових технологій та інноваційних методик навчанн Метою статті є огляд та опис контентного наповнення національної онлайн-платформи з цифрової грамотності, що може бути використано в процесі підготовки майбутніх фахівців спеціальності 081 «Право» у закладах вищої освіти. 1. ВСТУП − здатність використовувати бази даних органів юстиції та інформаційні технології необхідні під час здійснення юридичної діяльності; − здатність використовувати різноманітні інформаційні джерела для повного та всебічного встановлення певних обставин. Результати навчання − проводити збір і інтегрований аналіз матеріалів з різних джерел; − складати та узгоджувати план власного прикладного дослідження і самостійно збирати матеріали за визначеними джерелами; − вільно використовувати для правничої діяльності доступні інформаційні технології і бази даних; − використовувати комп’ютерні програмами, необхідні у правничій діяльності. р ; − вільно використовувати для правничої діяльності доступні інформаційні технології і бази даних − використовувати комп’ютерні програмами, необхідні у правничій діяльності. Тож заклади освіти, які здійснюють підготовку майбутніх фахівців у галузі права, повинні зважати на сучасні тенденції і вимоги та модернізувати підготовку майбутніх правників, які будуть здатні ефективно працювати та бути конкурентоспроможними на ринку праці. Зважаючи на вищезазначене, питання підготовки майбутніх правників до професійної діяльності в умовах цифровізації суспільства, розвиток їх цифрової грамотності, є надзвичайно актуальними. Зазначимо, що в межах нашого дослідження, під поняттям цифрова грамотність розуміємо «наявність навичок, необхідних для життя, навчання і роботи в суспільстві, де спілкування і доступ до інформації здійснюється за допомогою цифрових технологій (інтернет-платформи, соціальні мережі, мобільні пристрої тощо)» [4, с.54]. Аналіз останніх досліджень і публікацій. Питання застосування сучасних інформаційних технологій у професійній діяльності юристів, теоретичні та методичні основи їх впровадження в освітній процес під час підготовки здобувачів освіти юридичних спеціальностей неодноразово розглядалися у працях А.Г. Бочевар, Х.Б. Романів, В.Є. Ромашенко, Н.Г. Русіної, О.С. Федорчук, О.О. Храпенко, М.І. Шермана та ін. Останнім часом особливої популярності набуває національна онлайн-платформа з цифрової грамотності Веб-порталу «Дія. Цифрова освіта», на якій розміщено освітні серіали (курси), присвячені висвітленню новацій, зокрема, у сфері права та їх практичному використанню у юридичній діяльності. Однак, використання подібних курсів в освітньому процесі є малодослідженим, що і обумовлює актуальність обраної теми. Аналіз останніх досліджень і публікацій. Питання застосування сучасних інформаційних технологій у професійній діяльності юристів, теоретичні та методичні основи їх впровадження в освітній процес під час підготовки здобувачів освіти юридичних спеціальностей неодноразово розглядалися у працях А.Г. Бочевар, Х.Б. Романів, В.Є. Ромашенко, Н.Г. Русіної, О.С. Федорчук, О.О. Храпенко, М.І. Шермана та ін. Останнім часом особливої популярності набуває національна онлайн-платформа з цифрової грамотності Веб-порталу «Дія. Цифрова освіта», на якій розміщено освітні серіали (курси), присвячені висвітленню новацій, зокрема, у сфері права та їх практичному використанню у юридичній діяльності. Однак, використання подібних курсів в освітньому процесі є малодослідженим, що і обумовлює актуальність обраної теми. 2. РЕЗУЛЬТАТИ ДОСЛІДЖЕННЯ Розділ про проєкт містить основні відомості щодо мети та команди проєкту, інформацію про мережу хабів цифрової освіти в Україні, новини, результати дослідження цифрових навичок українців у різні роки та статистику щодо використання сервісу. 47 Сучасні інформаційні технології та інноваційні методики навчання в підготовці фахівців: методологія, теорія, досвід, проблеми Сучасні інформаційні технології та інноваційні методики навчання в підготовці фахівців: методологія, теорія, досвід, проблеми Випуск 66 Рис. 1. Зразок сертифікату цифрограму для вчителів Для повноцінного використання онлайн-платформи, користувачеві необхідно зареєструватися. Після цього він матиме власний електронний кабінет, у якому відображатиметься особиста інформація, переглянуті освітні серіали, сертифікати, отримані після проходження тестів, а також нагороди та місце в загальному рейтингу. Важливо зазначити, що весь контент безкоштовний та доступний як з мобільного пристрою, так і з персонального комп’ютера. Таким чином, немає жодних обмежень щодо використання даної онлайн-платформи. Крім самоосвіти вбачаємо перспективи у використанні матеріалів даної онлайн- Рис. 1. Зразок сертифікату цифрограму для вчителів Рис. 1. Зразок сертифікату цифрограму для вчителів Для повноцінного використання онлайн-платформи, користувачеві необхідно зареєструватися. Після цього він матиме власний електронний кабінет, у якому відображатиметься особиста інформація, переглянуті освітні серіали, сертифікати, отримані після проходження тестів, а також нагороди та місце в загальному рейтингу. Важливо зазначити, що весь контент безкоштовний та доступний як з мобільного пристрою, так і з персонального комп’ютера. Таким чином, немає жодних обмежень щодо використання даної онлайн-платформи. Крім самоосвіти, вбачаємо перспективи у використанні матеріалів даної онлайн- платформи безпосередньо в освітньому процесі. Зупинимося детальніше на освітніх серіалах (курсах), яких на даний час налічується понад 70. Широка тематика та зручні фільтри допоможуть знайти та ознайомитися з корисною та актуальною інформацією як для школярів та здобувачів вищої освіти, так і для уже практикуючих спеціалістів та батьків. На наш погляд, під час підготовки бакалаврів спеціальності 081 «Право» доцільно використовувати такі курси (освітні серіали). «Цифрові юристи» – курс, створений для надання відповіді, чому право та IT почали зближуватися. У ньому розглядаються: питання цифровізації у праві; сучасні технічні сервіси та чат-боти, які є інструментом вирішення типових завдань у діяльності юриста; сутність та приклади використання технології Blockchain в юридичній діяльності; юридичні професії майбутнього та ін. Запрошені експерти наводять приклади українських та світових юридичних стартапів та діляться порадами і способами залучення ресурсів для реалізації власних проєктів [6]. Серіал налічує 4 серії тривалістю 5-8 хвилин, кожна з яких передбачає виконання короткого тесту. По завершенню опанування курсу, з метою отримання сертифікату, необхідно скласти фінальний тест. Як зазначає В. 2. РЕЗУЛЬТАТИ ДОСЛІДЖЕННЯ Іонан, заступниця Міністра цифрової трансформації з питань євроінтеграції, «проєкт адресований насамперед спеціалістам юридичної галузі – теперішнім і майбутнім. Серіал допоможе студентам юридичних напрямків зрозуміти, які фахові компетенції вимагатиме від них ринок юридичних послуг після завершення навчання, а досвідченим юристам надасть вектор, в якому напрямку можна розвивати власну практику задля посилення конкурентоспроможності» [7]. Логічним продовженням попереднього є освітній серіал «Цифрові юристи 2.0» [8]. Він складається з 6 серій тривалістю 5-8 хвилин. Перша серія присвячена питанням людиноцентричного підходу у правосудді, друга – відкритим та персональним даним, третя – принципам роботи та напрямам використання штучного інтелекту у роботі юриста, четверта – юридичному дизайну, п’ята – онлайн-інструментам для дистанційної роботи 48 Методологічні проблеми впровадження цифрових технологій та інноваційних методик навчанн етодологічні проблеми впровадження цифрових технологій та інноваційних методик навчання юристів та альтернативним постачальникам юридичних послуг, шоста – огляду послуг, які можна отримати через платформу «Дія». Успішне виконання фінального тестування дає можливість отримати сертифікат обсягом 0,1 кредиту ЄКТС. юристів та альтернативним постачальникам юридичних послуг, шоста – огляду послуг, які можна отримати через платформу «Дія». Успішне виконання фінального тестування дає можливість отримати сертифікат обсягом 0,1 кредиту ЄКТС. Освітній серіал «Персональні дані» [9] допоможе розібратися з сутністю поняття «персональні дані» та їх захистом, політикою приватності, тонкощами безпечного використання онлайн-послуг і соціальних мереж без шкоди для особи. Програма курсу передбачає перегляд 7 коротких за тривалістю серій, у яких сертифікований експерт з питань інформаційної приватності Дмитро Корчинський у доступній формі пояснює матеріал, а запрошені гості ставлять актуальні запитання та перевіряють надані поради. Також у вільному доступі надаються шаблони запиту на доступ до персональних даних та скарги на порушення прав на захист персональних даних. Для отримання сертифікату проходження курсу необхідно дати правильні відповіді на запитання фінального тесту. Готовність постійно оновлювати знання і навички, вміння використовувати в практичній діяльності сучасні інструменти, які полегшують виконання завдань та посадових обов’язків, є важливою рисою сучасного фахівця будь-якої галузі. Сьогодні досить популярними є інструменти Google, використання яких може бути ефективним як у професійній діяльності, так і в повсякденному житті. Для ознайомленням із функціоналом деяких із них можна скористатися освітнім серіалом «Цифрова грамотність державних службовців 1.0. на базі інструментів Google» [10]. Він передбачає перегляд 15 серій, кожна з яких має практичне спрямування – вирішення конкретних ситуацій, що можуть виникати в професійній діяльності. 2. РЕЗУЛЬТАТИ ДОСЛІДЖЕННЯ Сертифікований тренер Google for Education, директор Академії цифрового розвитку Антоніна Букач допомагає розібратися з усім різноманіттям онлайн- інструментів, серед яких: − Gmail – безкоштовна служба електронної пошти; − Gmail – безкоштовна служба електронної пошти; − Google Calendar – веб-додаток і сервіс для планування зустрічей, подій і спра − Google Keep – сервіс, призначений для створення і зберігання нотаток; − Google Docs – безкоштовний хмарний офісний пакет, що включає в себе текстовий і табличний редактори та службу для створення презентацій; e Forms – інструмент, що надає можливість легко та швидко складати опитуванн и, а також збирати іншу інформацію з її подальшим аналізуванням; − Google Forms – інструмент, що надає можливість легко та швидко складат анкети, а також збирати іншу інформацію з її подальшим аналізуванням; − Google Sites – безкоштовний конструктор сайтів; g ру р − Google Scholar – пошукова система вільного доступу, яка індексує повний текст наукових публікацій всіх форматів і дисциплін; − Google Classroom – інструмент, за допомогою якого можна організувати та оптимізувати робочий процес (не лише освітній); − Google Public Data Explorer – інструмент, що надає та візуалізує загальнодоступні дані і прогнози від цілого ряду міжнародних організацій і наукових установ; − Google Alerts – інструмент, який надає можливість користувачеві налаштувати сповіщення щодо появи нової інформації в онлайн-просторі відповідно до зазначених ключових слів; − Google Мої карти – сервіс, який дозволяє легко створювати карту улюблених місць, а також дозволяє налаштувати їх перегляд, редагування та вбудовувати на сайт; −сервіси для скорочення посилань. Для отримання сертифікату про проходження даного курсу потрібно виконати підсумкове тестування. Для отримання сертифікату про проходження даного курсу потрібно виконати підсумкове тестування. В умовах сьогодення, для забезпечення доступу до отримання публічних послуг онлайн, використання цифрових сервісів та електронного документообігу, реалізації функції електронної демократії, необхідний електронний підпис. Щоб ознайомитися з даним поняттям та відповісти на всі питання, що з ним пов’язані, створено курс «Електронний підпис» [11], програма якого передбачає опрацювання 7 серій тривалістю 4-9 хвилин та отримання сертифікату. Користувач ознайомиться із сутністю та призначенням електронного цифрового підпису, дізнається у яких ситуаціях він потрібен, як отримати кваліфікований 49 Сучасні інформаційні технології та інноваційні методики навчання в підготовці фахівців: методологія, теорія, досвід, проблеми Випуск 66 електронний підпис, як використовувати його для підписання документів та перевіряти уже наявний, як пройти електронну ідентифікацію, а також як зробити усе це з мобільного телефону, щоб із легкістю отримувати більший спектр послуг. 2. РЕЗУЛЬТАТИ ДОСЛІДЖЕННЯ Поширення цифровізації в усі сфери життя та діяльності людини, активне використання девайсів та сервісів, які зберігають про нас багато особистої інформації, вимагають дотримання певних правил поведінки для убезпечення від загроз у кіберпросторі. На національній онлайн-платформі представлено освітні серіали, які ознайомлять та продемонструють на реальних прикладах як не стати жертвами кіберзловмисників. Одним із них є серіал «Основи кібергігієни», який створено в рамках проєкту «Посилення спроможностей українських державних органів у сфері кібергігієни та кібербезпеки», що реалізується Координатором проектів ОБСЄ в Україні за підтримки Міністерства закордонних справ і міжнародного розвитку Великобританії та Федерального міністерства закордонних справ Німеччини [12]. Метою курсу є засвоєння базових принципів кібергігієни та дій у випадку виявлення інформаційних атак. Програма курсу включає 8 серій тривалістю 4 7 і і 4-7 хвилин, під час перегляду яких можна дізнатися: − що таке «кібергігієна»; − види хакерських атак; − поняття «соціальної інженерії», її прийоми, методи та принципи; − рекомендації щодо організації безпечного використання мережі Інтернет; − особливості використання електронної пошти; − загальні рекомендації з використання програмного забезпечення та типи шкідливого ПЗ; − особливості налаштування конфіденційності та інших питань безпеки в соціальних мережах; загальні рекомендації з використання програмного забезпечення та типи шкідливого ПЗ; − особливості налаштування конфіденційності та інших питань безпеки в соціальних мережах; − правила безпечної роботи з мобільними пристроями; − загрози, які виникають під час використання змінних носіїв інформації; − види маніпуляцій з інформацією у кіберсфері та інше. Також до деяких серій подано додаткові матеріали, які доступні для завантаження. У них представлена корисна інформація або результати проведених досліджень з теми, про яку розповідали в серії. Курс завершується складанням тесту, з можливістю отримати сертифікат, що засвідчує успішне його проходження, обсягом 0,33 кредиту ЄКТС. Кібершахрайство з sim-картою стало поширеною проблемою, адже жертвою може бути будь-хто – від звичайного громадянина до власників компаній. З метою попередження та захисту від кібершахраїв Міністерством цифрової трансформації України за підтримки агентства з міжнародного розвитку США (USAID), Кіберполіції, Урядової команди реагування на комп’ютерні надзвичайні події України, яка функціонує в складі Державного центру кіберзахисту CERT-UA, та Національного банку України було створено освітній серіал «Обережно! Кібершахраї» [13]. Впродовж перегляду 4 серій користувачі дізнаються: як відновити номер телефону, якщо віддалено замінили сім-карту; як діяти в разі втрати або викрадення мобільного телефону; як розпізнавати й протидіяти фішингу та вішингу. Засвоєння курсу завершується фінальним тестуванням та отриманням сертифікату. Ще одним освітнім серіалом, присвяченим кібербезпеці, є «Кіберняні» [14]. 3. ВИСНОВКИ ТА ПЕРСПЕКТИВИ ПОДАЛЬШИХ ДОСЛІДЖЕНЬ Цифровізація впливає на функціонування юридичної сфери – змінюються цілі, пріоритети та навички юристів нового покоління, з’являються нові технологічні продукти, які здатні спростити, а інколи і замінити, роботу фахівця. Тому, підготовка конкурентоздатних спеціалістів галузі права вимагає не лише ґрунтовного освоєння фундаментальних та спеціалізованих знань, вільного володіння чинним нормативно- правовим забезпеченням, але й умілого використання ними сучасних технологій [15]. Одним із головних завдань юридичної освіти є формування у студентів цифрової грамотності, яка є важлим складником професійної компетентності. Уміння використовувати інформаційно-цифрові ресурси, впроваджувати в юридичну діяльність нові підходи та інструменти для вирішення поставлених завдань відкривають ширші професійні перспективи та підіймають правника на якісно новий фаховий рівень. Для цього необхідно модернізувати підготовку здобувачів освіти – знайомити їх з новаціями, технологічними рішеннями, що застосовуються у галузі права і набувають дедалі більшого поширення та популярності, а також прививати навики та бажання до постійного розвитку та самоосвіти. Тому, подальшого вивчення потребують методичні засади впровадження сучасних онлайн- інструментів та контенту освітніх платформ у підготовку майбутніх фахівців спеціальності 081 Право. Методологічні проблеми впровадження цифрових технологій та інноваційних методик навчання Також для формування цифрової грамотності сучасного правника, розширення обсягу знань, необхідних для складання цифрограму, корисними будуть освітні серіали (курси) – «Базові цифрові навички», «Доступ до публічної інформації», «Very Verified: онлайн-курс з медіаграмотності», «Медіаграмотність у часи пандемії», «Доступ до публічної інформації», «Відкриті дані для державних службовців» та інші. Користувачам варто слідкувати за оновленнями на платформі, оскільки освітні серіали з’являються майже щомісяця. ф р р Дані освітні серіали (курси) можуть бути рекомендовані викладачем у силабусі / робочій програмі навчальної дисципліни як різновид неформальної освіти, результати якої зараховуватимуться здобувачеві освіти у якості опанування певної її складової. 2. РЕЗУЛЬТАТИ ДОСЛІДЖЕННЯ Він містить 10 серій тривалістю 4-11 хвилин, з яких користувач дізнається: якими мають бути паролі; де перевірити чи є у хакерів саме ваші паролі; що таке багатофакторна автентифікація та як її налаштувати; важливість оновлення програмного забезпечення; особливості налаштування безпеки для популярних месенджерів; для чого та як здійснювати резервне копіювання даних; правила здійснення безпечних платежів в Інтернеті; яку інформацію можна, а яку не варто повідомляти в мережі, аби не стати черговою жертвою пограбування чи шантажу та багато іншого. До участі у серіалі долучилися експерти з кібербезпеки, які цікаво, доступно, за допомогою конкретних прикладів пояснюють складну, але надзвичайно важливу інформацію. Виконавши завдання фінального тестування користувач отримує сертифікат, що засвідчує успішне проходження курсу. 50 Slobodianiuk Iryna Yuriivna Candidate of Pedagogical Sciences (PhD), Teacher of the Department of Pedagogy, Psychology and Professional Methods Municipal Institution of Higher Education «Bar Humanitarian Pedagogical College named after Mykhailo Hrushevsky» Bar, Ukraine ORCID ID 0000-0002-1249-8729 islobodianuk@gmail.com Slobodianiuk Iryna Yuriivna Candidate of Pedagogical Sciences (PhD), Teacher of the Department of Pedagogy, Psychology and Professional Methods Municipal Institution of Higher Education «Bar Humanitarian Pedagogical College named after Mykhailo Hrushevsky» Bar, Ukraine ORCID ID 0000-0002-1249-8729 islobodianuk@gmail.com Slobodianiuk Iryna Yuriivna Candidate of Pedagogical Sciences (PhD), Teacher of the Department of Pedagogy, Psychology and Professiona Methods Municipal Institution of Higher Education «Bar Humanitarian Pedagogical College named after Mykhailo Hrushevsky» Bar, Ukraine ORCID ID 0000-0002-1249-8729 islobodianuk@gmail.com Slobodianiuk Iryna Yuriivna Candidate of Pedagogical Sciences (PhD), Teacher of the Department of Pedagogy, Psychology and Professional Methods Municipal Institution of Higher Education «Bar Humanitarian Pedagogical College named after Mykhailo Hrushevsky» Bar, Ukraine ORCID ID 0000-0002-1249-8729 islobodianuk@gmail.com Abstract. All spheres of human activity are being influenced by digitization and the legal industry is not an exception. Obviously, this should be taken into consideration for the training of future lawyers. The article substantiates the necessity to modernize the training program for Bachelors of Law to include the mastering of modern digital technologies and their further use in professional activities. Based on the analysis of The Standard of Higher Education of the First (Bachelor) Level of higher education in the Field of Study 08 (Law), Subject Area 081 (Law), we have defined competences and learning outcomes, the formation of which directly depends on the quality mastery of modern information and digital technologies. The main topics that should be included in the content of the discipline that will be responsible for the formation of digital literacy have been highlighted. The content of the national online platform for digital literacy of the web portal "Diia. Digital education" has been proposed to be used during the professional training of Bachelors of Law. The structure of the online platform and the guidelines to its use have been described. The recommended educational series (courses) for the training of the Law students have been singled out and the peculiarities of each course along with the list of topics covered have been outlined. We have prepared a list of educational series that are available through the online platform to help students prepare for the national digital literacy exam. Such educational series (courses) can be recommended by educators as a type of non-formal education. Slobodianiuk Iryna Yuriivna Candidate of Pedagogical Sciences (PhD), Teacher of the Department of Pedagogy, Psychology and Professional Methods Municipal Institution of Higher Education «Bar Humanitarian Pedagogical College named after Mykhailo Hrushevsky» Bar, Ukraine ORCID ID 0000-0002-1249-8729 islobodianuk@gmail.com We have determined the vectors for modernization of the training program for the Law students as well as the necessity for the further study of methodological approaches for implementation of online-tools and contents of educational platforms into the educational process. Key words: national online platform for digital literacy; web-portal "Diia. Digital education", training of Bachelors in the specialty 081 (Law); educational series (courses). Key words: national online platform for digital literacy; web-portal "Diia. Digital education", training Bachelors in the specialty 081 (Law); educational series (courses). СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ [1] Очеретяний В.В. Аспекти цифровізації професійних послуг юриста. Соціально-економічні трансформації у розвитку держави : зб. матер. Всеукр. наук.-практ. конф., м. Вінниця, 22-23 квітня 2021 р.: у 2-х т. Т.1. Ч.2. / За заг. ред. В.І. Мельник. Тернопіль: Крок, 2021. С. 55-57. р р р [2] Питання Єдиного державного веб-порталу цифрової освіти “Дія. Цифрова освіта”: Постанова Кабінету Міністрів України від 10.03.2021 р. №184. URL: https://zakon.rada.gov.ua/laws/show/184-2021- %D0%BF#n11. [3] Про затвердження стандарту вищої освіти зі спеціальності 081 Право для першого (бакалаврського) рівня вищої освіти: наказ Міністерства освіти і науки України від 20.07.2022 р. №644. URL: https://mon.gov.ua/storage/app/media/vishcha-osvita/zatverdzeni%20standarty/2022/07/21/081-pravo-bakalavr- 644-20.07.2022.pdf p [4] Тілікіна Н.В. Медіа-, інформаційна і комп’ютерна грамотність як компоненти цифрової грамотності. Наукові записки Львівського університету бізнесу та права. 2021. Том 29. С. 46-56. [5] Храпенко О.О. Застосування системи «Електронний суд» у підготовці студентів-юристів. Інформаційні технології і засоби навчання. 2019. №70(2). С. 155-164. ( ) [6] Цифрові юристи: освітній серіал. URL: https://osvita.diia.gov.ua/courses/digital-lawyers. [6] Цифрові юристи: освітній серіал. URL: https://osvita.diia.gov.ua/courses/digital-lawyers. [7] «Цифрові юристи»: Мінцифра презентувала освітній серіал про legal tech. Юридична газета online: всеукраїнське професійне юридичне видання. 14 грудня 2020 р. URL: https://yur- gazeta.com/golovna/cifrovi-yuristi-mincifra-prezentuvala-osvitniy-serial-pro-legal-tech.html. [8] Цифрові юристи 2.0: освітній серіал. URL: https://osvita.diia.gov.ua/courses/digital-lawyers-2. [9] Персональні дані: освітній серіал. URL: https://osvita.diia.gov.ua/courses/personaldata. [8] Цифрові юристи 2.0: освітній серіал. URL: https://osvita.diia.gov.ua/courses/digital lawyers 2. [9] Персональні дані: освітній серіал. URL: https://osvita.diia.gov.ua/courses/personaldata. [10] Ц ф і б і 1 0 б і і і G l і ій і URL [10] Цифрова грамотність державних службовців 1.0. на базі інструментів Google: освітній https://osvita.diia.gov.ua/courses/civil-servants. [11] Електронний підпис: освітній серіал. URL: https://osvita.diia.gov.ua/courses/digital-signature. [ ] р р p g y yg [13] Обережно! Кібершахраї: освітній серіал. URL: https://osvita.diia.gov.ua/courses/attention-cyber-fraudsters. р р р р p g [14] Кіберняні: освітній серіал. URL: https://osvita.diia.gov.ua/courses/cybernanny. [15] Маньгора В.В. Інформаційно-правове забезпечення юридичної освіти. Інформація і право. 2020. № 1(32). С. 138-144. 51 Сучасні інформаційні технології та інноваційні методики навчання в підготовці фахівців: методологія, теорія, досвід, проблеми Випуск 66 References (TRANSLATED AND TRANSLITERATED) [1] Ocheretianyi V.V. Aspects of digitalization of professional services of a lawyer. In Proc. of Conf. Social and economic transformations in state development, Vinnytsia, April 2021, pp. 55-57. (in Ukrainian) 1] Ocheretianyi V.V. Aspects of digitalization of professional services of a lawyer. In Proc. of Conf. Social and economic transformations in state development, Vinnytsia, April 2021, pp. 55-57. (in Ukrainian) p , y , p , pp ( ) [2] Pytannia Yedynoho derzhavnoho veb-portalu tsyfrovoi osvity “Diia. Tsyfrova osvita”: Postanova Kabinetu Ministriv Ukrainy vid 10.03.2021 r. №184. URL: https://zakon.rada.gov.ua/laws/show/184-2021-%D0%BF#n11. p , y , p , pp ( ) [2] Pytannia Yedynoho derzhavnoho veb-portalu tsyfrovoi osvity “Diia. Tsyfrova osvita”: Postanova Kabinetu Ministriv Ukrainy vid 10.03.2021 r. №184. URL: https://zakon.rada.gov.ua/laws/show/184-2021-%D0%BF#n11. [3] Pro zatverdzhennia standartu vyshchoi osvity zi spetsialnosti 081 Pravo dlia pershoho (bakalavrskoho) rivnia vyshchoi osvity: nakaz Ministerstva osvity i nauky Ukrainy vid 20.07.2022 r. №644. URL: https://mon.gov.ua/storage/app/media/vishcha-osvita/zatverdzeni%20standarty/2022/07/21/081-pravo-bakalavr-644- 20.07.2022.pdf [4] Tilikina N.V. Media, information and computer literacy as components of digital literacy. Scie University of Business and Law. 2021, V. 29, pp. 46-56. (in Ukrainian) [5] Khrapenko О.О. Use of the "Electronic Court" system in future lawyers training. Information Technologies and Learning Tools. 2019. №70(2). pp. 155-164. (in Ukrainian) Digital Lawyers: an educational series. URL: https://osvita.diia.gov.ua/courses/digital-lawyers. [6] Digital Lawyers: an educational series. URL: https://osvita.diia.gov.ua/courses/digital-lawyers. [6] Digital Lawyers: an educational series. URL: https://osvita.diia.gov.ua/courses/digital-lawyers. [7] «Digital lawyers»: the Ministry of Digital presented an educational series about legal tech. Legal newspaper online: All-Ukrainian professional legal publication. 14 December 2020. URL: https://yur-gazeta.com/golovna/cifrovi-yuristi- mincifra-prezentuvala-osvitniy-serial-pro-legal-tech.html. (in Ukrainian) [8] Digital Lawyers 2.0: an educational series. URL: https://osvita.diia.gov.ua/courses/digital-lawye [9] Personal Data: an educational series. URL: https://osvita.diia.gov.ua/courses/personaldata. [ ] p g p [10] Digital literacy of civil servants 1.0. based on Google tools: an educational series. URL: https://osvita.diia.gov.ua/courses/civil-servants. p g [11] Digital Signature: an educational series. URL: https://osvita.diia.gov.ua/courses/digital-signature. [12] Basics of Cyber Hygiene: an educational series. URL: https://osvita.diia.gov.ua/courses/cyber-hygiene [13] Attention! Cyber Fraudsters: an educational series. URL: https://osvita.diia.gov.ua/courses/attentio [14] Cybernanny: an educational series. URL: https://osvita.diia.gov.ua/courses/cybernanny. [14] Cybernanny: an educational series. URL: https://osvita.diia.gov.ua/courses/cybernanny. 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https://openalex.org/W3039704475	https://europepmc.org/articles/pmc7348043?pdf=render	English	null	Histone Deacetylase 11 Knockdown Blocks Larval Development and Metamorphosis in the Red Flour Beetle, Tribolium castaneum	Frontiers in genetics	2,020	cc-by	10,869	Keywords: juvenile hormone, epigenetics, histone deacetylase 11, histone H3, dsRNA ORIGINAL RESEARCH published: 03 July 2020 doi: 10.3389/fgene.2020.00683 Edited by: Edited by: Enrique Medina-Acosta, State University of the North Fluminense Darcy Ribeiro, Brazil Reviewed by: Rodrigo Nunes Da Fonseca, Federal University of Rio de Janeiro, Brazil Reviewed by: Rodrigo Nunes Da Fonseca, Federal University of Rio de Janeiro, Brazil Yoonseong Park, Kansas State University, United States Correspondence: Subba Reddy Palli rpalli@email.uky.edu; rpalli@uky.edu Correspondence: Subba Reddy Palli rpalli@email.uky.edu; rpalli@uky.edu Correspondence: Subba Reddy Palli rpalli@email.uky.edu; rpalli@uky.edu Specialty section: This article was submitted to Epigenomics and Epigenetics, a section of the journal Frontiers in Genetics Received: 27 March 2020 Accepted: 04 June 2020 Published: 03 July 2020 Smitha George and Subba Reddy Palli Post-translational modiﬁcations (PTM) such as methylation, acetylation, phosphorylation, and ubiquitination of histones and other proteins regulate expression of genes. The acetylation levels of these proteins are determined by the balance of expression of histone acetyltransferase (HATs) and histone deacetylases (HDACs). We recently reported that class I HDACs (HDAC1 and HDAC3) play important roles in juvenile hormone (JH) suppression of metamorphosis in the red ﬂour beetle, Tribolium castaneum. Here, we report on the function of a single class IV HDAC member, HDAC11. Injection of dsRNA targeting T. castaneum HDAC11 gene into newly molted last instar larvae induced knockdown of the target gene and arrested larval development and prevented metamorphosis into the pupal stage. Dark melanized areas were detected in larvae that showed developmental arrest and mortality. Developmental expression studies showed an increase in HDAC11 mRNA levels beginning at the end of the penultimate larval stage. These higher levels were maintained during the ﬁnal instar larval and pupal stages. A JH analog, hydroprene, suppressed HDAC11 expression in the larvae. Sequencing of RNA isolated from control and dsHDAC11 injected larvae identiﬁed several differentially expressed genes, including those involved in JH action, ecdysone response, and melanization. The acetylation levels of core histones showed an increase in TcA cells exposed to dsHDAC11. Also, an increase in histone H3 acetylation, speciﬁcally H3K9, H3K18 and H3K27, were detected in HDAC11 knockdown larvae. These studies report the function of HDAC11 in insects other than Drosophila for the ﬁrst time and show that HDAC11 inﬂuences the acetylation levels of histones and expression of multiple genes involved in T. castaneum larval development. Edited by: Enrique Medina-Acosta, State University of the North Fluminense Darcy Ribeiro, Brazil Reviewed by: Rodrigo Nunes Da Fonseca, Federal University of Rio de Janeiro, Brazil Yoonseong Park, Kansas State University, United States *Correspondence: Citation: George S and Palli SR (2020) Histone Deacetylase 11 Knockdown Blocks Larval Development and Metamorphosis in the Red Flour Beetle, Tribolium castaneum. Front. Genet. 11:683. doi: 10.3389/fgene.2020.00683 Two major insect hormones, ecdysteroids (20-hydroxyecdysone, 20E, is the most active form) and juvenile hormones (JH) regulate many aspects of insect life, including postembryonic development (Riddiford, 1996; Wyatt et al., 1996; Hartfelder, 2000; Singtripop et al., 2000; Min et al., 2004; Giray et al., 2005; Hrdy et al., 2006; Futahashi and Fujiwara, 2008). These hormones have been extensively studied because of their involvement in the regulation of multiple biological processes, July 2020 \| Volume 11 \| Article 683 Frontiers in Genetics \| www.frontiersin.org 1 HDAC11 Function in Tribolium George and Palli (Yang and Seto, 2008). HDAC11 depletion in neuroblastoma cell lines induces cell death mediated by apoptotic programs (Thole et al., 2017). HDAC11 knockout study in mice identiﬁed an age-dependent brain region-speciﬁc function in regulating FEZ1 (fasciculation and elongation protein zeta 1), a gene associated with schizophrenia (Bryant et al., 2017). HDAC11 knockout mice showed resistance to high-fat-diet-induced obesity and metabolic syndrome, suggesting that HDAC11 functions as a critical metabolic regulator (Sun et al., 2018). However, not much information is available on HDAC11 function in insects. Functions of D. melanogaster histone deacetylases were studied by RNA interference and microarrays and showed that HDAC1 and HDAC3 control expression of genes involved in multiple processes including lipid metabolism, cell cycle regulation and signal transduction (Foglietti et al., 2006). However, three other HDACs tested did not show any detectable functions (Foglietti et al., 2006). Also, overexpression of HDAC 3, 6 or 11 suppressed CGG repeat-induced neurodegeneration in D. melanogaster Fragile X Tremor Ataxia Syndrome model suggesting that HDAC activators might be used to repress transcription of fragile X syndrome gene (Todd et al., 2010). In the current studies, we employed RNAi, RNA sequencing, and RT-qPCR to elucidate the role of HDAC11 in T. castaneum. Knockdown of HDAC11 during the larval stage induced arrest in larval development, melanization, and mortality. RNA isolated from T. castaneum larvae injected with double-stranded RNA (dsRNA) targeting the gene coding for HDAC11 (dsHDAC11) or dsmalE (a control dsRNA targeting E. coli malE gene) was sequenced, and diﬀerential gene expression analysis was conducted. Genes involved in hormone action and multiple biological processes such as melanization were identiﬁed as diﬀerentially expressed genes in HDAC11 knockdown larvae. Citation: including diapause, reproduction, and polyphenism (Wigglesworth, 1934; Riddiford, 1994, 2012). Juvenile hormones are sesquiterpenoids secreted by the corpora allata that mediate a variety of functions in insects (Jindra et al., 2013). The JH signaling cascade is a complex molecular process that includes multiple players such as JH receptor, Methoprene- tolerant (Met) (Wilson and Fabian, 1986; Konopova and Jindra, 2007) and, its heterodimeric partner, SRC, steroid receptor co-activator homolog (Li et al., 2011; Zhang et al., 2011). The JH-receptor complex binds to the juvenile hormone response elements (JHRE) present in the promoters of JH- response genes and regulate their expression (Kayukawa et al., 2012; Cui et al., 2014). 20-hydroxyecdysone binds to a heterodimer of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP) and ecdysone response elements present in the promoters of ecdysone-induced transcription factors including E75, E74, Broad complex (BR-C) and E93 and regulate their expression (Palli et al., 2005). These ecdysone induced transcription factors, in turn, regulate expression of multiple genes important for growth, development, molting and metamorphosis (Riddiford et al., 2000). p ( , ) Hormones represent attractive targets for the development of environmentally friendly insect control methods. Hindering this eﬀort is the lack of knowledge on the molecular basis of hormone action. Research in epigenetics-based gene regulation has facilitated the discovery of various post-translational modiﬁcation mechanisms (PTM) such as methylation, acetylation, phosphorylation, and ubiquitination. The role of acetylation in the regulation of 20E induced gene expression in Drosophila melanogaster has been reported (Bodai et al., 2012). The CREB-binding protein (CBP) mediates acetylation of histone H3K27 and antagonizes “Polycomb” silencing in D. melanogaster (Tie et al., 2009). The CBP also functions in regulating the expression of hormone response genes in Tribolium castaneum (Roy et al., 2017; Xu et al., 2018) and Blattella germanica (Fernandez-Nicolas and Belles, 2016). Since acetylation is a key component in the regulation of gene expression, we decided to explore the function of histone deacetylases (HDACs) in the red ﬂour beetle, T. castaneum. Recent ﬁndings from our lab have demonstrated that class I HDACs (HDAC1 and HDAC3) play important roles in JH suppression of metamorphosis in T. castaneum (George et al., 2019; George and Palli, 2020). Here, we focused on the function of sole class IV HDAC member, HDAC11 (TC007473), to study its role in T. castaneum development. Insects and Cells Insects (T. castaneum, GA-1 strain) (Haliscak and Beeman, 1983) were maintained in a Percival incubator set at 30◦C and 65 ± 5% relative humidity with complete dark conditions on organic wheat ﬂour (Heartland Mill, Marienthal, KS) mixed with 10% baker’s yeast (MP biomedicals, Solon, OH, United States). The T. castaneum cells, BCIRL-TcA-CLG1 (TcA), were cultured in EX-CELL 420 (Sigma-Aldrich, St-Louis, MO, United States) medium supplemented with 10% Fetal Bovine Serum (FBS, VWR-Seradigm, Radnor, PA, United States) at 28◦C as described previously (Goodman et al., 2012). Human HDACs identiﬁed to date can be grouped into four classes; Class I-IV based on their structure, phylogeny, and function. Class I HDACs are ubiquitously expressed and play essential roles in proliferation, whereas classes II and IV have a tissue-speciﬁc function (Lehrmann et al., 2002). HDAC11 ﬁrst described in 2002 is a unique member class IV HDAC family since it is not homologous with RPD3 or HDA1 yeast enzymes (Gao et al., 2002). Selective/class-speciﬁc inhibitors targeting HDAC11 have been developed for treating patients with myeloproliferative neoplasms (MPN) (Yue et al., 2020). HDAC11 shows some sequence similarity to class I and II HDACs and is highly conserved in invertebrates and plants Frontiers in Genetics \| www.frontiersin.org Hormone Treatments Both S-Hydroprene (Ethyl 3, 7, 11-trimethyl-2, 4- dodecadienoate) and JH III were purchased from Sigma-Aldrich. The hydroprene was dissolved in cyclohexane at 2 µg/µl concentration and one microliter of this solution was applied on the integument of each last instar larva. One microliter of cyclohexane solvent alone was applied to each control larva. For cell culture experiments, JH III was prepared in DMSO at 10 mM concentration and one microliter per ml of culture medium was July 2020 \| Volume 11 \| Article 683 Frontiers in Genetics \| www.frontiersin.org 2 HDAC11 Function in Tribolium George and Palli added to achieve a ﬁnal concentration of 10 µM. Control cells were treated with the same volume of DMSO. antibodies were used to detect various lysine acetylation sites of histone H3. Band density was determined by Image-J software and normalized with loading control protein, ß-Actin. The Anti- rabbit IgG, HRP-linked antibody (Cell signaling #7074), was used for chemiluminescence detection. The blots were developed with SupersignalTM West Femto Maximum sensitivity Substrate (Thermo Fisher, Scientiﬁc, Rockford, IL, United States). Differential Gene Expression Analysis p y Total RNA extracted from three biological replicates of larvae at 12 h after treatment with dsHDAC11 or dsmalE using the TRI reagent-RT and used for RNA-seq library preparation following the protocol described previously (Ma et al., 2014; Hunt, 2015; Kalsi and Palli, 2017). Raw reads were analyzed following CLC genomic workbench pipeline (Version 11.0.1, Qiagen, United States). Blast2GO Pro Plugin in the CLC workbench was used to determine the GO terms. The GO terms were used for predicting functions of diﬀerentially expressed genes. The GO terms were represented using Web Gene Ontology Annotation Plot (WEGO) as described previously (Ye et al., 2006). FIGURE 1 \| TcHDAC11 catalytic domain and phylogeny. (A) Schematic representation of the TcHDAC11 with the catalytic domain marked. The numbers on the top show the position of amino acids. The data for this Figure is obtained from NCBI (Geer et al., 2002). (B) Phylogenetic tree demonstrating the relationship between HDAC classes. The tree was inferred using the Neighbor-joining method. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016). The tree was drawn to scale, with branch lengths and the evolutionary distances used to infer the phylogenetic tree as the same units. The identiﬁcation of HDACs in T. castaneum was reported previously (George et al., 2019). Tribolium castaneum HDAC11 T. castaneum HDAC11 contains a single Zn+ dependent catalytic domain surrounded by a short N- and C- terminus (Figure 1A). Phylogenetic analysis of HDACs in T. castaneum revealed that TcHDAC11 is closer to Class I deacetylases (HDAC1, HDAC3, and HDAC8) than to class II (Figure 1B). T. castaneum HDAC11 open reading frame consists of 314–331 residues with a molecular mass of 35.2–37.2 kDa and one catalytic Total RNA was isolated from treated and control insects using TRI reagent-RT (Molecular Research Center, Inc. Cincinnati, OH, United States). The RNA was converted to cDNA using M-MLV reverse transcriptase (Invitrogen-ThermoFisher Scientiﬁc). RT-qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, United States) in Applied Biosystems StepOnePlus Real-time PCR instrument. The qPCR mixture contained 2 µl of diluted cDNA (1:5), 0.4 µl gene- speciﬁc primer mix, 2.6 µl nuclease-free water, and 5 µl SYBR green in a 10 µl ﬁnal volume. The qPCR cycling conditions were: initial holding stage 95◦C (20 s), followed by 40 cycles of denaturation at 95◦C (5 s), annealing, and extension at 60◦C (30 s) along with melting curve. The relative mRNA levels were calculated using RP49 as a reference gene. FIGURE 1 \| TcHDAC11 catalytic domain and phylogeny. (A) Schematic representation of the TcHDAC11 with the catalytic domain marked. The numbers on the top show the position of amino acids. The data for this Figure i b i d f NCBI (G l 2002) (B) Ph l i d i Statistical Analysis T. castaneum HDAC11 ortholog was identiﬁed using the D. melanogaster HDAC11 sequence available at the FlyBase (Thurmond et al., 2019). Speciﬁc primers targeting two regions of HDAC11 and containing T7 polymerase promoter at the 5′ end (Supplementary Table S1) and genomic DNA isolated from T. castaneum were used to PCR amplify the fragment of HDAC11. The PCR fragment used as a template for dsRNA synthesis. Primers used to amplify a fragment of JH receptor, Met, from T. castaneum (TcMet) have been reported previously (Parthasarathy et al., 2008). The MEGAscript T7 kit (Invitrogen, United States) was used for dsRNA synthesis. Puriﬁcation, quality check, and quantiﬁcation of dsRNA were performed as described previously (George et al., 2019). dsRNA prepared using a fragment of Escherichia coli maltose-binding proteins (malE) was used as a control. The statistical analyses were performed using JMP Pro 14.0 (SAS Institute Inc., Cary, NC, United States) to test for statistical diﬀerences among treatments. Post-hoc tests were conducted using the Tukey-Kramer HSD method (α = 0.05). One-way ANOVA was performed for comparison between treatments. Western Blot Analysis We used Acetylated-Lysine (Ac-K2-100) MultiMabTM Rabbit mAb mix (Cell signaling #9814) to detect proteins post- translationally modiﬁed by acetylation. Histone H3 antibody sampler kit #9927 (Cell Signaling, Danvers, MA, United States) that includes Lys9, Lys14, Lys18, Lys27, and Lys56 speciﬁc Frontiers in Genetics \| www.frontiersin.org July 2020 \| Volume 11 \| Article 683 3 HDAC11 Function in Tribolium George and Palli E 2 \| Phenotypes and mortality induced by dsRNA mediated knockdown of HDAC11 in T. castaneum. (A) a: Control larvae injected with dsmalE pupated and erged as healthy adults. b: dsHDAC11 was injected into the newly molted last instar larvae. Phenotypes were photographed on the ﬁfth day after injection. own of the HDAC11 gene affected larval development resulting in pigmentation, growth retardation, and mortality. c: Light microscopic images of ation in HDAC11 knockdown larvae. A high degree of hard melanization was detected inside the body. (B) Newly molted last instar larvae, pupae or adults ected with dsHDAC11 or dsmalE. Total RNA was isolated from treated insects at 72 h after injection and used to determine relative mRNA levels of C11. Knockdown efﬁciency was calculated by comparing TcHDAC11 mRNA levels in dsTcHDAC11 and dsmalE treated insects. Mean ± SE (n = 3–5) are Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. (C) Newly molted last instar larvae, pupae or adults were with dsHDAC11 or dsmalE. The mortality was recorded until death or adult eclosion. Mean ± SE (n = 30) are shown. Means marked with different letters are ntly different from each other, P ≤0.05 by ANOVA. FIGURE 2 \| Phenotypes and mortality induced by dsRNA mediated knockdown of HDAC11 in T. castaneum. (A) a: Control larvae injected with dsmalE pupated and later emerged as healthy adults. b: dsHDAC11 was injected into the newly molted last instar larvae. Phenotypes were photographed on the ﬁfth day after injection. Knockdown of the HDAC11 gene affected larval development resulting in pigmentation, growth retardation, and mortality. c: Light microscopic images of melanization in HDAC11 knockdown larvae. A high degree of hard melanization was detected inside the body. (B) Newly molted last instar larvae, pupae or adults were injected with dsHDAC11 or dsmalE. Total RNA was isolated from treated insects at 72 h after injection and used to determine relative mRNA levels of TcHDAC11. Western Blot Analysis The data shown are mean ± SE (n = 4). C, cyclohexane; H, hydroprene. molted last instar larvae (day 0), freshly formed white-colored pupae and adults. The control insects injected with dsmalE (Figure 2Aa) pupated after 5–6 days after dsRNA injection and later emerged as healthy adults. However, developmental arrest and mortality were observed in 100% of dsHDAC11 injected larvae (Figure 2Ab). The arrested larvae showed dark pigmentation inside their body (Figure 2Ac). Dark melanized patches of tissues attached to the integument were detected in dissected larvae. Injection of dsHDAC11 reduced mRNA levels of the target gene by 97, 55, and 65% in larvae, pupae and adults, respectively, when compared to their levels in control insects domain (Figure 1A). Comparison between the T. castaneum full-length HDAC11 amino acid sequence with those from D. melanogaster and human HDAC11s amino acid sequences showed 54 and 58 percent amino acid identity, respectively (Supplementary Figure S1). Western Blot Analysis Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. (B) JH suppresses the expression of HDAC11 in T. castaneum larvae and TcA cells. S-Hydroprene (JH analog) dissolved in cyclohexane was topically applied to 48 h old last instar larvae (0.5 µL of 2 µg/µL). Total RNA was isolated from larvae collected at 6 h after treatment and subjected to RT-qPCR. Similarly, TcA cells were treated with 10 µm of JH III in DMSO or DMSO alone for 6 h. Total RNA isolated from larvae was used to quantify Kr-h1 and HDAC11 mRNA levels. Mean ± SE (n = 4) are shown. (C) Met is required for suppression of HDAC11 by hydroprene. dsMet or dsmalE was injected into day 0 last instar larvae. At 48 h after injection, the larvae were treated with hydroprene. Total RNA isolated from larvae was used to quantify Kr-h1, HDAC11 and, Met mRNA levels. The data shown are mean ± SE (n = 4). C, cyclohexane; H, hydroprene. FIGURE 3 \| Developmental expression proﬁle and JH induction of HDAC11 in T. castaneum determined by RT-qPCR. (A) HDAC11 mRNA levels were determined during the penultimate, last larval, and pupal stages at 24 h intervals. Total RNA was isolated from a pool of two larvae for each replication and subjected to RT-qPCR analysis to determine the relative mRNA levels. The HDAC11 mRNA levels were normalized using RP49. Results expressed as Mean ± SE (n = 4). Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. (B) JH suppresses the expression of HDAC11 in T. castaneum larvae and TcA cells. S-Hydroprene (JH analog) dissolved in cyclohexane was topically applied to 48 h old last instar larvae (0.5 µL of 2 µg/µL). Total RNA was isolated from larvae collected at 6 h after treatment and subjected to RT-qPCR. Similarly, TcA cells were treated with 10 µm of JH III in DMSO or DMSO alone for 6 h. Total RNA isolated from larvae was used to quantify Kr-h1 and HDAC11 mRNA levels. Mean ± SE (n = 4) are shown. (C) Met is required for suppression of HDAC11 by hydroprene. dsMet or dsmalE was injected into day 0 last instar larvae. At 48 h after injection, the larvae were treated with hydroprene. Total RNA isolated from larvae was used to quantify Kr-h1, HDAC11 and, Met mRNA levels. Western Blot Analysis Knockdown efﬁciency was calculated by comparing TcHDAC11 mRNA levels in dsTcHDAC11 and dsmalE treated insects. Mean ± SE (n = 3–5) are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. (C) Newly molted last instar larvae, pupae or adults were injected with dsHDAC11 or dsmalE. The mortality was recorded until death or adult eclosion. Mean ± SE (n = 30) are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. FIGURE 2 \| Phenotypes and mortality induced by dsRNA mediated knockdown of HDAC11 in T. castaneum. (A) a: Control larvae injected with dsmalE pupated and later emerged as healthy adults. b: dsHDAC11 was injected into the newly molted last instar larvae. Phenotypes were photographed on the ﬁfth day after injection. Knockdown of the HDAC11 gene affected larval development resulting in pigmentation, growth retardation, and mortality. c: Light microscopic images of melanization in HDAC11 knockdown larvae. A high degree of hard melanization was detected inside the body. (B) Newly molted last instar larvae, pupae or adults were injected with dsHDAC11 or dsmalE. Total RNA was isolated from treated insects at 72 h after injection and used to determine relative mRNA levels of TcHDAC11. Knockdown efﬁciency was calculated by comparing TcHDAC11 mRNA levels in dsTcHDAC11 and dsmalE treated insects. Mean ± SE (n = 3–5) are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. (C) Newly molted last instar larvae, pupae or adults were injected with dsHDAC11 or dsmalE. The mortality was recorded until death or adult eclosion. Mean ± SE (n = 30) are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. July 2020 \| Volume 11 \| Article 683 Frontiers in Genetics \| www.frontiersin.org George and Palli HDAC11 Function in Tribolium FIGURE 3 \| Developmental expression proﬁle and JH induction of HDAC11 in T. castaneum determined by RT-qPCR. (A) HDAC11 mRNA levels were determined during the penultimate, last larval, and pupal stages at 24 h intervals. Total RNA was isolated from a pool of two larvae for each replication and subjected to RT-qPCR analysis to determine the relative mRNA levels. The HDAC11 mRNA levels were normalized using RP49. Results expressed as Mean ± SE (n = 4). Developmental Expression and JH Suppression of TcHDAC11 The X and Y-axis represent the −log10 P-values and log2 fold change of mean normalized values, respectively. The red dots indicate the genes that showed a ≥2-fold difference in expression with a P ≤0.05. (C) RNA-seq data is veriﬁed and compared with RT-qPCR represented as a stacked bar graph. RT-qPCR conﬁrmed the expression of 20 selected genes from the up-regulated group (Supplementary Data Sheet_RNA-seq data). To test if JH suppresses HDAC11 gene expression, the last instar larvae were treated with JH analog hydroprene in cyclohexane or cyclohexane alone. As shown in Figure 3B, signiﬁcantly lower levels of HDAC11 mRNA were detected in larvae treated with hydroprene when compared to those in the control larvae treated with cyclohexane. Similarly, lower levels of HDAC11 mRNA were detected in TcA cells exposed to JH III, when compared to those in the control cells exposed to DMSO (Figure 3B). Also, the JH-response gene, Kr-h1, was induced by hydroprene in larvae and JH III in cells (Figure 3B). These data suggest that JH suppresses HDAC11 gene expression. To determine whether or not JH suppression of the HDAC11 gene requires the JH receptor, Met, T. castaneum last instar larvae were injected with dsMet or dsmalE and then treated with hydroprene or cyclohexane. As shown in Figure 3C, dsMet injected larvae showed signiﬁcantly lower levels of Met mRNA when compared to those in larvae injected with dsmalE. As expected, the HDAC11 mRNA levels decreased in dsmalE injected larvae treated with hydroprene but not in dsMet injected larvae treated with hydroprene. Also, the Kr-h1 gene was induced in dsmalE injected larvae but not in dsMet injected larvae. These data suggest that Met is required for JH suppression of HDAC11 gene expression. HDAC11 Knockdown Arrests Larval Development castaneum affects the expression of genes involved in JH action and response. The knockdown of HDAC11 in newly molted last instar larvae caused an upregulation of genes involved in JH action (SRC, CBP) and JH response (Kr-h1, 4EBP, G13402). dsHDAC11 or dsmalE was injected into newly molted last instar larvae. Total RNA was extracted at 12 h after treatment, and the mRNA levels of JH-response genes (Kr-h1, 4EBP, G13402), genes involved in JH action (Met, SRC, CBP), HSP90 and Actin were quantiﬁed. Mean ± SE (n = 4) are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. injected with dsmalE (Figure 2B). Injection of dsHDAC11 also induced 100, 38, and 80% mortality in larvae, pupae and adults, respectively (Figure 2C). In contrast, the control insects injected with dsmalE showed less than 20% mortality (Figure 2C). Frontiers in Genetics \| www.frontiersin.org HDAC11 Knockdown Arrests Larval Development To determine HDAC11 function in larval, pupal development, and metamorphosis, the dsHDAC11 was injected into newly July 2020 \| Volume 11 \| Article 683 Frontiers in Genetics \| www.frontiersin.org HDAC11 Function in Tribolium George and Palli FIGURE 5 \| HDAC11 knockdown in the last instar larvae of T. castaneum affects the transcription of genes involved in multiple pathways. (A) The heatmap of differentially expressed genes in dsHDAC11 and dsmalE treated insects. (B) Differentially expressed genes identiﬁed after HDAC11 knockdown represented as the volcano plot. The X and Y-axis represent the −log10 P-values and log2 fold change of mean normalized values, respectively. The red dots indicate the genes that showed a ≥2-fold difference in expression with a P ≤0.05. (C) RNA-seq data is veriﬁed and compared with RT-qPCR represented as a stacked bar graph. RT-qPCR conﬁrmed the expression of 20 selected genes from the up-regulated group (Supplementary Data Sheet_RNA-seq data). FIGURE 4 \| HDAC11 knockdown in the last instar larvae of T. castaneum affects the expression of genes involved in JH action and response. The knockdown of HDAC11 in newly molted last instar larvae caused an upregulation of genes involved in JH action (SRC, CBP) and JH response (Kr-h1, 4EBP, G13402). dsHDAC11 or dsmalE was injected into newly molted last instar larvae. Total RNA was extracted at 12 h after treatment, and the mRNA levels of JH-response genes (Kr-h1, 4EBP, G13402), genes involved in JH action (Met, SRC, CBP), HSP90 and Actin were quantiﬁed. Mean ± SE (n = 4) are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. FIGURE 4 \| HDAC11 knockdown in the last instar larvae of T. castaneum affects the expression of genes involved in JH action and response. The knockdown of HDAC11 in newly molted last instar larvae caused an upregulation of genes involved in JH action (SRC, CBP) and JH response (Kr-h1, 4EBP, G13402). dsHDAC11 or dsmalE was injected into newly molted last instar larvae. Total RNA was extracted at 12 h after treatment, and the mRNA levels of JH-response genes (Kr-h1, 4EBP, G13402), genes involved in JH action (Met, SRC, CBP), HSP90 and Actin were quantiﬁed. Mean ± SE (n = 4) are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. FIGURE 4 \| HDAC11 knockdown in the last instar larvae of T. Developmental Expression and JH Suppression of TcHDAC11 No HDAC11 mRNA was detected in T. castaneum larvae soon after molting into the penultimate larval stage (Figure 3A). Then the HDAC11 mRNA levels increased gradually, reaching the maximum levels by the end of the penultimate larval stage (Figure 3A). The HDAC11 mRNA was detected throughout the last instar larval and pupal stages, albeit with some ﬂuctuations in their levels (Figure 3A). In general, higher levels of HDAC11 mRNA were detected at the end of the penultimate and last instar larval stages when compared to those at the beginning of these stages. Also, higher levels of HDAC11 mRNA were detected in the pupae when compared to those in the larval stages. FIGURE 5 \| HDAC11 knockdown in the last instar larvae of T. castaneum affects the transcription of genes involved in multiple pathways. (A) The heatmap of differentially expressed genes in dsHDAC11 and dsmalE treated insects. (B) Differentially expressed genes identiﬁed after HDAC11 knockdown represented as the volcano plot. The X and Y-axis represent the −log10 P-values and log2 fold change of mean normalized values, respectively. The red dots indicate the genes that showed a ≥2-fold difference in expression with a P ≤0.05. (C) RNA-seq data is veriﬁed and compared with RT-qPCR represented as a stacked bar graph. RT-qPCR conﬁrmed the expression of 20 selected genes from the up-regulated group (Supplementary Data Sheet_RNA-seq data). FIGURE 5 \| HDAC11 knockdown in the last instar larvae of T. castaneum FIGURE 5 \| HDAC11 knockdown in the last instar larvae of T. castaneum affects the transcription of genes involved in multiple pathways. (A) The heatmap of differentially expressed genes in dsHDAC11 and dsmalE treated insects. (B) Differentially expressed genes identiﬁed after HDAC11 knockdown represented as the volcano plot. The X and Y-axis represent the −log10 P-values and log2 fold change of mean normalized values, respectively. The red dots indicate the genes that showed a ≥2-fold difference in expression with a P ≤0.05. (C) RNA-seq data is veriﬁed and compared with RT-qPCR represented as a stacked bar graph. RT-qPCR conﬁrmed the expression of 20 selected genes from the up-regulated group (Supplementary Data Sheet_RNA-seq data). FIGURE 5 \| HDAC11 knockdown in the last instar larvae of T. castaneum affects the transcription of genes involved in multiple pathways. (A) The heatmap of differentially expressed genes in dsHDAC11 and dsmalE treated insects. (B) Differentially expressed genes identiﬁed after HDAC11 knockdown represented as the volcano plot. Identiﬁcation of Genes Affected by HDAC11 Knockdown The eﬀect of Knockdown of HDAC11 on the expression of JH response genes in larvae were tested using RT-qPCR. T. castaneum last instar larvae were injected with dsHDAC11 or dsmalE A, the total RNA isolated from these larvae was used to determine mRNA levels of HDAC11 and genes known July 2020 \| Volume 11 \| Article 683 Frontiers in Genetics \| www.frontiersin.org 6 HDAC11 Function in Tribolium George and Palli TABLE 1 \| Expression changes of hormone-response genes identiﬁed in larvae treated with dsHDAC11. Gene symbol Gene description Fold Changea P-valueb Kr-h1 Krüppel homolog 1 4.68 0.00 LOC655028 ecdysone-induced protein 74EF 2.03 0.00 EcR ecdysone receptor 2.18 0.03 Tcjheh-r1 juvenile hormone epoxide hydrolase-like protein 1 28.14 0.01 Tcjheh-r2 juvenile hormone epoxide hydrolase-like protein 2 2.03 0.01 Tcjheh-r5 juvenile hormone epoxide hydrolase-like protein 5 2.40 0.00 LOC661705 Phosphoenol pyruvate carboxykinase [GTP] 3.14 0.00 LOC659239 Krueppel homolog 2 3.87 0.00 TcSRC nuclear receptor coactivator 1 2.24 0.02 LOC660434 broad-complex core protein isoform 6-like 2.59 0.02 LOC658929 nuclear hormone receptor FTZ-F1 2.03 0.04 USP ultraspiracle nuclear receptor 2.08 0.00 LOC664565 CREB-binding protein 7.73 0.00 LOC660626 Hairy 9.33 0.00 LOC658656 Apterous A 4.67 0.00 aExperiment – Fold Change (normalized values). bBaggerley’s test: normalized values. TABLE 1 \| Expression changes of hormone-response genes identiﬁed in larvae treated with dsHDAC11. FIGURE 6 \| RT-qPCR validation of RNA-seq data in Tribolium larvae and TcA cells. (A) Differentially expressed genes predicted by RNA-seq analysis are veriﬁed by RT-qPCR. Total RNA was extracted at 12 h after injection was used to quantify mRNA levels. Mean ± SE of four replications is shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by t-test. (B) TcA cells were treated with dsHDAC11 or dsmalE. Total RNA was extracted 72 h after dsRNA treatment was used to quantify mRNA levels. Mean ± SE of four replications is shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. FIGURE 6 \| RT-qPCR validation of RNA-seq data in Tribolium larvae and TcA cells. (A) Differentially expressed genes predicted by RNA-seq analysis are veriﬁed by RT-qPCR. Total RNA was extracted at 12 h after injection was used to quantify mRNA levels. Mean ± SE of four replications is shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by t-test. (B) TcA cells were treated with dsHDAC11 or dsmalE. Identiﬁcation of Genes Affected by HDAC11 Knockdown Gene symbol Gene description JH dsHDAC11 Fold change P-value Fold change P-value LOC660562 rho GTPase-activating protein 100F 115.45 0.020 29.80 0.002 Kr-h1 Krüppel homolog 1 29.91 0.000 4.68 0.000 LOC662658 4-hydroxyphenylpyruvate dioxygenase 3.46 0.000 4.00 0.000 LOC100141923 sodium-coupled monocarboxylate transporter 2-like 202.63 0.004 3.82 0.014 LOC660154 projectin 13.63 0.030 3.68 0.000 LOC659434 ﬁbroblast growth factor receptor homolog 1 95.55 0.030 3.63 0.000 LOC661127 uncharacterized LOC661127 4.02 0.030 3.38 0.000 LOC107398485 sodium-independent sulfate anion transporter-like 4.28 0.030 3.21 0.004 LOC658154 alpha-2C adrenergic receptor 8.08 0.001 2.90 0.012 LOC100142605 ankyrin-3 2.90 0.008 2.38 0.007 LOC103314138 voltage-dependent T-type calcium channel subunit alpha-1G 6.85 0.020 2.14 0.000 TABLE 2 \| JH-response genes that showed an increase in expression in dsHDAC11 treated larvae. TABLE 3 \| COG5048 domains identiﬁed in genes up-regulated in dsHDAC11 treated larvae. Gene symbol Gene description Locus tag Fold change P-value LOC658487 Zinc ﬁnger protein GLIS2 homolog TC000326 2.49 0.000 LOC659757 Zinc ﬁnger protein Gﬁ-1 TC031695 6.19 0.000 Kr-h1 Krüppel homolog 1 TC012990 4.68 0.000 LOC660309 Krueppel-like factor 8 TC006125 2.23 0.000 Pho Pleiohomeotic TC015577 3.69 0.000 LOC662411 Zinc ﬁnger protein 184 TC015048 2.92 0.040 LOC103313120 Zinc ﬁnger protein OZF N/A 3.99 0.050 LOC103314758 Zinc ﬁnger protein ZFP69 TC033495 3.11 0.010 LOC107397979 Gastrula zinc ﬁnger protein XlCGF26.1-like N/A 8.52 0.030 N/A, not available. TABLE 4 \| Genes involved in melanin biosynthesis that showed an increase in their expression in HDAC11 knockdown larvae. Gene symbol Gene description Locus Tag Fold Change P-value Ddc dopa decarboxylase TC013480 4.46 0.000 Dat dopamine N acetyltransferase TC008204, Nat 2.50 0.000 Lac1 laccase 1 TC000821, TcLac1 2.36 0.000 Th tyrosine hydroxylase TC002496 3.39 0.000 Y-2 yellow-2 TC003539 4.97 0.000 to be involved in JH-response. The mRNA levels of HDAC11 decreased and those of three JH-response genes (Kr-h1, 4EBP, G13402), SRC, and CBP increased signiﬁcantly in dsHDAC11 injected larvae when compared to those in larvae injected with dsmalE (Figure 4). However, the expression of Met and two housekeeping genes, Actin and HSP90, were not aﬀected by HDAC11 knockdown (Figure 4). These data suggest that HDAC11 may inﬂuence the expression of JH-response as well as genes coding for proteins involved in JH action. Figure 5A. The DEGs are shown as a volcano plot with red dots indicating statistically signiﬁcant genes after the EDGE test between treatment and control (Figure 5B). Frontiers in Genetics \| www.frontiersin.org Identiﬁcation of Genes Affected by HDAC11 Knockdown Total RNA was extracted 72 h after dsRNA treatment was used to quantify mRNA levels. Mean ± SE of four replications is shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0 05 by ANOVA 7 July 2020 \| Volume 11 \| Article 683 Frontiers in Genetics \| www.frontiersin.org HDAC11 Function in Tribolium George and Palli TABLE 2 \| JH-response genes that showed an increase in expression in dsHDAC11 treated larvae. Gene symbol Gene description JH dsHDAC11 Fold change P-value Fold change P-value LOC660562 rho GTPase-activating protein 100F 115.45 0.020 29.80 0.002 Kr-h1 Krüppel homolog 1 29.91 0.000 4.68 0.000 LOC662658 4-hydroxyphenylpyruvate dioxygenase 3.46 0.000 4.00 0.000 LOC100141923 sodium-coupled monocarboxylate transporter 2-like 202.63 0.004 3.82 0.014 LOC660154 projectin 13.63 0.030 3.68 0.000 LOC659434 ﬁbroblast growth factor receptor homolog 1 95.55 0.030 3.63 0.000 LOC661127 uncharacterized LOC661127 4.02 0.030 3.38 0.000 LOC107398485 sodium-independent sulfate anion transporter-like 4.28 0.030 3.21 0.004 LOC658154 alpha-2C adrenergic receptor 8.08 0.001 2.90 0.012 LOC100142605 ankyrin-3 2.90 0.008 2.38 0.007 LOC103314138 voltage-dependent T-type calcium channel subunit alpha-1G 6.85 0.020 2.14 0.000 TABLE 3 \| COG5048 domains identiﬁed in genes up-regulated in dsHDAC11 treated larvae. Gene symbol Gene description Locus tag Fold change P-value LOC658487 Zinc ﬁnger protein GLIS2 homolog TC000326 2.49 0.000 LOC659757 Zinc ﬁnger protein Gﬁ-1 TC031695 6.19 0.000 Kr-h1 Krüppel homolog 1 TC012990 4.68 0.000 LOC660309 Krueppel-like factor 8 TC006125 2.23 0.000 Pho Pleiohomeotic TC015577 3.69 0.000 LOC662411 Zinc ﬁnger protein 184 TC015048 2.92 0.040 LOC103313120 Zinc ﬁnger protein OZF N/A 3.99 0.050 LOC103314758 Zinc ﬁnger protein ZFP69 TC033495 3.11 0.010 LOC107397979 Gastrula zinc ﬁnger protein XlCGF26.1-like N/A 8.52 0.030 N/A, not available. TABLE 4 \| Genes involved in melanin biosynthesis that showed an increase in their expression in HDAC11 knockdown larvae. Gene symbol Gene description Locus Tag Fold Change P-value Ddc dopa decarboxylase TC013480 4.46 0.000 Dat dopamine N acetyltransferase TC008204, Nat 2.50 0.000 Lac1 laccase 1 TC000821, TcLac1 2.36 0.000 Th tyrosine hydroxylase TC002496 3.39 0.000 Y-2 yellow-2 TC003539 4.97 0.000 to be involved in JH-response. The mRNA levels of HDAC11 decreased and those of three JH-response genes (Kr-h1 4EBP Figure 5A. The DEGs are shown as a volcano plot with red dots indicating statistically signiﬁcant genes after the TABLE 2 \| JH-response genes that showed an increase in expression in dsHDAC11 treated larvae. Identiﬁcation of Genes Affected by HDAC11 Knockdown (B) Acetylated-Lysine (Ac-K2-100) MultiMabTM Rabbit mAb mix #9814 was used to detect acetylation levels of proteins extracted from TcA cells exposed to dsHDAC11 or dsmalE. The acetylation levels of histone H3K9, H3K18 and H3K27 increased in HDAC11 knockdown cells were detected as described in Figure 7A. The band densities were quantiﬁed and plotted as described in Figure 7A. The Mean + SE (n = 3) band densities are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. FIGURE 7 \| HDAC11 knockdown affects acetylation levels of histone H3 in T. castaneum. (A) The total protein extracted from dsHDAC11 or dsmalE injected larvae were resolved on SDS-PAGE gels, transferred to western blots, and probed with antibodies recognizing Acetyl-Histone H3 (Antibody Sampler Kit # 9927-Cell Signaling (H3K9, H3K14, H3K18, H3K27, and H3K56). ß-actin served as a loading control. The HRP-linked IgG (#7074-Cell Signaling) was used as a secondary antibody. Band densities were determined by Image-J software and normalized with loading control protein-ß-Actin. The Mean + SE (n = 3) band densities are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. (B) Acetylated-Lysine (Ac-K2-100) MultiMabTM Rabbit mAb mix #9814 was used to detect acetylation levels of proteins extracted from TcA cells exposed to dsHDAC11 or dsmalE. The acetylation levels of histone H3K9, H3K18 and H3K27 increased in HDAC11 knockdown cells were detected as described in Figure 7A. The band densities were quantiﬁed and plotted as described in Figure 7A. The Mean + SE (n = 3) band densities are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. in HDAC11 knockdown larvae (Table 3). Since dsHDAC1 (George et al., 2019) and dsHDAC11 induced 100% mortality, we compared DEGs between these two datasets and identiﬁed several common genes (Supplementary Data Sheet_dsHDAC1 vs. dsHDAC11). Notably, the expression of CBP was up- regulated by seven-fold in both treatments. Since dsHDAC11 larval phenotypes showed enhanced pigmentation, we searched for genes coding for enzymes involved in melanization in our RNA-seq data. Interestingly, several genes coding for enzymes known to function in melanin biosynthesis were up-regulated in dsHDAC11 injected larvae (Table 4). in their expression levels determined by the two methods (Figure 5C). Identiﬁcation of Genes Affected by HDAC11 Knockdown We identiﬁed 1913 DEGs (Supplementary Data Sheet), 95% (1815) of these genes are up-regulated, and 5% (98) are down- regulated. Hormone response genes Kr-h1, ecdysone induced protein 74EF, and ecdysone receptor are among the up-regulated genes (Supplementary Data Sheet, dsHDAC11-DEG, Table 1). Web-based GO analysis of diﬀerently expressed genes showed enrichment of GO terms for binding, especially nucleic acid and protein binding, biological regulation, pigmentation, and developmental process (Supplementary Figure S2). To conﬁrm the results from RNA-seq analysis, we selected 20 genes based on their predicted function and expression levels and veriﬁed by RT-qPCR using the RNA isolated from T. castaneum larvae. Eighteen out of 20 genes tested showed a positive correlation to be involved in JH-response. The mRNA levels of HDAC11 decreased and those of three JH-response genes (Kr-h1, 4EBP, G13402), SRC, and CBP increased signiﬁcantly in dsHDAC11 injected larvae when compared to those in larvae injected with dsmalE (Figure 4). However, the expression of Met and two housekeeping genes, Actin and HSP90, were not aﬀected by HDAC11 knockdown (Figure 4). These data suggest that HDAC11 may inﬂuence the expression of JH-response as well as genes coding for proteins involved in JH action. The RNA isolated from dsHDAC11 and dsmalE injected larvae at 12 h after treatment were sequenced and assembled into transcriptomes and used to identify other targets of HDAC11. Run summary and total read counts of sequencing output are shown in Supplementary Table S2. A heatmap representing the overall pattern of normalized mean expression values of diﬀerentially expressed genes (DEGs) is shown in July 2020 \| Volume 11 \| Article 683 8 HDAC11 Function in Tribolium George and Palli FIGURE 7 \| HDAC11 knockdown affects acetylation levels of histone H3 in T. castaneum. (A) The total protein extracted from dsHDAC11 or dsmalE injected larvae were resolved on SDS-PAGE gels, transferred to western blots, and probed with antibodies recognizing Acetyl-Histone H3 (Antibody Sampler Kit # 9927-Cell Signaling (H3K9, H3K14, H3K18, H3K27, and H3K56). ß-actin served as a loading control. The HRP-linked IgG (#7074-Cell Signaling) was used as a secondary antibody. Band densities were determined by Image-J software and normalized with loading control protein-ß-Actin. The Mean + SE (n = 3) band densities are shown. Means marked with different letters are signiﬁcantly different from each other, P ≤0.05 by ANOVA. DISCUSSION We recently reported on the function of class I HDACs in JH suppression of metamorphosis in T. castaneum (George et al., 2019). Here, we investigated the role of the sole member of class IV HDACs, HDAC11. HDAC11 knockdown induced complete lethality during the larval stage. Human HDAC11 is biochemically distinct from other HDACs but phylogenetically closely related to class I HDACs (De Ruijter et al., 2003). Similarly, phylogenetic analysis of HDACs in T. castaneum revealed that TcHDAC11 is close to class I than to class II deacetylases (Figure 1B). FIGURE 8 \| Schematic representation of HDAC11 action in the red ﬂour beetle. Here we summarized the results RNA sequencing and RT-qPCR analysis showed that knockdown in HDAC11 affects the expression of JH and 20E response genes as well as those coding for proteins involved in the action of these two hormones. Since both JH and 20E play critical roles in larval development and metamorphosis, HDAC11 may have blocked larval development and metamorphosis by affecting the action of these two hormones. The GO terms for pigmentation is signiﬁcantly enriched in HDAC11 knockdown samples compared to the control. RNA-seq data showed that the genes coding for enzymes (Tyrosine hydrolase, Ddc, Yellow-2, Laccase 1, Dopamine N acetyltransferase) involved in insect melanin biosynthesis are up-regulated. RNAi mediated knockdown of HDAC11 induces mortality in larvae, pupae, and adults. Mortality factor 4-like protein 1 (TC011468) and apterous A (TC003972) were identiﬁed as differentially expressed genes in HDAC11 knockdown larvae. HDAC11 knockdown resulted in an increase in acetylation levels of histones H3, especially H3K18 and H3K27. y ( g ) Interestingly, we observed that knockdown of the HDAC11 gene during the ﬁnal instar larval stage of the red ﬂour beetle, T. castaneum resulted in a dark-colored larval phenotype that eventually died. Human HDAC11 is reported to be involved in the regulation of diﬀerent inﬂammatory responses and diverse immune functions (Yanginlar and Logie, 2018). Dopa decarboxylase (Ddc) and phenoloxidase (PO) are necessary for insect cuticular melanization, and the molecular action of 20-hydroxyecdysone on various transcription factors leads to Ddc expression in Manduca sexta (Hiruma and Riddiford, 2009). Strict regulation of immune response and melanization is crucial for the proper development and survival of insects. RNA-seq data showed that the genes coding for enzymes (Tyrosine hydrolase, Ddc, Yellow-2, Laccase 1, Dopamine N acetyltransferase) known to be involved in insect melanin biosynthesis are up-regulated in HDAC11 knockdown larvae (Table 4). HDAC11 Knockdown Increases Acetylation Levels of Histone H3 Increase in acetylation levels of H3K9, H3K18, and H3K27 was detected in dsHDAC11 treated larvae compared to the levels in dsmalE treated larvae (Figure 7A). Also, TcA cells exposed to dsHDAC11 showed an increase in acetylation levels of core histones H3, H2, and H4 (Figure 7B) compared with control cells treated with dsmalE. An increase in acetylation levels of H3K9, H3K18 and H3K27 was detected in dsHDAC11 treated cells compared to control cells treated with dsmalE (Figure 7B). These data suggest that H3 is one of the targets for HDAC11 deacetylation. Identiﬁcation of Genes Affected by HDAC11 Knockdown RT-qPCR analysis of RNA isolated from larvae (Figure 6A) and TcA cells (Figure 6B) also veriﬁed an increase in expression levels of EcRA, E74, E93 Ftz-f1, TC011468 and ap A increase predicted by diﬀerential gene expression analysis of transcriptomes of larvae treated with dsHDAC11 or dsmalE. A comparison of up-regulated genes between JH III treated TcA cells (Roy and Palli, 2018), and dsHDAC11 treated larvae identiﬁed eleven common genes, including the Kr-h1 (Table 2). Nine genes that code for proteins containing zinc ﬁnger, COG5048 domains found in Kr-h1 are also up-regulated July 2020 \| Volume 11 \| Article 683 Frontiers in Genetics \| www.frontiersin.org 9 HDAC11 Function in Tribolium George and Palli Frontiers in Genetics \| www.frontiersin.org July 2020 \| Volume 11 \| Article 683 DISCUSSION RNA-seq analysis revealed several genes, including transcription factor sox-10, Hairy, CBP, SRC, chromatin remodeler SWI/SNF complex subunit SMARCC2, polycomb complex protein BMI-1-A, polycomb protein Scm, BTB/POZ, ultrabithorax, EcR, tramtrack, ecdysone- induced protein 74EF, and nuclear hormone receptor FTZ-F1 that showed an increase in expression in HDAC11 knockdown larvae. These data suggest that the deacetylation of histones and other proteins by HDAC11 may lead to a decrease in expression of these genes involved in JH and 20E action and response. However, the precise mechanisms involved in the arrest in larval development induced by HDAC11 knockdown remains to be elucidated. Further studies are required to uncover molecular mechanisms governing these phenotypes. lymphoma (Buglio et al., 2011). The protein “Eiger” is the close homolog for OX40L in T. castaneum and D. melanogaster eiger (egr) encodes the TNF superfamily ligand that activates the intracellular JNK pathway, which mediates cell death, tumor suppression, and growth regulation (Igaki et al., 2002; Shklover et al., 2015). Our studies revealed that HDAC11 is essential for survival, and RNAi mediated knockdown induce developmental arrest and mortality. lymphoma (Buglio et al., 2011). The protein “Eiger” is the close homolog for OX40L in T. castaneum and D. melanogaster eiger (egr) encodes the TNF superfamily ligand that activates the intracellular JNK pathway, which mediates cell death, tumor suppression, and growth regulation (Igaki et al., 2002; Shklover et al., 2015). Our studies revealed that HDAC11 is essential for survival, and RNAi mediated knockdown induce developmental arrest and mortality. y HDAC11 regulates oligodendrocyte-speciﬁc gene expression and cell development in the cell line of rats by deacetylation of histone H3K9/K14 (Liu et al., 2009). Our western blots analysis showed that HDAC11 regulates acetylation levels of H3, speciﬁcally H3K9, H3K18 and H3K27. As demonstrated for HDAC1 in T. castaneum (George et al., 2019), HDAC11 may also inﬂuence acetylation levels of histones, especially H3, and regulate promoter access and, consequently, the expression of genes involved in JH and 20E action and response. In conclusion, we showed that HDAC11 knockdown aﬀects hormone action and melanin biosynthesis and thereby arrest in development and metamorphosis of the red ﬂour beetle, T. castaneum (Figure 8). Apterous A (ap A, TC003972) and mortality factor 4- like protein 1 (TC011468) are signiﬁcantly up-regulated in dsHDAC11 treated larvae. RT-qPCR reconﬁrmed RNA-seq predictions (Figures 6A,B). In D. DATA AVAILABILITY STATEMENT We have deposited the short-read (Illumina HiSeq 4000) sequence data in the NCBI SRA (accession numbers PRJNA495026 and PRJNA612004). BioSample metadata are available in the NCBI BioSample database (http://www.ncbi.nlm.nih.gov/biosample/) under accession numbers SAMN10203356, SAMN10203357, SAMN14356366, and SAMN14356367). DISCUSSION melanogaster, apterous encodes a member of LIM (Lin11, Isl-1& Mec-3) homeobox transcription factor which contributes to the identity of wing cells, JH production, and neuronal pathﬁnding (Cohen et al., 1992). Mortality factor 4-like protein 1 (TC011468), also known as NuA4 complex subunit EAF3 homolog-like protein is a MORF- related gene and part of the Tip60 chromatin remodeling complex in D. melanogaster. Tip60 is involved in DNA repair by acetylating phosphorylated H2AV in D. melanogaster (Kusch et al., 2004). Mortality factor 4 like 1 protein regulates chromatin remodeling and mediates epithelial cell death in a mouse model of pneumonia (Zou et al., 2015). Interestingly, HDAC inhibitors activity leads to the modulation of expression of various genes and in turn, induces growth arrest, diﬀerentiation, and apoptotic cell death (Marks et al., 2000). Moreover, HDAC11 overexpression inhibits the cell cycle progression in ﬁbroblast of Balb/c-3T3 cells. Also, the HDAC11 transcript was identiﬁed as a platelet-derived growth factor (PDGF) target, and HDAC11 mRNA abundance correlates inversely with proliferative status (Bagui et al., 2013). We identiﬁed ﬁbroblast growth factor receptor homolog 1, ankyrin-3 as common genes up-regulated in HDAC11 knockdown larvae, or JH III treated cells (Table 2). Nuclear hormone receptors and their transcriptional coregulators were expressed in neural stem cells, and their expression was altered during diﬀerentiation induced by ﬁbroblast growth factor 2 (FGF2) withdrawal. FGF2 withdrawal strongly induced the mRNA expression of HDAC11 in mouse cells (Androutsellis-Theotokis et al., 2013). Chromatin modiﬁer, HDAC11, regulates lymph node metastasis development and dissemination in the breast cancer experimental model (Leslie et al., 2019). Human HDAC11 expression is limited to kidney, heart, brain, skeletal muscle, and testis, suggesting a tissue-speciﬁc function (Gao et al., 2002). Small interfering RNAs (siRNAs) that selectively inhibited HDAC11 expression, signiﬁcantly up-regulated OX40L, and induced apoptosis in Hodgkin lymphoma (HL) cell lines. Silencing HDAC11 increased the production of tumor necrosis- α (TNF-α) and IL-17 in the supernatants of HL cells. An HDAC inhibitor study in human cell lines revealed that HDAC11 plays an essential role in regulating OX40 ligand expression in Hodgkin ACKNOWLEDGMENTS We thank Najla Albishi for help with microscopic techniques and Hunt lab for RNA-seq analysis. FUNDING This work was supported by grants from the National Institute of Health (GM070559-14) and the National Institute of Food and Agriculture, US Department of Agriculture (HATCH project 2351177000). AUTHOR CONTRIBUTIONS SG and SP designed the experiments, and wrote the manuscript. SG carried out the experiments. Both authors contributed to the article and approved the submitted version. DISCUSSION In D. melanogaster, microbial infection triggers activation of phenoloxidases; serine proteases and serine protease inhibitors (serpins) control the sites of infection (Tang, 2009). Diﬀerential expression of genes coding for several serine proteases, toll-like receptors, and serpins were observed in HDAC11 knockdown samples (Datasheet_melanization terms). Melanization and toll pathway share similar serine proteases in D. melanogaster (Dudzic et al., 2019). caspase-dependent apoptosis (Thole et al., 2017). HDAC11 depletion in MYCN-driven neuroblastoma cell lines strongly induces cell death, mostly mediated by apoptotic programs (Thole et al., 2017). Inhibitor studies in mouse models showed that HDAC11 plays an important role in oncogene-induced hematopoiesis in myeloproliferative neoplasms (MPNs) (Yue et al., 2020). Based on these previous ﬁndings, it is tempting to hypothesize that HDAC11 knockdown may induce the death of some internal tissues resulting in the dark color detected in larvae. However, further work is required to identify speciﬁc tissues/cells involved and to uncover mechanisms behind the phenotype detected after HDAC11 knockdown in T. castaneum larvae. Another ﬁnding of this research is the discovery that HDAC11 is required for larval development and metamorphosis in T. castaneum. This is the ﬁrst report on the role of HDAC11 on the growth and development of insect larvae. RNA sequencing and RT-qPCR analysis showed that knockdown in HDAC11 aﬀects the expression of JH and 20E response genes as well as those coding for proteins involved in the action of these two hormones. Since both JH and 20E play critical roles in larval development and metamorphosis (Jindra et al., 2013); HDAC11 Inhibition of HDAC11 induced the expression of p53 in human liver cancer cells and promoted apoptosis (Gong et al., 2019). 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https://openalex.org/W2083842566	https://europepmc.org/articles/pmc4052002?pdf=render	English	null	Universal Grammar and Biological Variation: An EvoDevo Agenda for Comparative Biolinguistics	Biological theory	2,014	cc-by	10,719	Universal Grammar and Biological Variation: An EvoDevo Agenda for Comparative Biolinguistics Antonio Benı´tez-Burraco • Cedric Boeckx Received: 4 December 2013 / Accepted: 23 January 2014 / Published online: 15 March 2014 The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Recent advances in genetics and neurobiology have greatly increased the degree of variation that one ﬁnds in what is taken to provide the biological founda- tions of our species-speciﬁc linguistic capacities. In par- ticular, this variation seems to cast doubt on the purportedly homogeneous nature of the language faculty traditionally captured by the concept of ‘‘Universal Grammar.’’ In this article we discuss what this new source of diversity reveals about the biological reality underlying Universal Grammar. Our discussion leads us to support (1) certain hypotheses advanced in evolutionary develop- mental biology that argue for the existence of robust biological mechanisms capable of canalizing variation at different levels, and (2) a bottom-up perspective on comparative cognition. We conclude by sketching future directions for what we call ‘‘comparative biolinguistics,’’ specifying which experimental directions may help us succeed in this new research avenue. proper characterization of the biological foundations of language and, in so doing, to a rapprochement of the cognitive sciences with the biological sciences, an enter- prise that we refer to as biolinguistics (Di Sciullo and Boeckx 2011; Boeckx 2013; Boeckx and Grohmann 2013). Our main contention in this article is that although the comparative method has ﬁgured prominently in linguistics, the objects routinely compared (languages, dialects, so- ciolects) may not be the only, or indeed the most appro- priate ones to shed light on the biological foundations of our species-speciﬁc linguistic capacity. There are, we claim, deeper layers of variation to explore and to under- stand. Indeed, as we intend to show here, these deeper layers of variation beg questions regarding the proper biological interpretation of standard concepts in the ﬁeld of (bio)linguistics, such as Universal Grammar (Chomsky 1965). We believe that in part it is the failure to properly address these sources of variation that has rendered the adjective ‘‘universal’’ (and in fact the whole argument of language as a speciﬁc component of human biology) more controversial than it should be. Biol Theory (2014) 9:122–134 DOI 10.1007/s13752-014-0164-0 Biol Theory (2014) 9:122–134 DOI 10.1007/s13752-014-0164-0 LONG ARTICLE C. Boeckx Catalan Institute for Research and Advanced Studies (ICREA) & Department of Linguistics, University of Barcelona, Barcelona, Spain Universal Grammar and Biological Variation: An EvoDevo Agenda for Comparative Biolinguistics However, the recent emphasis on the biological underpin- nings of language—the return of biolinguistic concerns— has begun to reveal deeper layers of variation down to the genetic level that make these standard assumptions unrea- sonable, and beg the question of where the uniformity or universality of grammar comes from. At the same time, the human faculty for language has routinely been assumed to be uniform within the species (Chomsky 1965, 1980), an assumption captured by the term ‘‘Universal Grammar.’’ For many people, this assumption is a central tenet of the Chomskyan revolution in the language sciences. As is well known, this revolution brought about a radical shift of focus in language studies, a shift oriented towards biology, and away from behavior- ism; indeed, a shift that provides the foundations of modern biolinguistics. Early investigations of the biological foundations of language (see Lenneberg 1967) relied on evidence from a variety of sources, such as: We will then proceed to argue that in fact this deeper variation is problematic mainly for naive approaches to the biology of language. However, we ﬁnd that these approa- ches are still the majority within the ﬁeld, and should therefore be corrected to accommodate the variation we ﬁnd. (1) The way in which language is acquired by the child, which suggests that language ‘‘learning’’ mecha- nisms are biased or constrained in certain ways. (2) The fact that speciﬁc language deﬁcits recurrently appear whenever certain brain areas are impaired (either developmentally or after a stroke, a trauma, or a tumoral process). We will then suggest that new theoretical approaches coming from biology, paradigmatically, evolutionary developmental biology (EvoDevo), could help us deal with, and account for, all the observed variation, while offering sources of uniformity to reliably give rise to spe- cies-typical linguistic capacities. As a matter of fact, we claim that certain EvoDevo concepts could contribute signiﬁcantly to our understanding of the nature of language disorders. (3) Ultimately, the existence of language-related com- ponents in other extant or extinct species. These arguments in favor of Universal Grammar con- tinue to be made even by those who otherwise distance their positions from Chomsky’s in a number of ways (see, e.g., Jackendoff 2002, Chap. 4). Recent advances in neu- roscience and molecular biology have allowed us to gain a better understanding of the biological underpinnings of language. Universal Grammar and Biological Variation: An EvoDevo Agenda for Comparative Biolinguistics Keywords Biolinguistics Evolutionary developmental biology (EvoDevo) Genetics Language disorders Variation Keywords Biolinguistics Evolutionary developmental biology (EvoDevo) Genetics Language disorders Variation At the same time, we also think, and will argue in what follows, that linguists have asked questions regarding the locus of linguistic variation (traditionally construed) that would be useful to extend to the layers of variation we are about to highlight. But for this to be successful, it is important to carefully select, and properly conceptualize, the tools one uses to establish comparisons. Here, we think it is imperative to formulate these tools at the right level of granularity to allow for interdisciplinary exchange, in line with Poeppel and Embick (2005). It is our conviction that once this is done, these tools and questions about variation would enrich the range of studies that currently constitutes what we may want to call ‘‘comparative biolinguistics,’’ to be distinguished from the traditional label of comparative The present article is concerned with the amount and kind of variation that we think linguists and researchers in allied disciplines should wrestle with if they are to contribute to a A. Benı´tez-Burraco (&) Department of Spanish Philology and its Didactics, University of Huelva, Huelva, Spain e-mail: antonio.benitez@dfesp.uhu.es C. Boeckx Catalan Institute for Research and Advanced Studies (ICREA) & Department of Linguistics, University of Barcelona, Barcelona, Spain 123 A. Benı´tez-Burraco (&) Department of Spanish Philology and its Didactics, University of Huelva, Huelva, Spain e-mail: antonio.benitez@dfesp.uhu.es 3 123 Universal Grammar and Biological Variation linguistics, for reasons that will be spelled out in this article. can be accounted for by the right mixture of general prin- ciples governing cognition and statistical biases (see Baker 2001; Labov 2001; Yang 2006; Pearl 2007; Biberauer 2008; Culbertson 2010, for accessible overviews and concrete proposals to capture this variation). Our reﬂections are structured as follows. First, we will clarify why we think this new kind of linguistic variation is real, and problematic for standard characterizations of certain central concepts in the ﬁeld of (bio)linguistics. In a nutshell, linguists routinely acknowledge, and, we believe, have quite successfully dealt with, linguistic variation at the surface (languages, dialects, sociolects, and the like). At the same time, they have usually regarded the faculty of language giving rise to these variants as uniform across the species (pathologies aside), and genetically determined. Universal Grammar and Biological Variation: An EvoDevo Agenda for Comparative Biolinguistics For instance, we are now in a position to accu- rately know which brain areas and circuits are active during language processing (see Stemmer and Whitaker 2008; Friederici 2011; or Friederici and Gierhan 2013 for over- views). Similarly, we have identiﬁed many of the genes that contribute to the development and the initial wiring of these areas and circuits during growth (Benı´tez-Burraco 2009; Graham and Fisher 2012). Ultimately, the role of these brain areas and circuits, and of these genes in other extant (and even extinct) species are being studied, revealing (deep) homologues of (aspects of) the language faculty (see the contributions in Di Sciullo and Boeckx 2011; Boeckx and Grohmann 2013). Finally, we will sketch some concrete and novel ways in which linguists and other scientists in adjacent ﬁelds could contribute to a comparative biolinguistics. In this ﬁnal section, we are led to point out some important limitations of recent tools used in this domain. Layers of Variation 702), is assumed to be uniform across the species, pathologies aside. Moreover, we continue to come across many studies in which the ‘‘lin- guistic brain’’—i.e., the whole set of brain areas and circuits involved in language processing—is expected to be equally organized and sharply deﬁned in all individuals. For many, these ‘‘language areas’’ only process speciﬁc linguistic fea- tures and operations. Ultimately, the faculty of language is supposed to be equally implemented in all subjects as one of the modules encompassing their minds/brains. This module is further thought to be present ab initio, part of a genetic program of sorts (see, e.g., Wexler 1999 and Guasti 2002; and see Longa and Lorenzo 2008 for a critical overview of this ‘‘genocentric’’ literature). Our reading of the growing literature on the biological foundations of language suggests to us that the state of affairs described in the previous paragraph is quite an erroneous, and in fact simplistic, view of reality. In par- ticular, a fair amount of evidence exists that suggests that the human faculty for language is not really uniform. To be fair, some of this evidence is not new, but we feel that its signiﬁcance has not yet been properly appreciated. Doing this is the primary purpose of our article. Unfortunately, for linguists who confess a biological orientation, even those directly concerned with language development, this kind of variation ‘‘at the bottom’’ is ignored. They tend to idealize away from it, at their own peril. We say this because we think that the layers of variation just mentioned and the developmental dynamics that they involve lead to an important conclusion: it seems that there can be different ways of implementing a (more or less) functional faculty of language (see also Hancock and Bever 2013), and that talk of a ‘‘linguistic genotype’’ is fraught with difﬁculties. When one thinks about the possibility of a variable implementation of the language faculty, one is likely to think about facts like: (1) different linguistic modalities can coexist in the same subject, as people bilingual in oral and sign languages nicely exemplify (Emmorey and McCul- lough 2009); (2) psycholinguistic measures are varied across the normal population (Fenson et al. 2000); (3) language disorders, which plausibly represent different breakdowns of the faculty, are very diverse by nature, and, as the relevant literature has revealed, sometimes difﬁcult to distinguish from stages of normal language development. Layers of Variation Variation thoroughly pervades language. The human lan- guage faculty manifests itself in the form of many different languages, which are in turn (slightly) diverse across social groups, interactional contexts, geographical areas, and so on. Ultimately, differences can be found from one person to another, and even regarding the same person, for instance, when placed in different scenes. All of this is very familiar, and, we feel, linguists have fairly successfully coped with all this variation (which, of course, is not to say that everything is understood at this level). It is now clear that linguistic phenomena vary in systematic and constrained ways, and However, this revival of biolinguistic concerns has not yet substantially changed some of the concepts that remain at the center of the ﬁeld, such as Universal Grammar. In the vast majorities of studies in biolinguistics (we will return to exceptions), what is called the ‘‘linguistic genotype,’’ i.e., the whole set of genes involved in ultimately allowing for 12 3 3 124 A. Benı´tez-Burraco, C. Boeckx Rodenas-Cuadrado et al. 2013 for reviews). Additionally, it is quite difﬁcult to draw a precise map of the neural substrate of language, since the limits of the brain areas involved are rather changeable from one subject to another, and of course, in different clinical conditions (Fedorenko and Kanwisher 2009; Prat and Just 2011)—not to mention the additional problem that at the end of the day mapping is not explaining (Poeppel 2012). Ultimately, developmental tra- jectories followed by language acquisition, while displaying similar milestones, can be quite diverse, particularly at the cognitive/neurobiological levels (Bates et al. 1988; Dehaene et al. 1997). Language ontogeny in pathological populations is even more diverse, yet equally non-random (Thomas et al. 2009). It is now evident that similar cognitive proﬁles, in the normal population but also across pathologies, can rely on different brain architectures (Karmiloff-Smith 2010). ‘‘Modules are not born; they are made’’ (Bates et al. 1988, p. 284), although their basic wiring is achieved before birth, plausibly, under genetic guidance. This means that, even- tually, we will have to address the diverse sources of vari- ation (genetic, neurobiological, etc.) just mentioned in the context of a developmental perspective, allowing for dif- ferent trajectories that eventually converge phenotypically. language growth in the child, or ‘‘that part of our genetic endowment that is relevant to our linguistic development’’ (Anderson and Lightfoot 1999, p. Layers of Variation Our main point is that the naive depictions of the biology of language that continue to dominate the literature must be improved. In our opinion, all this variation ‘‘at the bottom’’ can be reconciled with a certain notion of universality, but only if biolinguists are willing to engage seriously, and comprehensively, with the biology literature; i.e., only by pursuing a program that has been called biolinguistics in the strong sense of the term in Boeckx and Grohmann (2007). We wish to stress that this is not a message exclusively directed towards linguists. As we show below, a productive comparative biolinguistics also needs to take into account lessons from linguistics about the nature of language in order to develop appropriate tools for comparison. All of this is appropriate for the lessons we want to draw in this article. But we think that the current revival of the biolinguistic approach has substantially expanded the realm of variation regarding language and linguistic phenomena. For example, it is now clear that it is not a handful but hundreds of genes that contribute to regulating the devel- opment and the functioning of the neural substrate of lan- guage (Benı´tez-Burraco 2009). Importantly, these ‘‘language genes’’ are polymorphic, with some variants giving rise to pathological conditions, but with others being present as well within the normal population. In fact, pathological alleles can be only regarded as such for certain populations and/or environmental conditions. Lastly, the same patho- genic allele can give rise to different language and/or cog- nitive disorders in different subjects. The celebrated ‘‘language gene’’ FOXP2 and its interactome nicely exem- plify this complex state of affairs (see Watkins 2011; Which Biology Does Universal Grammar Require? In our opinion, the root of the problem discussed in the previous section lies in the assumption that language 123 123 125 Universal Grammar and Biological Variation features are directly rooted in the genome. As the literature (reviewed in Longa and Lorenzo 2008, 2012; Lorenzo and Longa 2009) reveals, a ‘‘linguistic genotype,’’ uniform across the species, is explicitly postulated in numerous publications. This linguistic genotype is further equated to a Universal Grammar. Ultimately, nativism is conﬂated with geneticism. features are directly rooted in the genome. As the literature (reviewed in Longa and Lorenzo 2008, 2012; Lorenzo and Longa 2009) reveals, a ‘‘linguistic genotype,’’ uniform across the species, is explicitly postulated in numerous publications. This linguistic genotype is further equated to a Universal Grammar. Ultimately, nativism is conﬂated with geneticism. static depictions of language at all levels of analysis are inadequate, particularly at the biological level. In all fair- ness, we should point out that linguists are quite familiar with the idea that variation and change are tightly inter- woven. For instance, they are well aware of the fact that language change is always preceded by a phase of varia- tion, with different linguistic variants coexisting within the same community of speech (Weinreich et al. 1968 and much subsequent work). However, we do not ﬁnd this lesson always reﬂected in the literature on language acquisition and developmental disorders. For us, the right position to adopt is this one: ‘‘to understand developmental outcomes, it is vital to identify full developmental trajec- tories, to assess how progressive change occurs from infancy onwards, and how parts of the developing system may interact with other parts differently at different times across ontogenesis’’ (Karmiloff-Smith 2009, p. 58). However, as we have already pointed out, most (if not all) ‘‘language genes’’ are polymorphic, with some alleles affecting language development also in the ‘‘normal’’ population. In addition, genes do not code for cognitive properties. A direct link between the genotype and the phenotype is not only simplistic, but biologically untena- ble, given the way in which genes contribute to develop- mental processes, and how development actually takes place. Genes are not blueprints. Developmental processes also depend on non-genetic factors (Oyama et al. 2001; Newman et al. 2006; Bateson and Mamelli 2007). Which Biology Does Universal Grammar Require? In order to fully exploit the resources offered to bio- linguistics by EvoDevo, we think that it is just as important to stress that although we have insisted on variability so far, there are, of course, many sources of universality— over and above the genes. For example, at the neurobio- logical level we observe that anatomical variability is quite constrained. In this way, myelinization patterns, receptor maps, cytoarchitectonic probability maps, and other struc- tural features can be conﬁdently established (Zilles and Amunts 2009). Similarly, functional variability seems to be constrained as well, to the extent that regions of interest can be identiﬁed (Fedorenko et al. 2010). In sum, although variation is omnipresent, the brain still exhibits a robust structure when processing language (Grodzinsky 2010). At the molecular level, we observe that the initial wiring of the linguistic brain is similarly achieved in all subjects under the guidance of a core set of genetic cues (Benı´tez-Burraco 2009). When it comes to language growth in the child, we ﬁnd as well that developmental itineraries are also con- strained although not fully predetermined, as Lenneberg (1967) already noted. ) Concerning the neural substrate of language, it seems that brain areas actually perform basic kinds of computa- tions that are recruited for different, high-level cognitive functions. As Poeppel and Embick (2005, p. 112) state, ‘‘differently structured cortical areas are specialized for performing different types of computations, and… some of these computations are necessary for language but also for other cognitive functions.’’ Consequently, cognitive capacities such as language are very probably cross-mod- ular by nature. They result from the interplay of these diverse brain areas performing basic, low-level activities (Grifﬁths 2007). At the same time, it is only these struc- tures that are the ﬁnal output of genetically driven devel- opmental processes. In fact, it seems that it is only their basic architecture that is genetically encoded, while their functional speciﬁcities are environmentally driven in var- ious ways that remain to be elucidated. This is what ulti- mately supports the claim that modules are not born, but made, and that there is not just one way of implementing a functional language. Our main point here is that we must seriously study how developmental dynamics, of the sort that is at the heart of EvoDevo approaches in biology (Oyama et al. Which Biology Does Universal Grammar Require? (4) ( ) At the same time, our reading of the literature suggests to us that breakdowns and compensations, whenever they occur, do not proceed randomly. In reality, some aspects of language processing seem to be particularly vulnerable in all pathological conditions, while others seem to be preserved in all of them. For instance, inﬂectional morphology is problematic not just for people with speciﬁc language impairment (Marchman et al. 1999), but also for those suffering from speech- sound disorder (Mortimer and Rvachew 2010), Down’s syndrome (Eadie et al. 2002), or (a subtype of) autism (Roberts et al. 2004). Ultimately, only some pathological phenotypes have been described, while others have not been observed, a situation that we think could beneﬁt from being modeled in terms of morphospaces or adaptative landscapes (Svensson and Calsbeek 2012). It seems, then, that although there is not just one way of implementing a linguistic brain, it is also true that there are not so many ways of implementing a functional faculty of language. We think that this EvoDevo-inspired approach to vari- ation in language is bound to be of great interest for clinical linguistics. In particular, this distancing between the genotype and the phenotype has the potential to explain why in some people affected by a particular language disorder the sequence of the candidate genes is normal (phenocopy), but also why language can be preserved in individuals who are endowed with a pathogenic copy of one of these ‘‘language genes’’ (null penetrance). From a broader perspective, we are in fact tempted to argue that language disorders can be construed as conditions in which that process of canalization has failed to cope with the underlying variation (thus preventing reaching particular degrees of development). Similarly, they can be construed as decanalized states, following the model by Gibson (2009). According to this view, the pervasiveness and the high prevalence of complex genetic diseases among mod- ern populations is a consequence of the uncovering of cryptic genetic variation resulting from the evolution of the human genome, and environmental and cultural perturba- tions (see Benı´tez-Burraco and Boeckx 2013 for additional remarks concerning this possibility and also for its impli- cations regarding the evolution of language). We believe that key EvoDevo concepts like canaliza- tion, developmental plasticity, robustness, evolvability, or adaptative landscapes will greatly help in clarifying, understanding, and eventually explaining the problem, and the full scope of variation in language. Which Biology Does Universal Grammar Require? 2001; West- Eberhard 2003; Callebaut and Rasskin-Gutman 2005; Carroll 2005; Mu¨ller 2007), takes place if we really want to adequately deal with this issue of variation in language, and eventually, to achieve a real biological depiction of the language faculty. In a real sense it is the complex and changing interaction between the organism and its envi- ronment that shapes the ﬁnal cognitive architecture of the brain. Different factors, both internal and external, affect language development, to the extent that the different cognitive phenotypes can emerge from the same genotype (the reverse is also true: the same phenotypes can emerge from distinct genotypes). Put differently, it is clear that Arguably, it is the ontogeny of language disorders that more clearly reveals the real nature of the problem we want to urge linguists to wrestle with. What we recurrently observe in pathological populations is that: (1) Diffuse effects on the brain and on cognitive capacities/abilities are the norm. In fact, develop- mental disorders are better characterized by associ- ations across domains than by dissociations between them (Bishop 2002). (2) Deﬁcits in low-level, more generalized processes usually manifest as disturbances of upper, more specialized processes, which ultimately give rise to shortcomings in even higher-level, more speciﬁc cognitive capacities (Karmiloff-Smith 2009). 123 12 3 126 A. Benı´tez-Burraco, C. Boeckx spite of, and along with neurobiological and genetic diversity. (3) Importantly, impaired, delayed, or deviant systems are still adaptive. It is indeed worth bearing in mind that substantially preserved linguistic abilities can be achieved in spite of deeper cognitive impairments (Sirois et al. 2008; Parisse and Maillart 2009). Conceptually speaking, the state of affairs we have described in this section reminds us of the ‘‘embryonic hourglass’’ situation, discussed from an EvoDevo per- spective in Newman (2011). As Newman observes in the context of a discussion on the evolution of animal eggs (2011, p. 467), ‘‘why can taxa within a given phylum exhibit very different egg types, pass through a common intermediate morphology (the so-called ‘phylotypic stage’), only to diverge again’’ (hence the metaphor of the ‘‘hourglass’’)? Could the logic of the ‘‘self-organizing physical processes’’ that Newman relies on to answer this question also apply to situations like the one we have discussed here in the context of language? It is too early to know, but we think it is worth beginning to think about language development in this way. Tools for Comparative Biolinguistics In closing this section, we would like to ask why it should be the case that adaptability itself is limited or constrained in speciﬁc ways, to the extent that some perturbations cannot be eventually compensated by developmental dynamics. We’d like to suggest that cer- tain cognitive processes are more vulnerable per se than others to damage or to developmental disturbances because they rely on less resilient neural networks and thus have less robust compensatory mechanisms. This would be due, we think, to their evolutionary novelty (Toro et al. 2010; Mantini et al. 2013). In fact, the most noticeable outcome of the biological study of language is that the genetic, physiological, and even cognitive mechanisms underlying language are actually robust after thousands of years of stabilizing selection (in other words, because they have a long evolutionary history), while language itself is very delicate. Probably, as suggested by Gibson (2009), the stable equilibrium observed in pri- mates was disrupted by our evolutionary history as a species (in particular, by population bottlenecks and migratory movements; see Mellars 2006), by novel mutations, and by cultural changes. These changes brought about cognitive systems known as modern lan- guages. But at the same time these changes may well have uncovered all that cryptic variation, decanalized the whole system, and ultimately, made language so sensitive to damage (but, we want to stress again, only to some kinds of damage). In other words, the human language faculty is easy to disturb because it is an evolutionary novelty, but at the same time it relies on robust biological mechanisms that are able to compensate many kinds of damage because they are considerably older. Plausibly, this may shed light on why our reading of the literature suggests to us that the same components of language tend to be affected in many language disorders, and why many other aspects of the linguistic phenotype are always quite preserved, and eventually, why these conditions are so prevalent among humans. Nothing in biology makes sense except in the light of comparison. As the opening passages of Darwin’s two most famous books make clear, the business of biology is vari- ation, variation, variation. Without variation, there can’t be any meaningful selection, any descent with modiﬁcation, any origin of species. It is natural, then, to expect biolin- guists to be fans of the comparative method as well. Which Biology Does Universal Grammar Require? In all situations language development turns out to be sensitive to envi- ronmental changes, to the extent that different cognitive architectures may result from different linguistic input, as we observe in bilingual people (developmental plasticity). At the same time the language faculty has been shown to be remarkably resistant to (some sort of) environmental per- turbations (robustness) to the extent that it recurrently emerges in all individuals, even in some pathological conditions (canalization). Moreover, some components of the language faculty seem to be very resistant to damage and/or to evolutionary change (again, robustness); at the same time linguistic systems seem prone to change (evolvability). All these properties result from the modular organization of the biological substrate of the faculty at all levels of analysis (Wagner and Altenberg 1996; Bergman and Siegal 2002; Kitano 2004). In particular, we want to suggest that developmental dynamics strongly canalizes the existing variation, to the extent that the same pheno- type—i.e., a language faculty—can robustly emerge at the term of growth from diverse genotypes and brain archi- tectures. Phenotypic uniformity, then, may be achieved in Nonetheless, for us, the crucial point is the recurrent outcome of research that suggests that breakdowns and compensations in language disorders do not occur ran- domly, and ultimately, that it is only certain normal, impaired, delayed, or deviant faculties of language that emerge in the course of development. We think that this situation is similar to the one that linguists often stress in their studies on language comparison: variation, though large and substantial, is not random, and appears to be conﬁned to only certain components of grammar (Berwick and Chomsky 2011; Boeckx 2011; Boeckx and Leivada 123 Universal Grammar and Biological Variation 127 2013a). But as we have already argued, we think that lin- guists err in taking the genotype to be the source of uni- versality. And so we would like to encourage linguists to abandon this genocentric assumption and embrace the variation we ﬁnd ‘‘at the bottom’’ by developing adequate tools to characterize it, an issue we return to in the next section. The speciﬁcity of language would thus rely on the perva- sive tendency of the components of the language faculty to interface whenever growth takes place in the presence of a suitable amount of linguistic stimuli. Tools for Comparative Biolinguistics But as we have been at pains to point out above, a signiﬁcant aspect of variation has been ignored by many biolinguists. To make matters worse, for much of its (recent) history, biolinguistics has been contrastive, not comparative, in the following sense: biolinguists have emphasized that lan- guage is the exclusivity of humans (and that among humans, the language faculty is uniform). Recently, however, as De Waal and Ferrari (2010) have noted, a signiﬁcant shift of perspective seems to be under way in cognitive science: the sharp contrastive character of top-down approaches is progressively being replaced by an ‘‘increased appreciation that the basic building blocks of cognition might be shared across a wide range of species’’ (p. 201). This bottom-up approach, seeking to establish ‘‘cognitive phylogenies’’ (Fitch et al. 2010), focuses on the fundamental capacities underlying larger cognitive phe- nomena and is more in line with the Darwinian logic of descent (Hauser et al. 2002). We think that this shift of perspective, along with the appreciation of variation and non-genetic sources of universality we have urged biolin- guists to develop, provides the basis for a genuine, pro- ductive, comparative biolinguistic agenda. To advance this new comparative research program, it is, of course, crucially important to pay attention to the tools one uses to compare. For obvious reasons, not all the tools developed by linguists are equally useful in this respect. In fact, given the modular proclivities of classical cognitive science (Piattelli-Palmarini 2001), the difﬁculties in exporting linguistic technology outside of the comfort zone of comparative linguists were to be expected. Not surprisingly, progress on the genetic basis of language capacities has been called ‘‘a linguist’s nightmare’’ (Piat- telli-Palmarini and Uriagereka 2011), and although schol- ars have long been captivated by the parallels between birdsong and human speech and language, concrete, theo- retically-informed proposals capturing the differences and the similarities across vocal learning capacities are hard to come by. We think that this picture properly adjusts to current views of language evolution that embrace continuity and view novelty as the result of a reorganizational process rather than a product of innovative genes (West-Eberhard 2003; Mu¨ller 2010), but also with the view of language as a cognitive faculty resulting from the interface of compo- nents (cognitive, neural, genetic) otherwise not speciﬁcally linguistic (see Boeckx 2013 for review and discussion). 12 3 3 128 A. Benı´tez-Burraco, C. Tools for Comparative Biolinguistics Boeckx By resorting to the Chomsky hierarchy, Fitch and Hauser hoped to avoid ‘‘theory-internal’’ debates corre- sponding to the choice of a theoretical idiom (Head-driven Phrase Structure Grammar, Government-and-Binding, Lexical-Functional Grammar, etc.) and use computational primitives that were not so language-speciﬁc as to vitiate any cross-species, or cross-domain comparison. As Heinz (2011, p. 146; emphasis in original) writes, ‘‘since any pattern is a language [in the sense of the Chomsky hier- archy], a distinct advantage of the Chomsky Hierarchy is [that] it allows for the comparison of patterns from dif- ferent domains.’’ In the wake of Hauser et al. (2002) and the revival of biolinguistic concerns (Di Sciullo and Boeckx 2011), Fitch and Hauser (2004) were perhaps the ﬁrst to face this technical challenge, and chose to resort to the hierarchy of formal languages known as the Chomsky hierarchy, developed in the 1950s, to capture the ‘‘computational constraints on syntactic processing’’ in non-human prima- tes. As is well known, the Chomsky hierarchy classiﬁes logically possible patterns into sets of nested regions. Each region corresponds to patterns describable by means of ‘‘machines’’ (grammars), with smaller regions captured by increasingly less powerful machinery (see Fig. 1). As was made clear in O’Donnell et al. (2005, p. 284), the use of the Chomsky hierarchy in Fitch and Hauser (2004) was motivated by the concerns raised above: Building on Chomsky’s (1956, 1957) foundational results concerning the limitation of ﬁnite-state machines to capture natural language generalizations, Fitch and Hauser (2004) claimed that cotton-top tamarins could not detect structures in stimuli that went beyond the computational capacity of ﬁnite-state automata. The results proved con- troversial in more than one way (Goudarzi 2006; Liberman 2006; Anderson 2008), but so have the results (Gentner et al. 2006; Abe and Watanabe 2011) suggesting that some songbirds outperformed cotton-top tamarins, achieving learning results beyond the ﬁnite-state boundaries (Van Heijningen et al. 2009; Berwick et al. 2011, 2012; Ten Cate and Okanoya 2012). Apart from issues of experimental design, we believe that the overarching problem lies in the adequacy of the Chomsky hierarchy in assessing cognitive proﬁles. Although formal language theory can certainly help in designing experiments shedding light on mental Understanding developmental and evolutionary aspects of the language faculty requires comparing adult languages users’ abilities with those of non- verbal subjects, such as babies and non-human ani- mals. Tools for Comparative Biolinguistics For example, phonological patterns do not appear to require grammars that distinguish inﬁnitely many states, unlike some syntactic patterns, which appear to require formal grammars that do. This distinction between these two domains of the language faculty highlights the fact that the human language faculty is not monolithic. It is more like a mosaic, with all the implications this has for evolutionary studies. Heinz and Idsardi also comment on the hypothesis that natural lan- guage patterns are at most mildly context sensitive (Joshi 1985) and stress that we should not conclude from this that were it true, any mildly context-sensitive pattern is auto- matically a possible natural language one. g g y p g The third, and perhaps most important, lesson that we’d like to draw is that it is not at all clear what the expectations of the relevant experiments are, due to the inherent limitations of the Chomsky hierarchy in capturing the true nature (i.e., the constraints) of natural languages. This is not to say, of course, that such experiments are pointless. They can tell us many things, but it is not clear that they allow us to draw solid conclusions concerning the system linguists call natural languages. To make ourselves clear, suppose we found out that a non-human species were capable of mastering a mildly-context-sensitive language in the context of an arti- ﬁcial language experiment. What would we be able to con- clude from this? Our answer is, not much. In fact, we would only be able to conclude that they were able to learn this pattern. But it would not immediately tell us the underlying algorithm used. As reviewed in Ojima and Okanoya (2013), all the artiﬁcial grammar experiments to date suffer from this problem, as there is more than one way to acquire a particular pattern. (Remember that one of the strengths of the Chomsky hierarchy is that it allows for the comparison of patterns from different domains. But this is also its weakness—it is too nonspeciﬁc to exclude alternative cognitive ways of cap- turing a given pattern.) There are three lessons that we would like to draw from the use of the Chomsky hierarchy in comparative biolin- guistics. First, choosing to use this tool amounts to ignoring most of the research done on the nature of human languages over the past 50 years. Tools for Comparative Biolinguistics Classically, comparative work in this area has relied on the rich theoretical frameworks developed by linguists in the generative grammar tradition. However, the great variety of generative theories and the fact that they are models of language speciﬁcally makes it difﬁcult to know what to test in animals and children lacking the expressive abilities of normal, mature adults. We suggest that this problem can be mitigated by tapping equally rich, but more formal mathematical approaches to language. Fig. 1 Natural language patterns in the Chomsky hierarchy; reproduced from Heinz (2014) Fig. 1 Natural language patterns in the Chomsky hierarchy; reproduced from Heinz (2014) 123 123 Universal Grammar and Biological Variation 129 abilities (see the papers collected in Fitch and Friederici 2012), it suffers from a major problem in the context of biolinguistics. It is indeed well known that the Chomsky hierarchy is of limited use in characterizing human lin- guistic competence. As Berwick et al. (2012) correctly observe, the hierarchy is both ‘‘too weak and too strong,’’ failing as it does to cut natural language at its joints. the one hand and syntax-semantics on the other. To the best of our knowledge, none of the evidence for syntactic patterns falling outside the regular language class comes from ‘‘pure’’ syntactic patterns; rather, all of them involve patterns with (structural) semantic consequences. This is an important consideration for experiments because virtually all com- parative biolinguistic experiments using the Chomsky hier- archy are artiﬁcial language experiments that seek to target the learning of pure syntactic patterns (these languages don’t mean anything). But there are no such patterns in natural languages. Not surprisingly, when semantic cues are added to the experiment, as in Fedor et al. (2012), these were found to boost the learning of more complex formal grammars in humans. By ignoring semantics, artiﬁcial language experi- ments may well be removing the component that gives nat- ural language syntax its distinctive computational signature. Heinz and Idsardi (2011, 2013) and Heinz (2014) use- fully summarize important lessons that linguists have derived from applying the Chomsky hierarchy to the study of natural languages. They highlight the fact that not all natural language patterns fall exactly in the same range within the Chomsky hierarchy. In fact, as Fig. 1 reveals, patterns are quite scattered. Tools for Comparative Biolinguistics Such research has moved away from the Chomsky hierarchy, in large part because it became clear very quickly that it does not ‘‘uniquely’’ characterize human language in the sense that it does not identify any (sub)region of the hierarchy as the exclusive property of natural lan- guage. As such, it does not characterize precisely enough the capacity we as humans have. Consider the fact that, as numerous linguists have observed, no natural language has rules that require counting past two. But it is a logically possible language pattern, one that the Chomsky hierarchy can capture. In fact, as Heinz (2014) notes, it is a regular pattern,falling well below the attested power range of natural language syntax. But it is a constraint that signiﬁcantly shapes the human language capacity, one that we would want to ask comparative questions about. The Chomsky hierarchy does not allow us to do this. Only detailed theoretical lin- guistics work does. As should be clear by now, even if a non-human species were capable of mastering a mildly-context-sensitive lan- guage in the context of an artiﬁcial language experiment, we would not be able to conclude anything regarding the human language faculty. Because there is no (sub)region of the Chomsky hierarchy that is exclusively occupied by natural languages, we would not be able to conclude that the success of non-human primates shows that they have a component of the human language faculty. It is for this reason that we are skeptical about the use of the Chomsky hierarchy to identify language regions in the brain (Fitch and Friederici 2012; Moro and Chesi in 2014) or to draw linguistic inferences from artifacts in the fossil record (Camps and Uriagereka 2006; Balari and Lorenzo 2013; Longa 2013). Second, comparative biolinguistics experiments that rely on the Chomsky hierarchy tend to ignore the divide between phonology and syntax stressed by Heinz and Idsardi (2011, 2013), or rather, experimenters tend to take the notion of ‘‘syntax’’ too literally. The divide emphasized by Heinz and Idsardi (2011, 2013) is actually one between phonology on 123 12 23 130 A. Benı´tez-Burraco, C. Boeckx Buzsa´ki et al. (2013) may provide just what is needed in this context. They observe that despite the several-thou- sandfold increase of brain volume during the course of mammalian evolution, the hierarchy of brain oscillations (brain rhythms) remains remarkably preserved. Tools for Comparative Biolinguistics This con- served aspect of our biology is directly relevant for com- parative biolinguistics: it offers the possibility of conceiving of cross-species differences, or, as Buzsa´ki et al. (2013) discuss, of cognitive diseases, as slight variations (disrup- tions) within the preserved network constellation that would constitute a universal brain syntax (Buzsa´ki 2010)—dys- rhythmias and oscillopathies, as Buzsa´ki et al. (2013) call them. Put differently, the preservation of brain rhythms in mammals would be the cognitive scientist’s hox genes. As Boeckx (2013) observed, ﬁnding a substitute, or com- plement to the Chomsky hierarchy to construct cognitive phylogenies will be a serious challenge for the years to come. In the remainder of this section, we would like to sketch a possible research avenue that seems to us to have the right properties, and that connects to some EvoDevo concerns discussed above, as it draws on aspects of our biology that are conserved across species, and at the same time that are known to vary across human populations (language disorders). To begin with, we’d like to step back and consider what we believe was the main factor behind the renewed interest in the comparative method in linguistics at the end of the 1970s. The ‘‘new comparative syntax,’’ as it has been called, grew out of proposals articulated in Chomsky (1981). These proposals, as Chomsky acknowledged on numerous occasions (e.g., Chomsky 2009), grew out of reﬂections inspired by the work of Jacob and Monod (1961) on gene regulation. In the fullness of time the Jacob-Monod model developed into EvoDevo genomics (Carroll 2005). The most iconic ﬁnding of this ﬁeld is the hox gene set, which conﬁrmed Monod’s prediction that ‘‘what istrueforE. coli is alsotrue for the elephant.’’ As is now well established, the set of genes regulating development across a wide range of species is shared, which greatly enhances the possibilities of comparison. Against the back- ground of this deep conservation, species differences (varia- tions) can be thought of as little tweaks and nudges, like the 30 variations of the aria that Bach offered us in what is now known as the Goldberg variations. Arthur (2004), for instance, suggests that all variations reduce to instances of heterochrony (different timing of gene expression), heterotopy (different location of gene expression), heterometry (more of the gene product being made), and heterotypy (change in the nature of the gene product; e.g., switch on different target genes). Tools for Comparative Biolinguistics Obviously,toput thishypothesis tothe test inthe context of language, it is necessary for linguists to translate their ﬁndings concerning the properties of the human language faculty in terms of brain rhythms, to offer a mind/brain model on which to formulate parameters giving rise to distinct cognitive pro- ﬁles. This translation step may, in fact, be independently necessary to bridge the gap between mind and brain. David Poeppel has written eloquently and accessibly about the challenges neurolinguistics faces (Poeppel 2005, 2011, 2012; Poeppel and Embick 2005). The heart of the matter, according to Poeppel, is the ‘‘granularity mismatch’’ (or ‘‘mapping’’) problem: the objects of study in theoretical linguistics and in neuroscience don’t match. As a result, mapping one onto the other has proven impossible. Accordingly, Marr’s (1982) vision of cognitive neuroscience based on linking levels of analyses, to which biolinguistics should aspire, remains distant. Both theoretical linguistics and the neurosciences are to blame for this sorry state of affairs. For all the ‘‘bio’’ talk in linguistic circles, linguists have so far failed to distill what is known from linguistic theory into a set of computational primitives, and to try to link these with models and speciﬁc principles of neural computation. As has been said, g p g g g This model to understand variation was borrowed into linguistics, where it came to be known as the Principles-and- Parameters approach, with the principles providing the underlying uniformity and the parameters the sources of the surface variations (Chomsky 1981; Baker 2001). The analogy worked well for two decades, allowing for considerable empirical progress, but in recent years, the foundational assumptions of the Principles-and-Parameters model have been questioned (Newmeyer 2004, 2005; Boeckx 2011, 2014; Boeckx and Leivada 2013a). It appears that in order to capture the variation that comparative linguists focus on, something else is needed, perhaps something along the lines of Boeckx and Leivada (2013b). However, the logic of Principles and Parameters may be just what is needed in the context of comparative biolinguistics. Such a model need not require genes to provide the relevant parameters (even for biology, genes may be followers, not just leaders; Newman and Bhat 2009; Schwander and Leimar 2011), but its logic demands that one ﬁnd an aspect of deep conservation on which variants could be grafted. Tools for Comparative Biolinguistics we need linguistic models that are explicit about the computational primitives (structures and operations) they require, and that attempt to deﬁne linguistic problems at a ﬁne enough grain that one can discuss algorithmic and implementational approaches to their solution. We need a list of computations that lin- guistic theorists deem indispensable to solve their particular problem (e.g., in phonology, syntax, or semantics). (Fitch 2009, p. 298) Put another way, [l]inguists and psycholinguists owe a decomposition (or fractionation) of the particular linguistic domain in question (e.g., syntax) into formal operations that 123 123 Universal Grammar and Biological Variation 131 are, ideally, elemental and generic. Generic formal operations at this level of abstraction can form the basis for more complex linguistic representation and computation. (Poeppel 2005, p. 11) there are many more sources of variation than those linguists tend to study, there are also many more sources of univer- sality that canalize this variation and allow for languages to reliably develop in the individual than linguists tend to assume. We have furthermore suggested that lessons from comparative and theoretical linguistics may proﬁtably be extended to deeper layers of variation, and could offer a starting point for a new branch of comparative linguistics, one we have called comparative biolinguistics. This new subﬁeld may help correct the exuberant (genocentric) nativism that appears to be so problematic (Boeckx and Leivada 2013a), particularly for achieving a fruitful assim- ilation of linguistics to biology, allowing for a real, biolog- ically grounded biolinguistics to emerge. A rhythm-based model may have just the right kind of characteristic envisaged by Poeppel. In fact, direct evidence of the fruitfulness of this approach in the language domain comes from Poeppel’s own work, beginning with Poeppel (2003) and culminating with Giraud and Poeppel (2012). ( ) g pp ( ) What Poeppel and colleagues have shown is that by focusing on the endogenous rhythms generated by the cortex, it is possible to understand (as opposed to merely localizing) the cerebral specialization for speech perception and production, and to shed light on the nature of phrasal phonology. The main thesis is that neuronal oscillations contribute to cognition in several ways: for example, by segregating information and organizing spike timing. Speciﬁcally, a series of oscillations (in the delta, theta, and gamma ranges) appear to be able to track the dynamics of speech. In doing so, they ‘‘chunk’’ or ‘‘package’’ incoming information into units of the appropriate temporal granu- larity. This packaging corresponds to units of phrasal phonology (linking the algorithmic and computational levels). Put another way, Taking linguistic processes as involving multiple subprocesses with different characteristics, as opposed to being monolithic, each one associated with different fre- quencies of neural oscillations, may offer us the right component parts to identify what goes wrong in disorders, or how different orchestrations may give rise to cross- species cognitive differences, and endophenotypes. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. References Abe K, Watanabe D (2011) Songbirds possess the spontaneous ability to discriminate syntactic rules. Nat Neurosci 14:1067–1074 Anderson SR (2008) The logical structure of linguistic theory. 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https://openalex.org/W4311376710	http://edoc.mdc-berlin.de/22995/1/22995oa.pdf	English	null	Glucose-Induced Hemodynamic and Metabolic Response of Skeletal Muscle in Heart Failure Patients with Reduced vs. Preserved Ejection Fraction—A Pilot Study	Journal of cardiovascular development and disease	2,022	cc-by	8,029	Glucose-Induced Hemodynamic and Metabolic Response of Skeletal Muscle in Heart Failure Patients with Reduced vs. Preserved Ejection Fraction—A Pilot Study Michael Boschmann 1 , Lars Klug 1, Frank Edelmann 2,3, Anja Sandek 4,5, Stephan von Haehling 4,5, Hans-Dirk Düngen 2, Jochen Springer 6, Stefan D. Anker 2,3,6, Wolfram Doehner 2,3,6,* and Nadja Jauert 2,3,6,7,* Michael Boschmann 1 , Lars Klug 1, Frank Edelmann 2,3, Anja Sandek 4,5, Stephan von Haehling 4,5, Hans-Dirk Düngen 2, Jochen Springer 6, Stefan D. Anker 2,3,6, Wolfram Doehner 2,3,6,* and Nadja Jauert 2,3,6,7,* 1 Experimental & Clinical Research Center (ECRC), A Joint Cooperation between Charité-Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine, 10115 Berlin, Germany 2 Department of Cardiology, (Virchow Campus) Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany 3 DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10785 Berlin, Germany 4 Department of Cardiology and Pneumology, Universitätsmedizin Göttingen (UMG), 37075 Göttingen, Germany 5 German Centre for Cardiovascular Research (DZHK), Partner Site Göttingen, 37075 Göttingen, Germany 6 Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany 1 Experimental & Clinical Research Center (ECRC), A Joint Cooperation between Charité-Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine, 10115 Berlin, Germany 2 Department of Cardiology, (Virchow Campus) Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany 3 DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10785 Berlin, Germany 4 Department of Cardiology and Pneumology, Universitätsmedizin Göttingen (UMG), 37075 Göttingen, Germany 5 German Centre for Cardiovascular Research (DZHK), Partner Site Göttingen, 37075 Göttingen, Germany 6 Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany 7 7 Division of Physiology, Department of Human Medicine, MSB Medical School Berlin, Rüdesheimerstr 50, 14197 Berlin, Germany y * Correspondence: wolfram.doehner@bih-charite.de (W.D.); nadja.jauert@charite.de (N.J.) Abstract: (1) Background: Insulin resistance (IR) is a characteristic pathophysiologic feature in heart failure (HF). We tested the hypothesis that skeletal muscle metabolism is differently impaired in patients with reduced (HFrEF) vs. preserved (HFpEF) ejection fraction. (2) Methods: carbohydrate and lipid metabolism was studied in situ by intramuscular microdialysis in patients with HFrEF (59 ± 14y, NYHA I-III) and HFpEF (65 ± 10y, NYHA I-II) vs. healthy subjects of similar age during the oral glucose load (oGL); (3) Results: There were no difference in fasting serum and interstitial parameters between the groups. Blood and dialysate glucose increased signiﬁcantly in HFpEF vs. HFrEF and controls upon oGT (both p < 0.0001), while insulin increased signiﬁcantly in HFrEF vs. HFpEF and controls (p < 0.0005). Muscle tissue perfusion tended to be lower in HFrEF vs. Citation: Boschmann, M.; Klug, L.; Edelmann, F.; Sandek, A.; von Haehling, S.; Düngen, H.-D.; Springer, J.; Anker, S.D.; Doehner, W.; Jauert, N. Glucose-Induced Hemodynamic and Metabolic Response of Skeletal Muscle in Heart Failure Patients with Reduced vs. Preserved Ejection Fraction—A Pilot Study. J. Cardiovasc. Dev. Dis. 2022, 9, 456. https://doi.org/10.3390/ jcdd9120456 Keywords: insulin resistance; skeletal muscle; metabolism; microdialysis Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Journal of Cardiovascular Development and Disease Journal of Cardiovascular Development and Disease Glucose-Induced Hemodynamic and Metabolic Response of Skeletal Muscle in Heart Failure Patients with Reduced vs. Preserved Ejection Fraction—A Pilot Study HFpEF and controls after the oGL (p = 0.057). There were no differences in postprandial increases in dialysate lactate and pyruvate. Postprandial dialysate glycerol was higher in HFpEF vs. HFrEF and controls upon oGL (p = 0.0016); (4) Conclusion: A pattern of muscle glucose metabolism is distinctly different in patients with HFrEF vs. HFpEF. While postprandial IR was characterized by impaired tissue perfusion and higher compensatory insulin secretion in HFrEF, reduced muscle glucose uptake and a blunted antilipolytic effect of insulin were found in HFpEF. 1. Introduction Heart failure (HF) is complex clinical syndrome and global health burden with an increasing prevalence and growing health care costs [1]. Insulin resistance (IR) is a common feature in HF contributing to symptomatic severity and prognosis [2–4]. IR has been impli- cated in the development of exercise intolerance in HF, as skeletal muscle is a main target of physical exercises [5,6]. Skeletal muscle is a major organ of insulin-stimulated glucose uti- lization [7]; therefore, development of IR results in impaired glucose metabolism by skeletal muscle in non-diabetic patients with HF. Notably, IR develops intrinsically in patients with HF and independently of the presence of diabetic comorbidity [8]. Pathophysiological mechanism of IR development is complex and not yet clear in its details. Previously, we reported on reduced glucose transporter GLUT4 in skeletal muscle in non-diabetic patients with chronic HF [9]. Further, differences in IR by short insulin sensitivity test (SIST) have Copyright: © 2022 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/). https://www.mdpi.com/journal/jcdd J. Cardiovasc. Dev. Dis. 2022, 9, 456. https://doi.org/10.3390/jcdd9120456 J. Cardiovasc. Dev. Dis. 2022, 9, 456 2 of 10 2 of 10 been observed between the patients with HF with reduced (HFrEF) and patients with HF with preserved ejection fraction (HFpEF) [10]. However, the corresponding pathophysio- logical mechanisms of the different metabolic responses on injection of insulin in both HF phenotypes are not known. Different pathways including diminished tissue perfusion (i.e., impaired glucose delivery to muscle cells), impaired insulin signaling, insufﬁcient glucose phosphorylation within the cell, or inappropriate insulin availability might contribute to development of IR [11–13]. All these mechanisms affect the skeletal muscle energy metabolism and underlies physical endurance [2]. The technique of Microdialysis allows in vivo investigation of skeletal muscle or adipose tissue metabolism by assessing interstitial ﬂuid metabolites, either at steady state conditions or in dynamic proﬁles during metabolic loading. This technique has been successfully applied in patients with a wide range of medical conditions, including ischemic stroke [14], chronic HF [15], metabolic syndrome [16] or multiple sclerosis [17]. The aim of the present pilot study was to test the hypothesis, that there are speciﬁc differences in the glucose metabolism of skeletal muscle in patients with HFrEF versus HFpEF. 2.1. Patients Male patients with HFrEF and with HFpEF were prospectively studied in this pilot study. All patients were in stable ambulatory condition and on individually adjusted standard medical therapy in accordance with current guidelines (including beta-blockers, angiotensin-converting enzyme inhibitors, mineralocorticoid receptor antagonists, diuretics, and antiplatelet). None of the patients received any kind of antidiabetic therapy. All patients had a disease history for HF of at least 6 months and were in NYHA class I-III. Patients with HFrEF showed clinical symptoms and documented impaired left ventricular ejection fraction ([LVEF] ≤45%). Diagnosis of HFpEF was clinically documented and conﬁrmed by transthoracic echocardiography (TTE) according to the current guidelines [18]. Exclusion criteria were presence of medically treated diabetes mellitus type 2, anticoagulant therapy and novel anticoagulants (NOAC), acute decompensated heart failure within last 6 weeks, acute myocarditis or myocardial infarction, acute and chronic inﬂammatory diseases, immunosuppressive therapy, neurodegenerative disease or acute infection, and a history of cancer shorter than 5 years. Healthy male volunteers of similar age and body mass index served as controls. The Institutional Review Board of Charité-Universitätsmedizin Berlin, Germany ap- proved the study protocol (EA2/137/15). The study was conducted in accordance with Good Clinical Practice, and in compliance with the Declaration of Helsinki and applicable European Regulations. Written informed consent was obtained from all participants before study entry. 1. Introduction We applied the microdialysis technique in order to study the hemodynamic and metabolic response of the Vastus lateralis muscle to an oral glucose load (oGL) in patients with HFrEF and HFpEF versus healthy controls. 2.5.1. Transthoracic Echocardiography Participants underwent a Doppler echocardiographic evaluation (Vivid S5 with 3S-RS 1.5–3.6 MHz transducer, GE Medical Systems) according to a standardized protocol. Data regarding cardiac morphology, global ventricular function, and diastolic function were recorded by M-mode, two dimensional and Doppler echocardiography. Left ventricular ejection fraction (LVEF) was calculated from end-systolic and end-diastolic volume eval- uated from apical four-chamber and two-chamber view according to Simpson’s method. Peak E and A wave velocities and E wave deceleration time were measured from the mitral leaﬂet tip. Longitudinal time Doppler velocities, early diastolic wave (E’) and atrial wave (A’) were assessed by pulsed wave tissue Doppler imaging in the apical four-chamber view in septal and lateral ventricular wall. Diagnosis of HFpEF was conﬁrmed by echocardiog- raphy according to the ASE/EACI Guidelines 2016 [18]. 2.5. Clinical Assessments 2.5.1. Transthoracic Echocardiography 2.2. Study Protocol All clinical and metabolic assessments were performed under standardized conditions starting between 8:00 and 9:00 a.m. after an overnight fasting (≥12 h) after a resting period of at least 20 min in supine position in a quiet, air-conditioned room (metabolic ward). From the day before, patients were asked to abstain from (1) any kind of physical activity, (2) beverages containing caffeine and alcohol, and (3) from smoking. Fasting insulin resistance was assessed by homeostasis model assessment (HOMA) index as described previously [19]. Brieﬂy, the relative index of insulin resistance (IR, dimensionless or expressed in %) is calculated by the formula: (1) Insulin resistance = fasting glucose (mmol/L) × fasting insulin (µU/mL)/22.5 (1) J. Cardiovasc. Dev. Dis. 2022, 9, 456 3 of 10 3 of 10 HOMA-IR = 1 corresponds to normal insulin sensitivity, whereas values > 2 indicate the presence of insulin resistance. BMI was calculated as the ratio of weight (kg) and squared height (m2). q g Hemodynamic and metabolic responses of skeletal muscle were studied by the micro- dialysis technique at rest and after an oral 75-g glucose load (oGL) given as 300 mL drink (ACCUCHEK® Dextro®, O.G.T., Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany). Total time of testing was about 4 h. 2.3. Microdialysis Skeletal muscle microdialysis was used to monitor interstitial marker metabolites such as glucose for substrate supply, lactate und pyruvate for anaerobic and aerobic glucose metabolism, and glycerol for lipid mobilization [9]. Brieﬂy, a microdialysis probe (M71, µDialysis AB, Stockholm, Sweden) was inserted into the Vastus lateralis muscle after local anesthesia with lidocaine. After probe insertion, a 60 min period was allowed for tissue recovery from insertional trauma and for baseline calibration. Microdialysis probes were perfused with lactate-free Ringer’s solution supplemented with 50 mmol/L ethanol at ﬂow rate of 2 µL/min using a high precision microdialysis pump (M107, µDialysis AB, Stockholm, Sweden). Ethanol was added to assess changes in tissue perfusion by using the ethanol dilution technique based on Fick’s principle [9]. Dialysate samples were taken in 15-min intervals over 30 min before (baseline) and 120 min after the oGL. A vein catheter (Vasoﬁx® Safety, 20 G; B. Braun, Melsungen, Germany) was placed into the antecubital vein for repeated blood sampling in order to assess baseline and postprandial blood glucose and insulin levels. Blood samples were taken before and in 15-min intervals within the ﬁrst and 30-min intervals within the second hour after the oGL. 2.4. Assays All blood samples were processed immediately in a refrigerated centrifuge at 4.0 ◦C and aliquots (serum and plasma) were stored at −80 ◦C until analysis. Blood glucose and insulin as well as routine parameters were measured according to international standards; ethanol in the perfusate and dialysate with a standard spectrophotometric enzymatic assay; dialysate glucose, lactate, pyruvate and glycerol with an automated analyzer based on colorimetric principles (ISCUSﬂex, µDialysis, Stockholm, Sweden). In situ dialysate recovery for metabolites was about 50%, as assessed by near-equilibrium dialysis. 2.5. Clinical Assessments 3.1. Clinical Characteristics of Study Population Clinical characteristics of the study groups are presented in Table 1. All study groups were of similar age and BMI. There were no signiﬁcant differences in clinical lab parameters such as glucose, triglycerides, cholesterol, hemoglobin, HbA1c and creatinine between the study groups. However, there were a tendency of higher values for insulin and HOMA-IR in HFrEF and speciﬁcally in HFpEF vs. controls. LV end-diastolic diameter and LVEF as well as tricuspid annular plane systolic excursion were more severely impaired in patients with HFrEF compared to HFpEF (Table 2). Table 1. Baseline clinical characteristics of study groups. Table 1. Baseline clinical characteristics of study groups. Table 1. Baseline clinical characteristics of study groups. 2.5.2. Statistical Analysis All evaluations were done as descriptive statistics with no correction for multiple testing. Biochemical variables were tested for normal distribution using the Kolmogorov- Smirnov test. All data were presented as mean ± standard deviation (SD), median (in- terquartile range, IQR) or mean ± standard error of the mean (SEM) for repeated mea- surements after the oGL. T-test for paired samples, t-test for independent samples and J. Cardiovasc. Dev. Dis. 2022, 9, 456 4 of 10 4 of 10 analysis of variance (ANOVA) followed by Fisher’s post hoc test were used as appropriate. Global ﬁtting was used to compare the response curves for plasma glucose and insulin and skeletal muscle tissue dialysate concentrations of glucose, lactate, pyruvate and glycerol between treatment groups after the oGL. This test is a non-linear regression method in which one curve (healthy controls) is used as reference, allowing evaluations of discrepancy of the other curve (patient groups). All p-values given in the Figures refer to global ﬁtting. Non-normally distributed data were log-transformed to allow parametrical analysis. A value of p < 0.05 was considered statistically signiﬁcant. Statistical analysis was performed with the Statview 5.0 (SAS Institute, Cary, NC, USA) or GraphPad Prism (InStat, Version 6.0 Graphpad Software Inc., San Diego, CA, USA) software. ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; HOMA-IR, homeostasis model assessment— insulin resistance; IQR, interquartile range. Data are presented as mean ± SE if not otherwise indicated. * p < 0.05 vs. controls; † p < 0.05 vs. HFrEF. 3.1. Clinical Characteristics of Study Population Parameters Controls n = 8 HFrEF n = 12 HFpEF n = 6 p-Value Age, years 61 ± 10 58 ± 15 64 ± 10 0.6 Body mass index, kg/m2 25.5 ± 2.0 26.7 ± 3.5 26.6 ± 5.2 0.8 Systolic blood pressure, mmHg 128 ± 12 110 ± 20 * 132 ± 3 † 0.03 Diastolic blood pressure, mmHg 79 ± 7 67 ± 13 * 79 ± 7 † 0.03 Mean blood pressure, mmHg 96 ± 8 81 ± 15 * 98 ± 10 † 0.03 Glucose, mmol/L 5.3 ± 0.1 7.4 ± 3.1 6.5 ± 2.6 0.5 Insulin, µmol/mL, median (IQR) 4.7 (4.5–5.2) 7.3 (4.3–17.1) 10.2 (7.0–30.0) 0.1 HOMA-IR index, median (IQR) 1.1 (1.1–1.8) 1.8 (1.1–4.8) 4.9 (1.7–9.6) 0.08 Triglycerides, mg/dL 98 ± 25 123 ± 62 92 ± 31 0.4 Cholesterol, mg/dL 190 ± 25 146 ± 23 * 177 ± 60 0.07 High density lipoprotein, mg/dL 57 ± 13 45 ± 18 44 ± 16 0.2 Low density lipoprotein, mg/dL 113 ± 23 76 ± 13 * 105 ± 53 0.08 ASAT, U/L 21.7 ± 3.9 27.0 ± 8.8 26.8 ± 5.9 0.3 ALAT, U/L 23.7 ± 9.7 22.9 ± 12.4 31.2 ± 12.4 0.4 Hemoglobin, g/dL 14.4 ± 1.4 13.6 ± 1.0 12.7 ± 2.3 0.4 HbA1c, % 5.4 ± 0.6 5.5 ± 0.4 6.0 ± 0.9 0.3 Creatinine, mg/dL 0.94 ± 0.14 1.1 ± 0.2 1.0 ± 0.3 0.2 Midregional pro-ANP, nmol/L, median (IQR) 67 (23–85) 385 (145–1184) 177 (159–398) <0.05 Medication Angiotensin-converting enzyme inhibitor, n 9 3 0.3 Angiotensin receptor blocker, n 4 2 1.0 β-blocker, n 10 4 0.4 Diuretics, n 9 4 0.7 Aldosterone-antagonist, n 5 1 0.3 Statins, n 6 3 1.0 Antiplatelet, n 7 4 0.7 Novel oral anticoagulants, n 3 - ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; HOMA-IR, homeostasis model assessment— insulin resistance; IQR, interquartile range. Data are presented as mean ± SE if not otherwise indicated. * p < 0.05 vs. controls; † p < 0.05 vs. HFrEF. J. Cardiovasc. Dev. Dis. 2022, 9, 456 5 of 10 Table 2. Baseline cardiac characteristics of patient groups. 3.1. Clinical Characteristics of Study Population Parameters HFrEF n = 12 HFpEF n = 6 p-Value NYHA class I/II/III, n [/%] 1.9 ± 0.7 1.3 ± 0.5 0.2 Heart rate, beats per min 65 ± 10 69 ±8 0.9 LV ejection fraction, % 32 ± 10 52 ± 2 0.0003 LV end-diastolic diameter, mm 63.8 ± 12.7 52.6 ± 4.8 0.05 Intraventricular systolic septum thickness, mm 10.5 ± 1.3 11.2 ± 2.6 0.5 LV posterior wall thickness, mm 11.4 ± 1.3 11.0 ± 1.9 0.7 Left atrial diameter, mm 42.0 ± 8.6 46.7 ± 10.8 0.4 Maximal left atrial volume index, mL/m2 36.5 ± 6.2 39.7 ± 9.8 0.3 Mitral E-wave velocity, cm/s 62.5 ± 32.2 71.1 ± 14.9 0.7 Mitral A-wave velocity, cm/s 58.2 ± 33.6 63.8 ± 19.8 0.7 Septal e’ mitral annular velocity by TDI, cm/s 5.4 ± 2.6 6.3 ± 1.8 0.5 Lateral e’ mitral annular velocity by TDI, cm/s 7.5 ± 3.1 7.4 ± 1.9 0.6 Mitral E/e’ septal-lateral ratio 11.0 ± 5.5 13.2 ± 2.0 0.1 Aortic valve velocity, m/s 1.2 ± 0.1 1.7 ± 0.9 0.3 Right ventricular end-diastolic parameter, mm 36.4 ± 5.9 34.3 ± 6.8 0.6 Tricuspid annular plane systolic excursion, mm 14.7 ± 5.1 26.0 ± 5.2 0.02 LV, left ventricular; NYHA, New-York Heart Association; TDI, Tissue Doppler Imaging. Data are presented as mean ± SE if not otherwise indicated. Table 2. Baseline cardiac characteristics of patient groups. 3.2. Plasma Glucose and Insulin Proﬁles There were no signiﬁcant differences in fasting plasma glucose levels and HOMA-IR between the study groups (Figure 1, Table 1), although HOMA-IR tended to be higher in HFpEF vs. two other groups. Following the oGL, plasma glucose levels raised signiﬁcantly in all three groups within 60 min, but this increase was signiﬁcantly higher in HFpEF vs. HFrEF and healthy controls (15.2 ± 2.2 vs. 11.2 ± 0.9 and 10.3 ± 0.6 mmol/L, p < 0.0001, respectively; Figure 1, left panel). Plasma glucose levels remained elevated (>8 mmol/L) in HFpEF vs. HFrEF and controls even 120 min after the oGL (12.6 ± 2.4 vs. 9.9 ± 1.2 and 8.4 ± 0.9 mmol/L, p < 0.0001, respectively; Figure 1, left panel). Dev. Dis. 2022, 9, x FOR PEER REVIEW 6 of 12 Figure 1. 3.1. Clinical Characteristics of Study Population Plasma glucose and insulin levels in patients with heart failure with reduced ejection frac- tion (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; HFpEF vs. controls, p < 0.0001; HFrEF vs. controls, p = 0.0005. Figure 1. Plasma glucose and insulin levels in patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; HFpEF vs. controls, p < 0.0001; HFrEF vs. controls, p = 0.0005. Figure 1. Plasma glucose and insulin levels in patients with heart failure with reduced ejection frac- tion (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; HFpEF vs. controls, p < 0.0001; HFrEF vs. controls, p = 0.0005. Figure 1. Plasma glucose and insulin levels in patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; HFpEF vs. controls, p < 0.0001; HFrEF vs. controls, p = 0.0005. Figure 1. Plasma glucose and insulin levels in patients with heart failure with reduced ejection frac tion (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE HFpEF vs. controls, p < 0.0001; HFrEF vs. controls, p = 0.0005. Figure 1. Plasma glucose and insulin levels in patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; HFpEF vs. 3.3. Skeletal Muscle Metabolism 3.3. Skeletal Muscle Metabolism 3.1. Clinical Characteristics of Study Population controls, p < 0.0001; HFrEF vs. controls, p = 0.0005. Fasting plasma insulin levels tended to be higher in HFrEF and HFpEF patients vs. controls (13.0 ± 2.1 and 19.3 ± 6.2 vs. 6.4 ± 0.7 µU/mL, p = 0.1, respectively; Figure 1). Plasma insulin levels increased after the oGL in all three groups with the highest increase of insu- lin levels in HFrEF vs. HFpEF and controls 60 min after the oGL (91 ± 13 vs. 57 ± 17 and 65 ± 11 µmol/mL, p < 0.05, respectively). Even 120 min after the oGL, plasma insulin levels remained the highest in HFrEF vs. HFpEF and controls (92 ± 12 vs. 49 ± 32 and 54 ± 7 µmol/mL, p = 0.0005, respectively; Figure 1, right panel). 3 3 Skeletal Muscle Metabolism Fasting plasma insulin levels tended to be higher in HFrEF and HFpEF patients vs. controls (13.0 ± 2.1 and 19.3 ± 6.2 vs. 6.4 ± 0.7 µU/mL, p = 0.1, respectively; Figure 1). Plasma insulin levels increased after the oGL in all three groups with the highest increase of insulin levels in HFrEF vs. HFpEF and controls 60 min after the oGL (91 ± 13 vs. 57 ± 17 and 65 ± 11 µmol/mL, p < 0.05, respectively). Even 120 min after the oGL, plasma insulin levels remained the highest in HFrEF vs. HFpEF and controls (92 ± 12 vs. 49 ± 32 and 54 ± 7 µmol/mL, p = 0.0005, respectively; Figure 1, right panel). 6 of 10 J. Cardiovasc. Dev. Dis. 2022, 9, 456 3.3.1. Skeletal Muscle Perfusion and Glucose Uptake 3.3.1. Skeletal Muscle Perfusion and Glucose Uptake Muscle tissue perfusion as assessed by the ethanol ratio was similar in fasting con- dition between the patients with HFrEF, HFpEF and controls (0.21 ± 0.03, 0.18 ± 0.03, and 0.23 ± 0.04, n.s., respectively) (Figure 2, upper left panel). Whereas this ethanol ratio remained unchanged during the oGT in HFpEF patients and controls, it increased in HFrEF patients vs. baseline (p = 0.057) after glucose load, indicating lower postprandial muscle tissue perfusion in HFrEF (Figure 2, upper left panel). R PEER REVIEW 7 of 12 Figure 2. Skeletal muscle (Vastus lateralis) ethanol ratio and dialysate concentrations glucose, lactate, pyruvate, and glycerol of patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; upper right panel: HFpEF vs. controls, p < 0.0001; lower right panel: HFpEF vs. controls, p = 0.0016. Figure 2. Skeletal muscle (Vastus lateralis) ethanol ratio and dialysate concentrations glucose, lactate pyruvate, and glycerol of patients with heart failure with reduced ejection fraction (rEF, n = 12 men or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) a baseline and after an oral glucose load (75 g). Data are given as mean ± SE; upper right panel: HFpEF vs. controls, p < 0.0001; lower right panel: HFpEF vs. controls, p = 0.0016. Figure 2. Skeletal muscle (Vastus lateralis) ethanol ratio and dialysate concentrations glucose, lactate, pyruvate, and glycerol of patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; upper right panel: HFpEF vs. controls, p < 0.0001; lower right panel: HFpEF vs. controls, p = 0.0016. Figure 2. Skeletal muscle (Vastus lateralis) ethanol ratio and dialysate concentrations glucose, la pyruvate, and glycerol of patients with heart failure with reduced ejection fraction (rEF, n = 12 or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 me baseline and after an oral glucose load (75 g). 3.3.1. Skeletal Muscle Perfusion and Glucose Uptake Data are given as mean ± SE; upper right panel: H vs. controls, p < 0.0001; lower right panel: HFpEF vs. controls, p = 0.0016. Figure 2. Skeletal muscle (Vastus lateralis) ethanol ratio and dialysate concentrations glucose, lactate, pyruvate, and glycerol of patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; upper right panel: HFpEF vs. controls, p < 0.0001; lower right panel: HFpEF vs. controls, p = 0.0016. Figure 2. Skeletal muscle (Vastus lateralis) ethanol ratio and dialysate concentrations glucose, lactate, pyruvate, and glycerol of patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; upper right panel: HFpEF vs. controls, p < 0.0001; lower right panel: HFpEF vs. controls, p = 0.0016. Figure 2. Skeletal muscle (Vastus lateralis) ethanol ratio and dialysate concentrations glucose, lactate, pyruvate, and glycerol of patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; upper right panel: HFpEF vs. controls, p < 0.0001; lower right panel: HFpEF vs. controls, p = 0.0016. Figure 2. Skeletal muscle (Vastus lateralis) ethanol ratio and dialysate concentrations glucose, lactate, pyruvate, and glycerol of patients with heart failure with reduced ejection fraction (rEF, n = 12 men) or patients with preserved ejection fraction (pEF, n = 6 men) and healthy controls (n = 8 men) at baseline and after an oral glucose load (75 g). Data are given as mean ± SE; upper right panel: HFpEF vs. controls, p < 0.0001; lower right panel: HFpEF vs. controls, p = 0.0016. 7 of 10 7 of 10 J. Cardiovasc. Dev. Dis. 2022, 9, 456 Fasting dialysate glucose levels were significantly higher in HFpEF but not HFrEF patients vs. controls (2.95 ± 0.45 vs. 2.36 ± 0.24 and 1.75 ± 0.12 mmol/L, p < 0.0001). 3.3.2. Aerobic and Anaerobic Glycolysis Fasting dialysate lactate as an indicator of anaerobic glucose utilization (about 1 mmol/L) and pyruvate as an indicator of aerobic glucose utilization (about 30 µmol/L) levels were similar in all three groups (Figure 2, middle left and middle right panels). A similar increase of about 1.5-fold in lactate and about 2.5-fold in pyruvate levels was observed 120 min after the oGL in all study groups. y g p Because of the identical changes in dialysate lactate and pyruvate after the oGL in all three groups, lactate-to-pyruvate ratio decreased similarly by about 50% in all three groups. This indicates a proportional increase in aerobic and anaerobic glycolysis in HFpEF, HFrEF and controls. (Figure 2, lower left panel). 3.3.1. Skeletal Muscle Perfusion and Glucose Uptake After the oGL, dialysate glucose increased significantly in HFpEF vs. HFrEF and controls. Sixty minutes after the oGL, dialysate glucose levels achieved 4.77 ± 0.82, 2.83 ± 0.32 and 3.59 ± 0.29 mmol/L in HFpEF, HFrEF and controls, respectively (p < 0.0001, Figure 2, upper right panel). 3.3.3. Lipid Mobilization/Lipolytic Activity Fasting dialysate glycerol levels were similar in all three groups (about 45 µmol/L). Postprandial dialysate glycerol levels, the marker of tissue lipid mobilization, decreased in all study groups Figure 2, lower right panel). However, the slope of glycerol levels after oGT was lower in HFpEF and at the end of the test, dialysate glycerol was highest in HFpEF (33 ± 4 µmol/L) vs. controls (21 ± 3 µmol/L, p = 0.0016), vs. HFrEF (28 ± 3 µmol/L). This suggests a blunted anti-lipolytic effect of insulin in HFpEF vs. HFrEF and controls. 4. Discussion 2022, 9, 456 8 of 10 8 of 10 consequently results in scarce energy supply of the muscle and impaired energy metabolic balance, and leads to skeletal muscular weakness and exercise intolerance. consequently results in scarce energy supply of the muscle and impaired energy metabolic balance, and leads to skeletal muscular weakness and exercise intolerance. In patients with HFpEF, elevated HOMA-IR index > 4 under the fasting conditions and elevated postprandial blood glucose levels present perhaps another pathophysiological mechanism of IR as in HFrEF. Notably, fasting plasma insulin levels were the highest in HFpEF, but during the oGL, they have been reduced to values even lower than in the controls. Further, in fasting conditions as well as during the oGL, dialysate glucose levels were elevated in HFpEF suggesting truncated glucose uptake by skeletal muscle cells. In parallel, there was a higher degree of lypolytic activity, indicated by higher dialysate glycerol levels. These ﬁndings might suggest an inappropriate insulin production under the oGL in HFpEF. However, insulin fulﬁlled its peripheral anabolic functions by its canonical phosphokinase PI3K-Akt signaling pathway that has two main effects: (1) it activates glycolysis by activating the glucose transporter 4 (GLUT 4) in the skeletal muscle, and (2) it inhibits postprandial lipolysis, as indicated by a reduction of tissue glycerol—a marker of lipolysis [26]. Both effects seem to be impaired in HFpEF, which clearly supports the presence of IR in HFpEF. p p The pathophysiology of HFpEF is still not fully understood. The growing evidence suggests phenotypical differences between the HFrEF and HFpEF regarding the metabolic alterations [27,28]. Our data indicate that the metabolic effects of insulin are impaired in both, HFpEF and HFrEF patients, although at different levels. Interestingly, muscle glucose utilization itself is likely to be intact in patients with HFpEF and HFrEF, as indicated by comparable decreases of the lactate/ pyruvate ratio vs. controls within a physiological range after oGL. This ratio indicates the relation between anaerobic (lactate-production) and aerobic (pyruvate- production) glycolysis. Our ﬁndings are in line with previous clinical and experimental reports by our group and others. Glucose intolerance is an established risk factor in the development and pro- gression of both HF phenotypes [29]. Previously, a relationship between insulin resistance, higher NYHA, and impaired prognosis has been shown in patients with chronic HF [4]. 5. Study Limitations This is a pilot study in a small cohort of patients. However, the number of subjects per subgroup is similar to previous studies using the microdialysis technique for intra- muscular in vivo dynamic metabolic assessment and seemed also to be sufﬁcient [14,30]. Furthermore, patients on anticoagulant therapy could not be included into the trial due to elevated bleeding risks from intramuscular placing of the microdialysis probe. It is unlikely, however, that patients on oral anticoagulation may present different characteristics of glucose metabolism. Only male subjects were investigated in this pilot study in order to exclude gender-related confounding variables of metabolic control; therefore, potential sex-dependent differences of the metabolic state cannot be addressed. This will be an issue for upcoming studies. A relation between the muscle functional capacity and insulin resistance in heart failure has been shown previously [31] and was not part of the current study protocol. 4. Discussion In addition, differences between HFpEF and HFrEF in insulin resistance in non-diabetic patients who underwent a short insulin sensitivity test have been reported [10]. In this study, patients with HFrEF showed a lower glucose clearance during the in vivo insulin stimulation compared to patients with HFpEF and healthy controls. This might be ex- plained by impaired hemodynamics and lower skeletal muscle perfusion in HFrEF. A pattern similar to the controls of glucose response to insulin stimulation in parallel with elevated plasma glucose levels might indicate insufﬁcient insulin production in HFpEF. 4. Discussion The main ﬁndings of the present study are distinctly different patterns of skeletal muscle insulin resistance (IR) in both HFrEF and HFpEF patients. In HFpEF patients, we found higher postprandial blood and dialysate glucose values at rather normal insulin levels and postprandial dynamics, indicating an impaired glucose uptake not yet com- pensated by an increased insulin response. A reduced decrease in dialysate glycerol after the oGL also indicates a blunted anti-lipolytic effect of insulin in HFpEF. By contrast, in HFrEF patients, we found postprandial blood and dialysate glucose values within a rather high normal range, at signiﬁcantly higher postprandial insulin secretion, indicating tissue insulin resistance. The postprandial decrease in dialysate glycerol was similar to the healthy controls, indicating a compensated anti-lipolytic effect upon higher insulin secretion. In HFrEF patients, elevated blood glucose levels above 8 mmol/L two hours after the oGL and postprandial insulin levels above 100 µU/mL clearly indicate the presence of IR. Lower postprandial muscle tissue perfusion as indicated by an elevated ethanol ratio, suggests a proportionally lower glucose supply into the muscle. However, postprandial dialysate glucose levels are close to the levels of the controls, which could be an indicator of an impaired muscle glucose uptake in patients with HFrEF. In accordance, we observed elevated glucose plasma levels. Impaired hemodynamics secondary to reduced LVEF, reduced peripheral blood ﬂow [20] and peripheral endothelial dysfunction [21,22] may contribute to insufﬁcient organ and tissue perfusion in HFrEF [23,24]. Indeed, we observed lower systemic blood as well as a high-degree reduced LVEF and dilated left ventricle by echocardiography in these patients. At the same time, low postprandial skeletal muscle perfusion may be responsible for truncated delivery of insulin to muscle cells as well. Although the insulin concentrations have not been measured in dialysates in this study, we might assume this by elevated plasma levels of the insulin. Insufﬁcient stimulation of muscle cells by insulin would result in lower expression of the glucose transporter GLUT4 at the cell membrane and in lower glucose uptake [25]. Indeed, previously we have shown a reduced expression of GLU4 in the skeletal muscle [9]. Thus, impaired skeletal muscle tissue perfusion in HFrEF might cause low interstitial glucose and insulin supply that J. Cardiovasc. Dev. Dis. References und, L.H.; Seferovic, P.; Rosano, G.M.C.; Coats, A.J.S. Global burden of heart failure: A comprehensive demiology. Cardiovasc. Res. 2022, cvac013. [CrossRef] [PubMed] 1. Savarese, G.; Becher, P.M.; Lund, L.H.; Seferovic, P.; Rosano, G.M.C.; Coats, A.J.S. Global burden of he and updated review of epidemiology. Cardiovasc. Res. 2022, cvac013. [CrossRef] [PubMed] 2. Son, T.K.; Toan, N.H.; Thang, N.; Tuong, H.L.T.; Tien, H.A.; Thuy, N.H.; Van Minh, H.; Valensi, P. Prediabetes and insulin resistance in a population of patients with heart failure and reduced or preserved ejection fraction but without diabetes, overweight or hypertension. Cardiovasc. Diabetol. 2022, 21, 75. [CrossRef] [PubMed] yp 3. Yang, C.D.; Pan, W.Q.; Feng, S.; Quan, J.W.; Chen, J.W.; Shu, X.Y.; Aihemaiti, M.; Ding, F.H.; Shen, W.F.; Lu, L.; et al. Insulin Resistance Is Associated with Heart Failure with Recovered Ejection Fraction in Patients without Diabetes. J. Am. Heart Assoc. 2022, 11, e026184. [CrossRef] [PubMed] 4. Doehner, W.; Rauchhaus, M.; Ponikowski, P.; Godsland, I.F.; von Haehling, S.; Okonko, D.O.; Leyva, F.; Proudler, A.J.; Coats, A.S.; Anker, S.D. Impaired Insulin Sensitivity as an Independent Risk Factor for Mortality in Patients with Stable Chronic Heart Failure. J. Am. Coll. Cardiol. 2005, 46, 1019–1026. [CrossRef] J [ ] 5. Jia, D.; Zhang, J.; Liu, X.; Andersen, J.-P.; Tian, Z.; Nie, J.; Shi, Y. Insulin Resistance in Skeletal Muscle Selectively Protects the Heart in Response to Metabolic Stress. Diabetes 2021, 70, 2333–2343. [CrossRef] 6. Kinugawa, S.; Takada, S.; Matsushima, S.; Okita, K.; Tsutsui, H. Skeletal Muscle Abnormalities in Heart Failure. Int. Heart J. 2015, 56, 475–484. [CrossRef] 7. AlZadjali, M.A.; Godfrey, V.; Khan, F.; Choy, A.; Doney, A.S.; Wong, A.K.; Petrie, J.R.; Struthers, A.D.; Lang, C.C. Insulin resistance is highly prevalent and is associated with reduced exercise tolerance in nondiabetic patients with heart failure. J. Am. Coll. Cardiol. 2009, 53, 747–753. [CrossRef] 8. Doehner, W.; Frenneaux, M.; Anker, S.D. Metabolic impairment in heart failure: The myocardial and systemic perspective. J. Am. Coll. Cardiol. 2014, 64, 1388–1400. [CrossRef] 9. Doehner, W.; Gathercole, D.; Cicoira, M.; Krack, A.; Coats, A.J.; Camici, P.G.; Anker, S.D. Reduced glucose transporter GLUT4 in skeletal muscle predicts insulin resistance in non-diabetic chronic heart failure patients independently of body composition. Int. J. Cardiol. 2010, 138, 19–24. [CrossRef] 10. Scherbakov, N.; Bauer, M.; Sandek, A.; Szabó, T.; Töpper, A.; Jankowska, E.A.; Springer, J.; von Haehling, S.; Anker, S.D.; Lainscak, M.; et al. 6. Conclusions Our study has demonstrated distinctly different patterns of skeletal muscle insulin resistance in patients with HFrEF vs. HFpEF. In HFrEF patients, insulin resistance might be linked to impaired tissue perfusion secondary to hemodynamic failure and to impaired J. Cardiovasc. Dev. Dis. 2022, 9, 456 9 of 10 9 of 10 blood glucose transport, while in HFpEF, insulin resistance might be caused by an insufﬁ- cient insulin cellular signaling. Furthermore, the anti-lipolytic effect of insulin seems to be blunted in HFpEF vs. HFrEF. These ﬁndings provide novel insights into distinct metabolic characteristics of HFrEF and HFpEF. It supports the use of antidiabetic agents in patients with HF without diabetes mellitus in order to reduce systemic glucose levels. Better under- standing of speciﬁc mechanisms of impaired glucose metabolism in separate categories of HF may contribute to a speciﬁc therapeutic concept to improve energy utilization in patients with heart failure. Author Contributions: Conceptualization, N.J., J.S. and W.D.; methodology, M.B., L.K. and N.J.; validation, S.v.H., S.D.A. and J.S.; formal analysis, N.J. and M.B.; investigation, M.B. and N.J.; resources, N.J., F.E., H.-D.D. and A.S.; data curation, N.J. and M.B.; writing—original draft preparation, N.J., M.B. and A.S.; writing—review and editing, N.J., M.B., L.K., A.S., H.-D.D., F.E., J.S., S.v.H., S.D.A. and W.D.; visualization, M.B. and N.J.; project administration, N.J.; funding acquisition, N.J. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, N.J., J.S. and W.D.; methodology, M.B., L.K. and N.J.; validation, S.v.H., S.D.A. and J.S.; formal analysis, N.J. and M.B.; investigation, M.B. and N.J.; resources, N.J., F.E., H.-D.D. and A.S.; data curation, N.J. and M.B.; writing—original draft preparation, N.J., M.B. and A.S.; writing—review and editing, N.J., M.B., L.K., A.S., H.-D.D., F.E., J.S., S.v.H., S.D.A. and W.D.; visualization, M.B. and N.J.; project administration, N.J.; funding acquisition, N.J. All authors have read and agreed to the published version of the manuscript. Funding: N.J. received a grant from the DZHK (German Centre for Cardiovascular Research)— cooperation with Shared Expertise within the Translational Pipeline (15-073 SE). Funding: N.J. received a grant from the DZHK (German Centre for Cardiovascular Research)— cooperation with Shared Expertise within the Translational Pipeline (15-073 SE). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Charité-Universitätsmedizin Berlin, Germany (EA2/137/15, 12.11.2015). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. 6. Conclusions Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicity available due to privacy. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Is metabolic ﬂexibility altered in multiple sclerosis patients? PLoS ONE 2012, 7, e43675. [CrossRef] 18. Pieske, B.; Tschöpe, C.; de Boer, R.A.; Fraser, A.G.; Anker, S.D.; Donal, E.; Edelmann, F.; Fu, M.; Guazzi, M.; Lam, C.S.P.; et al. 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https://openalex.org/W2900912459	https://europepmc.org/articles/pmc6260410?pdf=render	English	null	Successfully Treated Norovirus- and Sapovirus-Associated Diarrhea in Three Renal Transplant Patients	Case reports in infectious diseases	2,018	cc-by	2,915	Hindawi Hindawi Hindawi Case Reports in Infectious Diseases Volume 2018, Article ID 6846873, 4 pages https://doi.org/10.1155/2018/6846873 Correspondence should be addressed to Noha Ghusson; nghusson@gmail.com Received 27 September 2018; Accepted 17 October 2018; Published 12 November 2018 Academic Editor: Tomoyuki Shibata Copyright © 2018 Noha Ghusson and Gustavo Vasquez. Tis 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. Objectives. To examine the burden of norovirus- and sapovirus-related diarrhea in renal transplant patients and to propose the use of nitazoxanide as a therapeutic option for treatment. Methods. We reviewed three renal transplant patients with viral diarrhea requiring hospitalization due to acute renal failure and signs of graft rejection. All three patients were treated with nitazoxanide. We examined their clinical courses after therapy and compared time to resolution of symptoms and viral shedding. Results. In all three renal transplant patients, improvement of diarrheal illness was witnessed within one week of nitazoxanide initiation. Conclusions. Infectious diarrhea remains an underestimated yet signiﬁcant cause of morbidity in solid organ transplant patients. Norovirus and sapovirus are often responsible for this presentation. Nitazoxanide was used as a treatment modality with success in reduction of symptoms, decreased duration of illness, and cessation of viral shedding. Noha Ghusson and Gustavo Vasquez Sidney Kimmel Medical College at Tomas Jeﬀerson University, Division of Infectious Diseases, 1015 Chestnut Street, Suite 1020, Philadelphia, PA 19107, USA orrespondence should be addressed to Noha Ghusson; nghusson@gmail.com 3. Discussion Sapovirus and norovirus are small, nonenveloped, single- stranded RNA viruses within the Caliciviridae family [2]. Tese enteric infections are typically community acquired and easily transmitted [1] via food-borne contact, person to person, or contaminated environmental surfaces [3]. While norovirus is the most common cause of viral gastroenteritis in adults [4], accounting for 90% of viral gastroenteritis worldwide [2], sapovirus also remains a major cause of enteric infections within the solid organ transplant pop- ulation. Despite the signiﬁcance of these infections, there are no data on the exact incidence of norovirus and sapovirus infections in the transplant population. RT-PCR is the gold standard for diagnosis and is typically made through use of a stool infectious panel [5]. Patients often present with abdominal cramping and watery diarrhea that is nonbloody and can be associated with vomiting [3]. Fever is un- common, and symptoms are usually self-limited in the healthy adult patient. In transplant patients, however, symptoms can last for several months [5], with a mean duration of 8.7 months [1]. As a result, patients are at in- creased risk for severe dehydration and acute kidney injury. 81% of patients hospitalized with these infections ex- perience acute renal failure [1] as a result of severe de- hydration due to diarrhea. Other causes besides dehydration include oxalic nephropathy and villous atrophy [1] as a di- rect result of gastrointestinal viral invasion. Such side eﬀects greatly increase rates of hospitalization and ultimately graft injury in this group of patients [5]. Te last patient is a 78-year-old male who underwent renal transplant in 2014 due to ESRD as a result of severe diabetic nephropathy. Te transplant was complicated by interstitial ﬁbrosis and tubular atrophy, requiring high doses of immunosuppressant medications. Tese medications included mycophenolate mofetil 500 mg twice daily, tacrolimus 4 mg twice daily, and prednisone 10 mg daily. Te patient presented to our hospital 3 years after renal trans- plant with an evidence of altered mental status, lethargy, and progressive weight loss. Per his family, he was having sig- niﬁcant diarrhea associated with agitation and confusion. On admission, he had severe metabolic acidosis and an elevated creatinine level of 7.8 mg/dL from baseline of 2.7 mg/dL, which required urgent hemodialysis. His meta- bolic derangements improved, but he remained confused. Another important issue in management of these in- fections includes the reduction of immunosuppressant medications once a diagnosis of norovirus or sapovirus has been made. Case Reports in Infectious Diseases Case Reports in Infectious Diseases 2 Te patient was having signiﬁcant watery stool output. Infectious workup was negative for Clostridium diﬃcile infection but conﬁrmed norovirus on a stool infectious panel. Initially, the immunosuppressant medications, pri- marily mycophenolate mofetil, were held in hopes of im- proving his diarrhea. When symptoms did not dissipate, he was started on oral nitazoxanide 500 mg twice daily em- pirically. Te patient’s diarrhea did improve within 72 hours of nitazoxanide initiation, and he was treated with a full 14- day course. Simultaneously, the patient developed acute transplant rejection which was conﬁrmed on renal ultra- sound. Tis was thought to be due to decrease in immu- nosuppressant medications. Since that time, hemodialysis has been reinitiated. Unfortunately, a repeat stool infectious panel after therapy was not collected as the patient was lost to follow-up from both the infectious disease and transplant nephrology clinics. Te patient was having signiﬁcant watery stool output. Infectious workup was negative for Clostridium diﬃcile infection but conﬁrmed norovirus on a stool infectious panel. Initially, the immunosuppressant medications, pri- marily mycophenolate mofetil, were held in hopes of im- proving his diarrhea. When symptoms did not dissipate, he was started on oral nitazoxanide 500 mg twice daily em- pirically. Te patient’s diarrhea did improve within 72 hours of nitazoxanide initiation, and he was treated with a full 14- day course. Simultaneously, the patient developed acute transplant rejection which was conﬁrmed on renal ultra- sound. Tis was thought to be due to decrease in immu- nosuppressant medications. Since that time, hemodialysis has been reinitiated. Unfortunately, a repeat stool infectious panel after therapy was not collected as the patient was lost to follow-up from both the infectious disease and transplant nephrology clinics. patient was treated initially with intravenous ﬂuids and antidiarrheal medications. Mycophenolate mofetil dose was decreased gradually and eventually discontinued. Azathio- prine was started in place of mycophenolate mofetil, and the patient did have some improvement in the consistency of her stools but the overall large volume output persisted. Nitazoxanide 500 mg orally (PO) twice daily was initiated, and the patient’s diarrhea improved within 3 days. She was treated for 7 days total with this medication, and a repeat stool infectious panel 1 month after therapy demonstrated a negative sapovirus PCR. Our second patient is a 70-year-old female with end- stage renal disease (ESRD) secondary to after streptococcal glomerulonephritis who received a kidney transplant in 2011. Case Reports in Infectious Diseases Te patient was maintained on tacrolimus 4 mg twice daily, prednisone 5 mg daily, and mycophenolate mofetil 500 mg daily. She presented four years after transplant with intermittent diarrhea for 4 months duration and signiﬁcant weight loss. On presentation, her creatinine was 3.4 mg/dL from a baseline of 1.2 mg/dL on prior studies. Te patient was found to have a positive stool infectious panel PCR for norovirus during an inpatient hospital evaluation. Clos- tridium diﬃcile study was negative, and fecal fat content was within normal limits. Mycophenolate mofetil was dis- continued due to concern that her symptoms may be a result of medication side eﬀect. She was subsequently started on azathioprine 50 mg daily for immunosuppression. With no real improvement in symptoms, she was treated with a 3-day course of 500 mg PO nitazoxanide twice daily with modest improvement. Two months later, the patient’s symptoms reoccurred, and she received a second, longer course of nitazoxanide for 3 weeks. Her symptoms resolved within a week of treatment but again relapsed over the subsequent 3 months. Stool bioﬁre PCRs were persistently positive for norovirus when checked monthly over the following 5- month time period. Finally, she was started on a 3-week course of nitazoxanide high dose (500 mg PO every eight hours). Initially, this was diﬃcult to tolerate due to gas- trointestinal distress, and the frequency of administration was decreased to twice daily. Te patient completed the three weeks of nitazoxanide with resolution of her gastrointestinal symptoms. She has been asymptomatic since the prolonged course, and repeat stool PCR for norovirus 10 days after completion of therapy is negative. 1. Introduction Our ﬁrst patient is a 30-year-old female with a medical history of systemic lupus erythematosus (SLE) complicated by stage V lupus nephritis. She underwent deceased donor renal transplant in 2005 and has been on chronic immu- nosuppression with tacrolimus 2 mg twice daily and mycophenolate mofetil 500 mg twice daily for approximately 10 years prior to presentation. Ten years after transplant, the patient presented with a chief complaint of diarrhea con- sisting of 4-5 loose, watery bowel movements daily. Her symptoms were accompanied by mild nausea, anorexia, and abdominal cramping, as well as an unintentional 15-pound weight loss. On admission, she did not have leukocytosis (WBC 4.5 B/L), but she was anemic with a hemoglobin count of 6.1 g/dL and had an elevated creatinine level of 1.6 mg/dL from 1.1 mg/dL baseline. An extensive workup for diarrhea was performed which revealed a positive fecal lactoferrin, normal fecal fat content, and antitissue transglutaminase antibody, a negative Clostridium diﬃcile stool toxin, and a positive stool infectious panel PCR for sapovirus. Te Sapovirus and norovirus are frequent causes of self-limited diarrheal illnesses in healthy hosts. In solid organ trans- plant patients, however, these viral infections are a fre- quent cause of chronic and intermittent diarrhea [1], causing signiﬁcant morbidity and often requiring hospi- talization in this population. With that comes an increased risk for serious disease and an inherent risk of de- hydration, acute renal failure, and lower graft/patient survival [1]. Traditionally, immunosuppressant medica- tions are tapered in this clinical scenario as a means to resolve these viral infections. However, decreasing im- munosuppressants increase the risk of acute transplant rejection. If an immunosuppressed patient does overcome the diarrheal illness, they remain at risk of prolonged asymptomatic viral shedding [2], thus putting themselves and others at risk of reinfection. Here we discuss three cases of renal transplant patients with diarrhea and the use of nitazoxanide in the treatment of norovirus or sapovirus gastroenteritis. 5. Limitations Despite the successful outcomes demonstrated amongst our patients with the use of nitazoxanide, our study has limitations that will beneﬁt from further investigation. First, the exact prevalence of norovirus and sapovirus is unknown due to lack of appropriate testing. Diarrhea in renal transplant patients is a common result of medication side eﬀect. Mycophenolate mofetil is a known culprit, for example, and therefore, a PCR stool infectious panel may not be sent in time for diagnosis. Tus, the prevalence of viral diarrhea in this population is grossly underestimated and underdetected. p p p Nitazoxanide is a thiazolide anti-infective agent [7] that works against anaerobic bacteria, protozoa, and viruses [2]. Te proposed mechanism of antiviral activity is the direct targeting of “cellular pathways involved in the synthesis of viral proteins,” thus inhibiting viral replication [4]. Te “drug modulates the host antiviral pathway by potentiating the protein kinase activated by double stranded RNA (PKR), an interferon induced eﬀector of cellular antiviral immunity. Activated PKR will halt viral protein synthesis” [8]. As a result, the medication is taken orally and passed through the gastrointestinal tract, addressing viral replication within the intestinal mucosa [4]. Nitazoxanide is noted to have a benign side eﬀect proﬁle, sitting abdominal pain, headache, diarrhea, and nausea [4] as the most common symptoms. It is a pregnancy category B drug [4] and overall is very well tolerated. Te most commonly cited dose of nitazoxanide used is 500 mg tablet twice a day. Te median time from ﬁrst dose to resolution of symptoms is 1.5 days [4] in the lit- erature. Amongst our renal transplant patients, all had signiﬁcantly reduced duration of illness as well as consis- tency and frequency of bowel movements within 3 days of drug initiation. All had complete resolution of diarrhea by the end of their therapeutic course. Aside from prolonged diarrheal illness, these viruses can continue to be shed within the stool of infected patients long after disappearance of clinical symptoms [9]. Repeat testing in 2 of our 3 patients shows that nitazoxanide also terminated the asymptomatic shedding. Unfortunately, our third patient was lost to follow-up, but obtaining this information can further sup- port use of this proposed therapy. As such, nitazoxanide is a promising safe, therapeutic option for treatment of viral gastroenteritis in renal transplant patients. Te outcomes of our 3 patients support the beneﬁt of nitazoxanide for therapy of norovirus- and sapovirus- associated diarrhea. 4. Conclusion Infectious diarrhea remains an underestimated yet signiﬁ- cant cause of morbidity in solid organ transplant patients. Viral etiologies, such as norovirus and sapovirus make up a high percentage of severe, chronic diarrhea and de- hydration requiring hospitalization in this population. Preemptive decrease in immunosuppressant medications further puts patients at risk of graft rejection and acute renal failure. 3. Discussion Medications such as mycophenolate mofetil and tacrolimus inherently increase the susceptibility to these infections [1]. As such, they are often tapered in attempt to “promote immune system reconstitution to treat the in- fection” [6], which ultimately puts patients at signiﬁcant risk for graft rejection and failure. While it is diﬃcult to maintain a balance between managing infection and preventing re- jection, [6] our hope is to eliminate this dichotomy by Case Reports in Infectious Diseases 3 providing an alternative management plan with use of oral nitazoxanide. bloating, and dehydration. Each patient was found to have a positive stool bioﬁre PCR for either norovirus or sap- ovirus in the years following their transplant surgery and suﬀered acute renal failure as a result. Nitazoxanide was used as a treatment modality with success in reduction of symptoms, decreased duration of illness, and cessation of viral shedding. Viral gastroenteritis is often managed by replacement of ﬂuids and correction of electrolyte disturbances [4]. Other methods of managing these infections include decreasing immunosuppressant medications which, as stated above, increases risk of graft rejection. Ribavirin has been oﬀered as a potential therapy for norovirus and sapovirus but has shown no signiﬁcant promise in reduction of symptoms or viral shedding. As a result, it is important to investigate therapeutic alternatives when reduction of immunosup- pressants is not a feasible choice. Tere is anecdotal evidence supporting the beneﬁt of nitazoxanide for management of norovirus and sapovirus in immunocompromised patients. 5. Limitations Te time to resolution of symptoms and clearance of virus remains unknown. Although im- provement of diarrhea can be easily reported, immuno- compromised patients with viral gastroenteritis can continue to shed the virus in stool for several weeks after resolution of symptoms. Tis poses the question as to whether we have fully eradicated the infection or if an asymptomatic balance state (or colonization) between host and virus is possible. By deﬁning clearance of infection, we will be able to determine a clear duration of nitazoxanide therapy in future cases of norovirus or sapovirus diarrhea. A third question we pose is whether there is an addi- tional beneﬁt to decreasing mycophenolate mofetil dose while concurrently treating these infections with nitazox- anide. All three of our patients had some modiﬁcation in their immunosuppression regimen prior to initiation of nitazoxanide. Although this may simply be a confounding variable, there is room for future research to determine the role of nitazoxanide with or without deescalating immu- nosuppressants during acute gastroenteritis in transplant patients. Lastly, there is opportunity for future eﬀorts in vaccine development for viral diarrhea caused by norovirus and sapovirus. With a vaccine, solid organ transplant patients will be placed at even lower risk of all the complications associated with these enteric illnesses. A preventive ap- proach to this common occurrence could be beneﬁcial. In the meantime, nitazoxanide proves to be a promising therapy for norovirus and sapovirus infectious diarrhea in renal transplant, and potentially all, transplant patients. Conflicts of Interest Noha Ghusson, the author of this article, declares that there are no conﬂicts of interest regarding the publication of this paper. All three renal transplant patients experienced severe, persistent diarrhea associated with abdominal discomfort, Case Reports in Infectious Diseases 4 References [1] D. Roos-Weil, K. Ambert-Balay, F. Lanternier et al., “Impact of norovirus/sapovirus-related diarrhea in renal transplant re- cipients hospitalized for diarrhea,” Transplantation, vol. 92, no. 1, pp. 61–69, 2011. [2] R. I. Glass, U. D. Parashar, and M. K. Estes, “Norovirus gas- troenteritis,” New England Journal of Medicine, vol. 361, no. 18, pp. 1776–1785, 2009. [3] D. Siddiq, H. L. Koo, J. A. Adachi, and G. M. Viola, “Norovirus gastroenteritis successfully treated with nitazoxanide,” Journal of Infectious Diseses, vol. 63, no. 5, pp. 394–397, 2011. [4] J. F. Rossingnol and M. El-Gohary, “Nitazoxanide in the treatment of viral gastroenteritis: a randomized double-blind placebo-controlled clinical trial,” Alimentary Pharmacology and Terapeutics, vol. 24, pp. 1423–1430, 2006. p pp [5] M. Koopmans, “Progress in understanding norovirus epide- miology,” Current Opinios in Infectious Diseases, vol. 21, no. 5, pp. 544–552, 2008. [6] P. T. Jurgens, L. A. Allens, A. V. Ambardekar, and C. K. McIlvennan, “Chronic norovirus infection in cardiac transplant patients: considerations for evaluation and man- agement,” Progress in Transplantation, vol. 21, no. 1, pp. 69–72, 2017. [7] E. B. Keeﬀe and J. F. Rossignol, “Treatment of chronic viral hepatitis with nitazoxanide and second generation thiazolides,” World Journal of Gastroenterology, vol. 15, no. 5, pp. 1805– 1808, 2009. [8] W. D. Yeubang Yin, M. P. Peppelnbosch, and Q. Pan, “Op- posing eﬀects of nitazoxanide on murine and human nor- ovirus,” Journal of Infectious Diseases, vol. 216, no. 6, pp. 780–782, 2017. [9] A. Steyer, T. Konte, M. Sagadin et al., “Intrahost norovirus evolution in chronic infection over 5 years of shedding in a kidney transplant recipient,” Frontiers in Microbiology, vol. 9, no. 371, pp. 1–12, 2018.
https://openalex.org/W2772956693	https://vestnik.donstu.ru/jour/article/download/256/257	Russian	null	Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving	Vestnik Donskogo gosudarstvennogo tehničeskogo universiteta	2,017	cc-by	17,145	Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* nt and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* Y. O. Chernyshev1, A. S. Sergeev2, A. N. Ryazanov3, E. O. Dubrov4 1,2 Don State Technical University, Rostov-on-Don, Russian Federation 3 “711 Voenproekt” JSC, Rostov-on-Don, Russian Federation 4 Rostov Scientific Research Institute for Radiocommunication, Rostov-on-Don, Russian Federation Y. O. Chernyshev1, A. S. Sergeev2, A. N. Ryazanov3, E. O. Dubrov4 1,2 Don State Technical University, Rostov-on-Don, Russian Federation 3 “711 Voenproekt” JSC, Rostov-on-Don, Russian Federation 4 Rostov Scientific Research Institute for Radiocommunication, Rostov-on-Don, Russian Federation Introduction. The research area of “natural calculation” is now widely used for the solution to optimization NP-complete problems including combinatorial tasks of cryptanalysis. A quick overview of the publications devoted to the application of the natural (bioin- spired) methods for cryptanalysis is provided. The main work ob- jective is to investigate a possibility of applying bee colony algo- rithms to the realization of block cipher cryptanalysis. Introduction. The research area of “natural calculation” is now widely used for the solution to optimization NP-complete problems including combinatorial tasks of cryptanalysis. A quick overview of the publications devoted to the application of the natural (bioin- spired) methods for cryptanalysis is provided. The main work ob- jective is to investigate a possibility of applying bee colony algo- rithms to the realization of block cipher cryptanalysis. Materials and Methods. The known bee colony techniques belong- ing to a relatively new class of the bioinspired optimization methods that simulate the processes occurring in wildlife are applied to solve this optimization problem. The description and the block diagram of the bee colony algorithm for the solution to a cryptanalysis task are provided; basic operations performed in parallel at the global level are noted. In the following, a set of independent operators allowing for the concurrent execution is defined. For this purpose, infor- mation-logical flowgraphs of the algorithm with the input control and information links are built, as well as matrices of succession, logical incompatibility, and independence are formed. This matrix of independence allows the definition of a set of algorithm operators admitting parallel execution. At that, the dimensionality of the max- imal internally stable sets defines the maximum number of the pro- cessors used for the algorithm implementation. Введение. Научное направление «природные вычисления» в последнее время широко используется для решения оптимиза- ционных NP-полных задач, в том числе комбинаторных задач криптоанализа. y @ , g @ , _ @ , @ *** The research is done with the financial support from RFFI (project 14-01-00634). Работа выполнена при финансовой поддержке РФФИ (проект 14-01-00634). Работа выполнена при финансовой поддержке РФФИ (проект 14-01-00634). E-mail: myvnn@list.ru, sergeev00765@mail.ru, alexandr_r89@mail.ru, dubrov@spark-mail.ru * The research is done with the financial support from RFFI (project 14-01-00634). 2017, №1(88), 144-159 2017, №1(88), 144-159 Вестник Донского государственного технического университета SCIENCE, AND MANAGEMENT УДК 004.056.55 10.23947/1992-5980-2017-17-1-144-159 Разработка и исследование параллельной модели алгоритмов пчелиных колоний для решения задач криптоанализа* Ю. О. Чернышев1, А. С. Сергеев2, А. Н. Рязанов3, Е. O. Дубров4 1,2 Донской государственный технический университет, г. Ростов-на-Дону, Российская Федерация 3 Открытое акционерное общество «711 Военпроект», г. Ростов-на-Дону, Российская Федерация 4 Ростовский научно-исследовательский институт радиосвязи, г. Ростов-на-Дону, Российская Федерация Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* It should be noted (and it was observed in the previous works) that the distinctive feature of applying the bioinspired meth- ods of cryptanalysis is the applicability of the encryption-decryption algorithm as a criterion function for the evaluation of the key ac- ceptability defined by the bioinspired method operations. Thus, it can be affirmed that when using the bioinspired methods, the secret key definition process depends not so much on the complexity of the encryption transformations, as on the bioinspired method itself which should provide a sufficient diversity of the key generation. Обсуждение и заключения. Основными результатами исследо- вания являются: разработка алгоритма колонии пчел, использу- емого для решения задачи криптоанализа; описание его струк- турной схемы и основных параллельно выполняемых этапов; построение матрицы независимости; оценка числа процессоров для реализации алгоритма. Следует заметить (и это отмечалось в предыдущих работах), что отличительной особенностью применения биоинспирированных методов криптоанализа яв- ляется возможность использования самого алгоритма шифро- вания (или расшифрования) в качестве целевой функции для оценки пригодности ключа, определенного с помощью опера- ций биоинспирированного метода. Поэтому можно утверждать, что при использовании биоинспирированных методов процесс определения секретного ключа зависит не столько от сложно- сти шифрующих преобразований, сколько от самого биоинспи- рированного метода, который должен обеспечивать достаточ- ное разнообразие генерации ключей. времени реализации алгоритма Keywords: cryptanalysis, bee colony algorithm, bee foragers, scout bees, information-logical graph, matrix of independence. Ключевые слова: криптоанализ, пчелиный алгоритм, пчелы- фуражиры, пчелы-разведчики, информационно-логический граф, матрица независимости. Введение. Научное направление «природные вычисления», объединяющее математические методы, в кото- рых заложен принцип природных механизмов принятия решений, в последние годы получает все более широкое рас- пространение при решении оптимизационных задач, в том числе задач криптоанализа. В данных методах и моделях основным определяющим элементом является построение начальной модели и правил, по которым она может изме- няться (эволюционировать). В течение последних лет были предложены разнообразные схемы эволюционных вычис- лений: генетический алгоритм, генетическое программирование, эволюционные стратегии, эволюционное програм- мирование, модели поведения роя пчел, стаи птиц и колонии муравьев, модели отжига или потока и другие конкури- рующие эвристические алгоритмы. В [1] рассмотрены методы решения задачи криптоанализа, относящейся к пере- борным задачам с экспоненциальной временной сложностью, для традиционных симметричных криптосистем, ис- пользующих шифры перестановки и замены, а также для шифров гаммирования с использованием генетических алго- ритмов. Среди последних разработок эвристических методов, используемых для решения задачи параметрической оптимизации технических объектов, можно отметить стохастический алгоритм, основанный на модели поведения роя светлячков, рассмотренный в [2]. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* В статье приводится краткий обзор публика- ций, посвященных применению природных (биоинспириро- ванных) методов для криптоанализа. Основной целью работы является исследование возможности применения алгоритмов пчелиных колоний для реализации криптоанализа блочных шифров. Материалы и методы. Для решения данной оптимизационной задачи применяются известные методы пчелиных колоний, относящиеся к сравнительно новому классу биоинспирирован- ных оптимизационных методов, имитирующих процессы, про- текающие в живой природе. Приводится описание и структур- ная схема алгоритма колонии пчел для решения задачи крипто- анализа, отмечаются основные операции, выполняемые парал- лельно на глобальном уровне. Далее определяется множество независимых операторов, допускающих параллельное выпол- нение. С этой целью строятся информационно-логические граф-схемы алгоритма с введенными связями по управлению и по информации, а также формируются матрицы следования, логической несовместимости и независимости. По данной мат- рице независимости возможно определение множества опера- торов алгоритма, которые допускают параллельное выполне- ние. При этом размерность максимального внутренне устойчи- вого множества определяет максимальное число процессоров, используемых для реализации алгоритма. Research Results. Theoretical estimates of time complexity of the bee colony algorithm are given as the key data. Besides, the prob- lem solution is provided: to find the required smallest number of processors of the homogeneous parallel computing systems with distributed memory, and a uniform plan for the implementation of operators for them, for the cryptanalysis algorithm based on the constructed information-logical graph data-logical graph, and for the preset time. The assessment of the wanted smallest number of processors for the cryptanalysis algorithm implementation, and the Результаты исследования. Основными результатами являются теоретические оценки временной сложности алгоритма пчели- ных колоний. Кроме того, приводится решение задачи: для алгоритма криптоанализа на основе построенного информаци- онно-логического графа и для заданного времени найти необ- ходимое наименьшее число процессоров однородной вычисли- тельной системы и план выполнения операторов на них. При- водится оценка необходимого минимального числа процессо- ров для реализации алгоритма криптоанализа, а также оценка Чернышев Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний evaluation of the total time of the algorithm realization are given. Discussion and Conclusions. The basic research results are: the development of the bee colony algorithm used for the cryptanalysis task solution; the description of its flowchart and the principal par- allel executed stages; the construction of a matrix of independence; the evaluation of the number of processors for the algorithm imple- mentation. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* Таким образом, итерационный процесс поиска решений при реализации алгоритма криптоанализа включает: — последовательное перемещение агентов-пчел на новые позиции в пространстве поиска; — последовательное перемещение агентов-пчел на новые позиции в пространстве поиска; — формирование соответствующих вариантов текста с последующей проверкой их оптимальности; — формирование соответствующих вариантов текста с последующей проверкой их оптимальности; ф р р у щ р ду щ р р выбор соответствующего оптимального (или квазиоптимального) варианта ключа [8]. выбор соответствующего оптимального (или квазиоптимального) варианта ключа [8]. В соответствии с [6, 8, 12] алгоритм колонии пчел включает следующие основные операции. вии с [6, 8, 12] алгоритм колонии пчел включает следующие основные операции. В соответствии с [6, 8, 12] алгоритм колонии пчел включает следующие основные операци 1. Формирование пространства поиска и создание популяции пчел. ование пространства поиска и создание популяции пчел. 2. Оценка целевой функции (ЦФ) пчел в популяции путем определения ЦФ, обусловливающей оптимальность исходного текста. ование перспективных участков для поиска в их окрестности. 3. Формирование перспективных участков для поиска в их окрестности. л с лучшими значениями ЦФ с каждого участка. 5. Выбор пчел с лучшими значениями ЦФ с каждого участка. 5. Выбор пчел с лучшими значениями ЦФ с каждого участка. 6. Отправка рабочих пчел (пчел-фуражиров) для случайного поиска и оценка их ЦФ. 7. Формирование новой популяции пчел. 8. Проверка условия окончания работы алгоритма. Если они выполняются, переход к 9, иначе — к 2. 9. Конец работы алгоритма. Структурная схема алгоритма колонии пчел приведена в [12]. Рассмотрим описание данного алгоритма для реализации криптоанализа [8, 9]. На первом этапе пчелиного алгоритма осуществляется формирование пространства поиска. Предположим, что каждая позиция as пространства поиска представляет собой размещенный в пространстве символ алфавита текста. При этом каждая пчела-агент содержит в памяти упорядоченный список Es = {esi, i = 1,2,…,n} посещенных символов. Этот список Es, поставленный в соответствие каждому символу, посещенному пчелой в про- странстве поиска, фактически представляет решение  текст, для которого могут быть определены секретный ключ и ЦФ (например, с помощью функции Якобсена [1, 3, 8]). Следующим этапом пчелиного алгоритма является формирование перспективных участков и поиск в их окрестности. Как и в [8], будем предполагать, что пространство поиска, в котором размещено m символов алфавита шифртекста, представляет собой квадратную матрицу А размером mхm. Для каждой позиции as определена окрест- ность размера λ для поиска, то есть множество позиций asi, находящихся на расстоянии (определяемом как количество элементов матрицы), не превышающем λ, от позиции as. Применительно к решению задачи криптоанализа этапы данного алгоритма реализуются в следующей форме. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* B [3] описаны методы криптографических атак на симметричные и ассиметричные криптосистемы с использованием биоинспирированных методов (алгоритмов муравьиных и пчелиных колоний). В [4, 5] исследована возможность применения методов генетического поиска для реализации криптоанализа блочных крип- тосистем. Данные задачи криптоанализа в большинстве случаев являются NP-полными и имеют комбинаторную слож- ность. В связи с этим, как отмечено в [6], основным мотивом для разработок новых алгоритмов решения комбинатор- ных задач являются возникшие потребности в решении задач большой размерности. В то же время, как отмечено в [7], недостатком методов эволюционной адаптации и генетических алгоритмов является наличие «слепого» поиска, что в ряде случаев приводит к увеличению времени поиска, генерации большого количества одинаковых и плохо приспо- собленных решений и увеличивает вероятность попадания в локальный оптимум. В этом плане представляет интерес применение эвристических методов, инспирированных природными системами, в которых осуществляется поэтапное построение решения задачи (то есть добавление нового оптимального частичного решения к уже построенному ча- стичному оптимальному решению). Одной из последних разработок в области роевого интеллекта является алгоритм пчел, который довольно успешно используется для нахождения экстремумов сложных многомерных функций [8, 9]. Отметим, что в [8] приводится обзор некоторых публикаций, посвященных применению алгоритмов пчелиных коло- ний для решения комбинаторных теоретико-графовых задач (задача разбиения графа, раскраска графа, сравнение с другими биоинспирированными методами), решению задачи размещения, задачи разложения составных чисел на про- стые сомножители, используемoй при криптоанализе асимметричных алгоритмов. Материалы и методы. В данной работе исследуется возможность параллельной реализации алгоритма пчели- ных колоний, применение которого для реализации методов криптоанализа (симметричных и асимметричных крипто- систем) описано ранее в [8–11]. При описании алгоритма криптоанализа воспользуемся методами и терминологией, используемыми в [6, 8]. Как отмечено в [6, 8], поведение пчелиного роя основано на самоорганизации, обеспечиваю- Вестник Донского государственного технического университета 2017, №1(88), 144-159 щей достижение общих целей роя при двухуровневой стратегии поиска. На первом уровне с помощью пчел- разведчиков формируется множество перспективных областей-источников. На втором уровне с помощью рабочих пчел-фуражиров исследуются окрестности данных областей. При этом основная цель колонии пчел  найти источник с максимальным количеством нектара. щей достижение общих целей роя при двухуровневой стратегии поиска. На первом уровне с помощью пчел- разведчиков формируется множество перспективных областей-источников. На втором уровне с помощью рабочих пчел-фуражиров исследуются окрестности данных областей. При этом основная цель колонии пчел  найти источник с максимальным количеством нектара. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* Положить значение ЦФ R равным малому положительному числу. 4. Сформировать множество nb базовых решений и соответствующее множество базовых позиций Аb = {abi} с лучшими значениями ЦФ R. 4. Сформировать множество nb базовых решений и соответствующее множество базовых позиций Аb = {abi} с лучшими значениями ЦФ R. 5. Задание номера агента-фуражира f = 1. 6. Выбор базовой позиции аiАb. 7. Выбор позиции аs(l), расположенной в окрестности базовой позиции аi, не совпадающей с ранее выбранны- ми на данной итерации позициями, и соответствующего решения (списка Es). 8. Включить позицию аs в множество Оi (где Оi  множество позиций, выбранных агентами-фуражирами в окрестности позиции аi). 9. Для всех вновь включенных позиций рассчитать и поставить им в соответствие решения Es и соответству- ющие значения ЦФ R. 10. f = f + 1, если f > nf, переход к п. 11, иначе — к п. 6. 11. Сформировать для каждой базовой позиции ai области Di = ai ΥОi. 12. В каждой области Di выбрать лучшую позицию аi* с лучшим значением ЦФ Ri. * 13. Среди всех значений Ri* выбрать лучшее значение R* и соответствующее решение (список позиций Е). 13. Среди всех значений Ri выбрать лучшее значение R* и соответствующее решение (список позиций Е). 14 Если значение R(l) предпочтительней значения R(l 1) то сохранить значение R(l) в противном случае 13. Среди всех значений Ri* выбрать лучшее значение R* и соответствующее решение (список позиций Е). 14. Если значение R(l) предпочтительней значения R(l-1), то сохранить значение R(l), в противном случае охраненным остается значение R(l 1) 13. Среди всех значений Ri выбрать лучшее значение R и соответствующее решение (список позиций Е ). 14. Если значение R(l) предпочтительней значения R(l-1), то сохранить значение R(l), в противном случае сохраненным остается значение R(l-1). р ( ) 15. Если l < L (не все итерации пройдены), l = l + 1, переход к п. 16, иначе — к п. 20. 16. Начать формирование множества базовых позиций. Во множество Аb1 включается nb1 лучших позиций, найденных агентами среди позиций аi в каждой из областей Di на итерации l-1. 17. Разместить nrl агентов-разведчиков случайным образом в пространстве поиска для выбора nrl позиций в пространстве поиска. 18. Включить в множество Аb2 nb2 лучших позиций из множества nrl новых позиций, найденных агентами- разведчиками на итерации l (nb2 + nb1 = nb). 19. Определить множество базовых позиций на итерации l как Аb = Аb1 Υ Аb2. Перейти к п. 5. 20. Конец работы алгоритма. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving*** На последующих итерациях алгоритма nrl агентов-разведчиков отправляются на поиск новых позиций (nrl < nr). Множество базовых позиций Аb(l) формируется из двух частей Аb1(l) и Аb2(l), при этом: На последующих итерациях алгоритма nrl агентов-разведчиков отправляются на поиск новых позиций (nrl < nr). Множество базовых позиций Аb(l) формируется из двух частей Аb1(l) и Аb2(l), при этом: у р р р р (nrl < nr). Множество базовых позиций Аb(l) формируется из двух частей Аb1(l) и Аb2(l), при этом: ) ( ) ф р ру у ( ) ( ) р — часть Аb1(l) содержит nb1 лучших решений а, найденных в каждой из областей на итерации l-1; ( ) ( ) ф р ру у ( ) ( ) р — часть Аb1(l) содержит nb1 лучших решений а, найденных в каждой из областей на итерации — часть Аb2(l) содержит nb2 лучших решений из nrl позиций, найденных пчелами-разведчиками на итерации l (nb1 + nb2 = nb). — часть Аb2(l) содержит nb2 лучших решений из nrl позиций, найденных пчелами-разведчиками на итерации l (nb1 + nb2 = nb). Определяется число агентов-фуражиров, отправляемых в окрестности каждой базовой позиции. Каждым агентом-фуражиром nfi выбирается базовая позиция аi(l), а также позиция аs(l), расположенная в окрестности этой ба- зовой позиции. Формируются области Di(l). В каждой области Di(l) выбирается лучшая позиция аi* c лучшей оценкой ЦФ Ri, и среди оценок Ri выбирается лучшая R. Если R(l) предпочтительней, чем R(l-1), то соответствующее реше- ние запоминается, и осуществляется переход к следующей итерации. Таким образом, алгоритм криптоанализа на основе пчелиной колонии, приведенный в [8, 9], можно сформу- лировать следующим образом. 1. Определить начальные параметры алгоритма: — количество пчел-агентов N; — количество итераций L; — количество агентов-разведчиков nr; — количество агентов-фуражиров nf ; — значение максимального размера окрестности λмакс; — количество базовых позиций nb; — nb1  количество базовых позиций, формируемых из лучших позиций а, найденных на l-1 ите — nrl  количество агентов-разведчиков, выбирающих случайным образом новые позиции на итерациях 2,3,..,L; — nb2  количество базовых позиций, формируемых из nrl новых лучших позиций, найденных агентами- разведчиками на l итерации. 2. Задать номер итерации l = 1. 2. Задать номер итерации l = 1. 3. Разместить nr агентов-разведчиков случайным образом в пространстве поиска, то есть выбрать произволь- ным образом nr символов в матрице А. Положить значение ЦФ R равным малому положительному числу. 3. Разместить nr агентов-разведчиков случайным образом в пространстве поиска, то есть выбрать произволь- ным образом nr символов в матрице А. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving*** Начальными параметрами алгоритма являются значение максимального размера окрестности для поиска λмакс и коли- чество: — пчел-агентов N, — итераций L, — агентов-разведчиков nr, — агентов-фуражиров nf. фур р f На l = 1 итерации алгоритма nr агентов-разведчиков случайным образом размещаются в пространстве поиска, то есть выбирается произвольным образом nr символов в матрице А. Значение ЦФ R на начальном этапе полагается равным малому положительному числу. Далее в соответствии с [6, 12] выбирается nb лучших (базовых) решений, у которых значения ЦФ R не хуже, чем значения ЦФ у любого другого решения. На начальной итерации этот выбор может быть осуществлен случайным образом. В пространстве поиска формируется множество базовых позиций Аb = {abi}, соответствующих базовым ре- шениям. На следующем шаге алгоритма в окрестности каждой базовой позиции направляется заданное число рабочих пчел (фуражиров), имитирующих поиск нектара [8, 9]. а агентом-фуражиром nfi базовой позиции ai реализуется случайный выбор позиции аs, располо- λ в границах 1 ≤ λ ≤ λмакс базовой позиции ai. После выбора агентом-фуражиром nfi базовой позиции ai реализуется случайный выбор женной в окрестности λ в границах 1 ≤ λ ≤ λмакс базовой позиции ai. После выбора агентом-фуражиром nfi базовой позиции ai реализуется случайный выбор позиции аs, располо- женной в окрестности λ в границах 1 ≤ λ ≤ λмакс базовой позиции ai. Таким образом, каждой пчеле-агенту ставится в соответствие упорядоченный список Es посещенных символов пространства поиска с определенной для этого списка ЦФ. Данная последовательность ставится в соответствие по- следнему посещенному пчелой-агентом символу пространства поиска. Таким образом, каждой пчеле-агенту ставится в соответствие упорядоченный список Es посещенных символов пространства поиска с определенной для этого списка ЦФ. Данная последовательность ставится в соответствие по- следнему посещенному пчелой-агентом символу пространства поиска. Аналогично [6, 8] вводится понятие области Di, представляющей собой Di = ai Υ Оi, где Оi  множество пози- ций, выбранных агентами-фуражирами в окрестности позиции аi. В каждой области Di выбирается позиция а* с луч- Чернышев Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний шей оценкой ЦФ Ri* (оценка области Di). Среди всех оценок областей Ri* выбирается лучшая оценка Ri* и соответ- ствующее решение (список Es). Вариант исходного текста с лучшим значением ЦФ запоминается, и осуществляется переход к следующей итерации. шей оценкой ЦФ Ri* (оценка области Di). Среди всех оценок областей Ri* выбирается лучшая оценка Ri* и соответ- ствующее решение (список Es). Вариант исходного текста с лучшим значением ЦФ запоминается, и осуществляется переход к следующей итерации. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving*** Список Е*  вариант исходного текста с лучшим значением ЦФ R. 20. Конец работы алгоритма. Список Е  вариант исходного текста с лучшим значением ЦФ R. Пример реализации данного алгоритма криптоанализа приведен в [4]. 20. Конец работы алгоритма. Список Е  вариант исходного текста с лучшим Пример реализации данного алгоритма криптоанализа приведен в [4]. Пример реализации данного алгоритма криптоанализа приведен в [4]. Пример реализации данного алгоритма криптоанализа приведен в [4]. 2017, №1(88), 144-159 Вестник Донского государственного технического университета ник Донского государственного технического университета 2017, №1(88), 1 Структурная схема данного алгоритма представлена на рис. 1. Сформировать множество nb базовых решений и соответствующее множество базовых пози- ций Ab = {abi} с лучшими значениями ЦФ R. F = 1 (задание номера агента- фуражира) 2 Номер итерации l = 1 Размещение nr агентов-разведчиков случайным образом в пространстве поиска (выбор про- извольным образом nr символов в матрице А). Определить значение ЦФ R равным малому положительному числу. Начало Определение начальных параметров алгоритма: количество пчел-агентов N; количество ите- раций L; количество агентов-разведчиков nr; количество агентов-фуражиров nf ; значение максимального размера окрестности макс; количество базовых позиций nb; nb1 — количество базовых позиций, формируемых из лучших позиций a, найденных на l-1 итерации; nrl — количество агентов-разведчиков, выбирающих случайным образом новые позиции на итера- циях 2,3,..,L; nb2 — количество базовых позиций, формируемых из nrl новых лучших позиций, найденных агентами-разведчиками на l итерации. Структурная схема данного алгоритма представлена на рис. 1. Начало Определение начальных параметров алгоритма: количество пчел-агентов N; количество ите- раций L; количество агентов-разведчиков nr; количество агентов-фуражиров nf ; значение максимального размера окрестности макс; количество базовых позиций nb; nb1 — количество базовых позиций, формируемых из лучших позиций a, найденных на l-1 итерации; nrl — количество агентов-разведчиков, выбирающих случайным образом новые позиции на итера- циях 2,3,..,L; nb2 — количество базовых позиций, формируемых из nrl новых лучших позиций, найденных агентами-разведчиками на l итерации. Выбор базовой позиции aiAb Сформировать множество nb базовых решений и соответствующее множество базовых пози- ций Ab = {abi} с лучшими значениями ЦФ R. F = 1 (задание номера агента- фуражира) 2 Номер итерации l = 1 Размещение nr агентов-разведчиков случайным образом в пространстве поиска (выбор про- извольным образом nr символов в матрице А). Определить значение ЦФ R равным малому положительному числу. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving*** Определение начальных параметров алгоритма: количество пчел-агентов N; количество ите- раций L; количество агентов-разведчиков nr; количество агентов-фуражиров nf ; значение максимального размера окрестности макс; количество базовых позиций nb; nb1 — количество базовых позиций, формируемых из лучших позиций a, найденных на l-1 итерации; nrl — количество агентов-разведчиков, выбирающих случайным образом новые позиции на итера- циях 2,3,..,L; nb2 — количество базовых позиций, формируемых из nrl новых лучших позиций, найденных агентами-разведчиками на l итерации. 1 Номер итерации l = 1 Размещение nr агентов-разведчиков случайным образом в пространстве поиска (выбор про- извольным образом nr символов в матрице А). Определить значение ЦФ R равным малому положительному числу. Сформировать множество nb базовых решений и соответствующее множество базовых поз ций Ab = {abi} с лучшими значениями ЦФ R. Сформировать множество nb базовых решений и соответствующее м ций Ab = {abi} с лучшими значениями ЦФ R Выбор базовой позиции aiAb ций Ab = {abi} с лучшими значениями ЦФ R. F = 1 (задание номера агента- фуражира) 2 1 F = 1 (задание номера агента- фуражира) Выбор базовой позиции aiAb Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний Чернышев Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний Чернышев Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний Сохраняется значение R(l-1) Сохраняется значение R(l) Выбор позиции as (l), расположенной в окрестности базовой позиции ai, не совпадающей с раннее вы- бранными на данной итерации позициями, и соответствующего решения (списка ES). Включение позиции as в Oi (множество позиций, выбранных агентами-фуражирами в окрестности по- зиции ai). Формирование для каждой базовой позиции ai области Di = aiΥOi. Выбор в каждый области Di лучшей позиции ai с лучшим значением ЦФ Ri. Выбор среди всех значений Ri лучшего значения R* и соответствующего решения (списка позиций Е). Определение для всех вновь включенных позиций решения Es и соответствующих значений ЦФ R. Значение R(l) предпочтительней значе- ния R(l-1) f = f + 1 f > nf 1 Да Не ор позиции as (l), расположенной в окрестности базовой позиции ai, не совпадающей с раннее вы- бранными на данной итерации позициями, и соответствующего решения (списка ES). ние позиции as в Oi (множество позиций, выбранных агентами-фуражирами в окрестности по- зиции ai). Сохраняется значение R(l-1) Сохраняется значение R(l) 3 Формирование для каждой базовой позиции ai области Di = aiΥOi. Выбор в каждый области Di лучшей позиции ai с лучшим значением ЦФ Ri. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving** Выбор среди всех значений Ri* лучшего значения R* и соответствующего решения (списка позиций Е). Определение для всех вновь включенных позиций решения Es и соответствующих значений ЦФ R. Значение R(l) предпочтительней значе- ния R(l-1) f = f + 1 f > nf Да Не Определение для всех вновь включенных позиций решения Es и соответствующих значений ЦФ R. f > nf Формирование для каждой базовой позиции ai области Di = aiΥOi. Выбор в каждый области Di лучшей позиции ai с лучшим значением ЦФ Ri. Выбор в каждый области Di лучшей позиции ai с лучшим значением ЦФ Ri. Сохраняется значение R(l-1) Сохраняется значение R(l) 3 Выбор среди всех значений Ri лучшего значения R* и соответствующего решения (списка позиций Е). Значение R(l) предпочтительней значе- ния R(l-1) Да Не Выбор среди всех значений Ri лучшего значения R* и соответствующего решения (списка позиций Значение R(l) предпочтительней значе- ния R(l-1) Не Сохраняется значение R(l-1) Сохраняется значение R(l) Вестник Донского государственного технического университета 2017, №1(88), 144-159 Рис. 1. Структурная схема криптоанализа на основе алгоритма колонии пчел Fig. 1. Block scheme of cryptanalysis based on bee colony algorithm Формирование множества базовых позиций Ab1. Включение в него nb1 лучших позиций, найденных агентами среди позиций ai* в каждой из областей Di на итерации l-1. Размещение nrl агентов-разведчиков случайным образом в пространстве поиска для выбора nrl позиций в простран- стве поиска. Включение во множество Ab2 nb2 лучших позиций из множества nrl новых позиций, найденных агентами- разведчиками на итерации l (nb2 + nb1 = nb). Формирование множества базовых позиций на итерации l как Ab = Ab1 Υ Ab2. 2 3 l = l + 1 l < L Конец Список Е* — вариант исходного тек- ста с лучшим значением ЦФ R* 3 l = l + 1 l < L Конец Список Е* — вариант исходного тек- ста с лучшим значением ЦФ R* Конец Список Е* — вариант исходного тек- ста с лучшим значением ЦФ R* Конец — вариант исходного тек- шим значением ЦФ R* l < L Формирование множества базовых позиций Ab1. Включение в него nb1 лучших позиций, найденных агентами среди позиций ai* в каждой из областей Di на итерации l-1. Размещение nrl агентов-разведчиков случайным образом в пространстве поиска для выбора nrl позиций в простран- стве поиска. Включение во множество Ab2 nb2 лучших позиций из множества nrl новых позиций, найденных агентами- разведчиками на итерации l (nb2 + nb1 = nb). Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving*** Формирование множества базовых позиций на итерации l как Ab = Ab1 Υ Ab2. 2 Формирование множества базовых позиций Ab1. Включение в него nb1 лучших позиций, найденных агентами среди позиций ai* в каждой из областей Di на итерации l-1. агентов-разведчиков случайным образом в пространстве поиска для выбора nrl позиций в простран стве поиска. 2 Как и ранее в [4, 5], в соответствии с данной структурной схемой на глобальном уровне можно отметить сле- дующие параллельно выполняемые этапы: Как и ранее в [4, 5], в соответствии с данной структурной схемой на глобальном уровне можно отметить сле- дующие параллельно выполняемые этапы: р параллельное размещение nr пчел-разведчиков случайным образом в пространстве поиска; — параллельный выбор базовых позиций, позиций, расположенных в их окрестности, получение решений Es и соот- ветствующих значений ЦФ R каждым агентом-фуражиром; — параллельное формирование областей Di и выбор в них лучших позиций аi* с лучшим значение б параллельное формирование областей Di и выбор в них лучших позиций аi с лучшим значением ЦФ Ri ; — параллельное размещение nrl агентов-разведчиков в пространстве поиска для выбора nrl позиций. — параллельное размещение nrl агентов-разведчиков в пространстве поиска для выбора nrl позиций. Таким образом, с учетом данных преобразований структурную схему пчелиного алгоритма можно предста- вить в виде, показанном на рис. 2. Для упрощения будем предполагать, что: 5 Таким образом, с учетом данных преобразований структурную схему пчелиного алгоритма можно предста- вить в виде, показанном на рис. 2. Для упрощения будем предполагать, что: — число агентов-разведчиков nr = 5; число базовых решений n = 4; — число агентов-разведчиков nr = 5; — число базовых решений nb = 4; — число агентов-фуражиров nf = 5; — nb1 = 2  количество базовых позиций, формируемых из лучших позиций а, найденных на l-1 итерации; — nb2 = 2  количество базовых позиций, формируемых из nrl новых лучших позиций, найденных агентами- разведчиками на l итерации. Чернышев Ю. О. и др. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving** Разработка и исследование параллельной модели алгоритмов пчелиных колоний р р р р р 1 Размещение агента- разведчика 3 5 Размещение агента- разведчика 2 4 Размещение агента- разведчика 4 6 Размещение агента- разведчика 5 7 Размещение агента- разведчика 1 3 Начало Определение начальных параметров алгоритма: N; L; nr; nf; макс; nb; nb1; nri; nb2 1 Номер итерации l = 1 2 Выбор базовой позиции аген- том-фуражиром 1, получение результата E1 8 Выбор базовой позиции агентом- фуражиром 2, получение резуль- тата E2 9 Выбор базовой позиции агентом- фуражиром 5, получение резуль- тата E5 1 Формирование области D1 и выбор позиции a1 с лучшим решением R1 1 Формирование области D1 и выбор позиции a2 с лучшим решением R2 1 Формирование области D4 и выбор позиции a4 с лучшим решением R4 1 2 Начало Определение начальных параметров алгоритма: N; L; nr; nf; макс; nb; nb nri; nb2 Выбор базовой позиции аген- том-фуражиром 1, получение результата E1 8 Выбор базовой позиции агентом- фуражиром 5, получение резуль- тата E5 1 Выбор базовой позиции агентом- фуражиром 2, получение резуль- тата E2 9 8 9 Формирование области D1 и выбор позиции a2 с лучшим решением R2 1 Формирование области D4 и выбор позиции a4 с лучшим решением R4 1 Формирование области D1 и выбор позиции a1 с лучшим решением R1 1 2017, №1(88), 144-159 Вестник Донского государственного технического университета Выбор среди всех значений Ri лучшего значения R и соответствующего решения (списка E) 17 ор среди всех значений Ri лучшего значения R* и соответствующего решения (списка E) 17 Рис. 2. Структурная схема криптоанализа на основе алгоритма пчел с учетом параллельно выполняемых этапов Fig. 2. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving** Block scheme of cryptanalysis based on bee colony algorithm with account of concurrent stages Для дальнейшего определения множества независимых операторов, допускающих параллельное выполн дем как и ранее в [4 5] использовать методы описанные в [13] Для данной структурной схемы показанно Значение R(l) предпочтительней значения R(l – 1) 18 Сохраняется значение R(l) 19 Сохраняется значение R(l – 1) 20 Выбор среди всех значений R i лучшего значения R и соответствующего решения (списка E ) 17 l = l + 1 21 l < L 22 Конец Список E* — вариант исходного текста с лучшим значением ЦФ R* 23 Формирование множества позиций Ab1 путем включения nb1 = 2 лучших пози- Размещение агента- разведчика 1 25 Размещение агента- разведчика 3 27 Размещение агента- разведчика 2 26 Размещение агента- разведчика 4 28 Размещение агента- разведчика 5 29 Выбор nb2 = 2 лучших позиций из множества nrl новых позиций, найденных агентами- разведчиками на итерации l, и формирование множества Ab2 30 Формирование множества базовых позиций на итерации l как Ab1ΥAb2 31 2 24 Значение R(l) предпочтительней значения R(l – 1) 18 Сохраняется значение R(l – 1) 20 Сохраняется значение R(l) 19 Конец Список E* — вариант исходного текста с лучшим значением ЦФ R* 23 22 Формирование множества позиций Ab1 путем включения nb1 = 2 лучших пози- 24 Формирование множества позиций Ab1 путем включения nb1 = 2 лучших пози- 24 Рис. 2. Структурная схема криптоанализа на основе алгоритма пчел с учетом параллельно выполняемых этапов Fig. 2. Block scheme of cryptanalysis based on bee colony algorithm with account of concurrent stages ис. 2. Структурная схема криптоанализа на основе алгоритма пчел с учетом параллельно выполняемых эта Fig. 2. Block scheme of cryptanalysis based on bee colony algorithm with account of concurrent stages Для дальнейшего определения множества независимых операторов, допускающих параллельное выполнение, будем, как и ранее в [4, 5], использовать методы, описанные в [13]. Для данной структурной схемы, показанной на рис. 2, составим информационно-логическую граф-схему G, отобразив в ней связи по управлению (двойная линия) и по информации (одинарная линия) (рис. 3). На рис. 3 двойной линией отмечены связи 18–19, 18–20 и 22–23, 22–24. Чернышев Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний Рис 3 Информационно логическая граф схема алгоритма криптоанализа Рис. 3. Информационно-логическая граф-схема алгоритма криптоанализа Рис. 3. Информационно-логическая граф-схема алгоритма криптоанализа Рис. 3. Информационно-логическая граф-схема алгоритма криптоанализа Fig. 3. Information-logical flowgraph of cryptanalysis algorithm Fig. 3. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving*** Information-logical flowgraph of cryptanalysis algorithm Для данного графа введем в рассмотрение матрицу следования S. В соответствии с [13] элемент Sij = , если существует связь по управлению, и Sij = 1, если существует связь по информации (рис. 4). 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 1 2 3 1 1 4 1 1 5 1 1 6 1 1 7 1 1 8 1 1 1 1 1 9 1 1 1 1 1 10 1 1 1 1 1 11 1 1 1 1 1 12 1 1 1 1 1 13 1 1 1 1 1 14 1 1 1 1 1 15 1 1 1 1 1 16 1 1 1 1 1 17 1 1 1 1 18 1 19 20 * 21 1 1 22 1 23 * 24 * 25 1 26 1 27 1 28 1 29 1 30 1 1 1 1 1 31 1 Рис. 4. Матрица следования алгоритма пчелиных колоний Fig. 4. Succession matrix of bee colony algorithm Рис. 4. Матрица следования алгоритма пчелиных колоний Fig. 4. Succession matrix of bee colony algorithm 2017, №1(88), 144-159 Вестник Донского государственного технического университета Далее, используя алгоритмы, описанные в [13], дополним матрицу S транзитивными связями (рис. 5), обозна- чив все элементы Sij = 1. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 1 2 3 1 1 4 1 1 5 1 1 6 1 1 7 1 1 8 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 12 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1 14 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 5. Матрица следования алгоритма пчелиных колоний, дополненная транзитивными связями Рис. 5. Матрица следования алгоритма пчелиных колоний, дополненная транзитивными связями Fig. 5. Succession matrix of bee colony algorithm supplemented with transitive relations Также сформируем симметричную матрицу следования S` (рис. 6) и введем в рассмотрение матрицу L логиче- ской несовместимости операторов. Данная матрица L очевидным образом будет содержать следующие ненулевые элементы, соответствующие логически несовместимым операторам: L(19,20) = L(20,19) = L(23,24) = L(23,25) =...= L(23,31) = L(24,23) = L(25,23) =…= L(31,23) = 1. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Чернышев Ю. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* Симметричная матрица следования алгоритма пчелиных колоний Fi 6 S t i i t i f b l l ith 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. 6. Симметричная матрица следования алгоритма пчелиных колоний Fi 6 S t i i t i f b l l ith Рис. 6. Симметричная матрица следования алгоритма пчелиных колоний Fig.6. Symmetric succession matrix of bee colony algorithm Fig.6. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* Symmetric succession matrix of bee colony algorithm Путем дизъюнктивного сложения этих матриц S` и L получим матрицу независимости M, показанную на рис. 7. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 7. Матрица независимости M алгоритма пчелиных колоний 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. 6. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 7. Матрица независимости M алгоритма пчелиных колоний Fig.7. Matrix of independence M of bee colony algorithm Путем дизъюнктивного сложения этих матриц S` и L получим матрицу независимости M, показанную на рис. 7. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний Чернышев Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. 6. Симметричная матрица следования алгоритма пчелиных колоний Fig.6. Symmetric succession matrix of bee colony algorithm Путем дизъюнктивного сложения этих матриц S` и L получим матрицу независимости M, показанную на рис. 7. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 7. Матрица независимости M алгоритма пчелиных колоний Fig.7. Matrix of independence M of bee colony algorithm 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. 6. Симметричная матрица следования алгоритма пчелиных колоний Fig.6. Symmetric succession matrix of bee colony algorithm Путем дизъюнктивного сложения этих матриц S` и L получим матрицу независимости M, показанную на рис. 7. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 17 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 22 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 23 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 27 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 30 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Рис. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 7. Матрица независимости M алгоритма пчелиных колоний Fig.7. Matrix of independence M of bee colony algorithm Fig.7. Matrix of independence M of bee colony algorithm Результаты исследования. Итак, по данной матрице независимости М можно очевидным образом опреде- лить множества операторов алгоритма, которые допускают параллельное выполнение. Размерность максимального внутренне устойчивого множества определяет максимальное число процессоров, используемых для реализации алго- ритма. Отметим, что теоретические оценки временной сложности алгоритма пчелиных колоний приведены в [12]. В лучшем случае временная сложность пчелиных алгоритмов Т составляет Т ≈ О (nlgn), в худшем случае Т ≈ О (n3). Как отмечено в [5], для повышения быстродействия и эффективности алгоритма за счет минимизации времени работы Т возможна организация процесса распараллеливания как на глобальном уровне (параллельная обработка Р элементов популяции на n процессорах), так и на локальном (параллельная реализация процесса оценки одного элемента попу- 2017, №1(88), 144-159 Вестник Донского государственного технического университета ляции). Таким образом, для повышения эффективности реализации алгоритма пчелиных колоний на локальном уровне в соответствии с [4] актуальной является задача: для алгоритма криптоанализа на основе построенного инфор- мационно-логического графа G и для заданного времени Тзад найти необходимое наименьшее число процессоров од- нородной вычислительной системы и план выполнения операторов на них. ляции). Таким образом, для повышения эффективности реализации алгоритма пчелиных колоний на локальном уровне в соответствии с [4] актуальной является задача: для алгоритма криптоанализа на основе построенного инфор- мационно-логического графа G и для заданного времени Тзад найти необходимое наименьшее число процессоров од- нородной вычислительной системы и план выполнения операторов на них. Для решения этой задачи, как и ранее, воспользуемся методами, описанными в [13]. При этом в качестве вре- мени Тзад примем, как и ранее, время Ткр — длину критического пути в информационно-логическом графе G. На пер- воначальном этапе при рассмотрении однородных вычислительных систем необходимо определение скалярных весов вершин в информационно-логическом графе, отражающих время выполнения операторов, составляющих схему на рис. 2. р Как и в [4, 5], для решения данной задачи воспользуемся методами, изложенными в [13]. Веса операторов, пока- зывающие время их выполнения и определенные в соответствии с основными правилами анализа программ, описан- ными [14], приведены на рис. 3. Отметим, что данные веса определены в соответствии с отмеченными выше допуще- ниями, что nr = 5; nb = 4; nf = 5; nb1 = 2; nb2 = 2, длина обрабатываемой строки текста (аналогично [8]) Т = 5. Легко убе- диться, что критический путь в графе G Ткр = 35. В предположении, что Тзад = Ткр для представленного на рис. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* 3 ин- формационно-логического графа и матрицы следования найдем ранние τрi и поздние сроки τпi окончания выполнения операторов с помощью алгоритмов, представленных в [13]. Ранние сроки: τр1 = 9, τр2 = 1, τр3 = τр4 = τр5 = τр6 = τр7 = 10, τр8 = τр9 = τр10 = τр11 = τр12 = 14, τр13 = τр14 = τр15 = τр16 = 18, τр17 = 22, τр18 = 23, τр19 = τр20 = 24, τр21 = 25, τр22 = 26, τр23 = 31, τр24 = 28, τр25 = τр26 = τр27 = τр28 = τр29 = 29, τр30 = 31, τр31 = 35. Поздние сроки: τр1 = 9, τр2 = 1, τр3 = τр4 = τр5 = τр6 = τр7 = 10, τр8 = τр9 = τр10 = τр11 = τр12 = 14, τр13 = τр14 = τр15 = τр16 = 18, τр17 = 22, τр18 = 23, τр19 = τр20 = 24, τр21 = 25, τр22 = 26, τр23 = 31, τр24 = 28, τр25 = τр26 = τр27 = τр28 = τр29 = 29, τр30 = 31, τр31 = 35. Поздние сроки: τп31 = 35, τп30 = 31, τп29 = τп28 = τп27 = τп26 = τп25 = 29, τп24 = 28, τп23 = 35, τп22 = 26, τп21 = 25, τп20 = τп19 = 24, τп18 = 23, τп17 = 22, τп16 = τп15 = τп14 = τп13 = 18, τп12 = τп11 = τп10 = τп9 = τп8 = 14, τп7 = τп6 = τп5 = τп4 = τп3 = 10, τп2 = τп1 = 9. τп31 = 35, τп30 = 31, τп29 = τп28 = τп27 = τп26 = τп25 = 29, τп24 = 28, τп23 = 35, τп22 = 26, τп21 = 25, τп20 = τп19 = 24, τп18 = 23, τп17 = 22, τп16 = τп15 = τп14 = τп13 = 18, τп12 = τп11 = τп10 = τп9 = τп8 = 14, τп7 = τп6 = τп5 = τп4 = τп3 = 10, τп2 = τп1 = 9. В соответствии с методикой, описанной в [13], в матрице независимости найдем внутренне устойчивые множе- ства, представляющие множества взаимно независимых операторов (ВНО). Это множества (1, 2), (3, 4, 5, 6, 7), (8, 9, 10, 11, 12), (13, 14, 15, 16), (25, 26, 27, 28, 29). В соответствии с методикой, описанной в [13], в матрице независимости найдем внутренне устойчивые множе- ства, представляющие множества взаимно независимых операторов (ВНО). Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving* Это множества (1, 2), (3, 4, 5, 6, 7), (8, 9, 10, 11, 12), (13, 14, 15, 16), (25, 26, 27, 28, 29). Используя значения τрi и τпi, как и ранее в [4, 5], оценим минимальное число процессоров для выполнения алго- ритма за время Ткр. Для этого построим диаграммы ранних и поздних сроков окончания выполнения операторов и найдем такое распределение временных границ операторов для всех ВНО, при котором число используемых процес- соров (функция n) минимально [13]. Легко убедиться, что у операторов, входящих в данные множества ВНО, ранние и поздние сроки окончания выполнения равны, поэтому максимальное значение n = 5 имеет место для ВНО (3, 4, 5, 6, 7), ВНО (8, 9, 10, 11, 12), ВНО (25, 26, 27, 28, 29). Таким образом, получена оценка числа процессоров n = 5, позволяющая выполнить алгоритм криптоанализа на основе метода пчелиных колоний за минимальное время Т = Ткр при отмеченных выше допущениях. Данная оценка является решением задачи, так как в соответствии с [13] в матрице независимости нет множеств ВНО, содержащих число операторов r > n. а очевидным образом следует утверждение. Отсюда очевидным образом следует утверждение. Отсюда очевидным образом следует утверждение. Утверждение. При реализации описанного выше параллельного алгоритма криптоанализа на основе метода пчелиных колоний, представленного информационно-логическим графом G на рис. 3 (в соответствии с технологией распараллеливания, описанной в [13]), необходимое минимальное число процессоров может быть определено как max(nr; nf ; nb). При этом общее время реализации алгоритма в общем случае составляет Т = Q × Tкр, Q р, где Q — количество итераций (в общем случае не превышающее длину блока текста), Ткр — длина критического пути в информационно-логическом графе G, определенная в соответствии с правилами анализа программ, описанными в [14]. Q р, где Q — количество итераций (в общем случае не превышающее длину блока текста), Ткр — длина критического пути в информационно-логическом графе G, определенная в соответствии с правилами анализа программ, описанными в [14]. ие и заключение. Таким образом, в данной работе: Обсуждение и заключение. Таким образом, в данной работе: Обсуждение и заключение. Таким образом, в данной работе: Обсуждение и заключение. Development and investigation of parallel model of bee colony algorithms for cryptanalysis problem solving*** Таким образом, в данной работе: — представлено описание алгоритма колонии пчел, используемого для реализации криптоанализа, его структурная схема; писание алгоритма колонии пчел, используемого для реализации криптоанализа, его структурн новные параллельно выполняемые этапы алгоритма, и на их основе построена информационн хема алгоритма; — определены основные параллельно выполняемые этапы алгоритма, и на их основе постро логическая граф-схема алгоритма; новные параллельно выполняемые этапы алгоритма, и на их основе построена информационн хема алгоритма; — построены матрицы следования и независимости, позволяющие определить основные параллельно выполняемые операции алгоритма; ; ка числа процессоров, необходимых для реализации алгоритма. — приведена оценка числа процессоров, необходимых для реализации алгоритма. В качестве заключения может быть представлен вывод, сделанный в публикациях [4, 5, 15, 16]. Основной от- личительной особенностью применения биоинспирированных методов криптоанализа является возможность исполь- зования самого алгоритма шифрования (или расшифрования) в качестве целевой функции для оценки пригодности Чернышев Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний ключа, определенного с помощью генетических операций. Вследствие этого при использовании биоинспирированных методов криптоанализа процесс определения секретного ключа (например, при криптоанализе 2-го типа) зависит не столько от сложности шифрующих преобразований, сколько от самого биоинспирированного метода, который должен обеспечивать достаточное разнообразие генерации ключей. Это свидетельствует об актуальности задачи исследования возможности применения биоинспирированных алгоритмов для криптоанализа блочных криптосистем. Также следует заметить, поскольку отличительной особенностью как блочных методов шифрования, так и биоинспирированных ме- тодов (в частности, генетических алгоритмов), является их внутренний параллелизм [4], то задача разработки алго- ритма криптоанализа на основе параллельной реализации составляющих этапов является актуальной. ключа, определенного с помощью генетических операций. Вследствие этого при использовании биоинспирированных методов криптоанализа процесс определения секретного ключа (например, при криптоанализе 2-го типа) зависит не столько от сложности шифрующих преобразований, сколько от самого биоинспирированного метода, который должен обеспечивать достаточное разнообразие генерации ключей. Это свидетельствует об актуальности задачи исследования возможности применения биоинспирированных алгоритмов для криптоанализа блочных криптосистем. 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[Application of bioinspired optimization methods for implementation of cryptanalysis of classical symmetric and asymmetric cryptosystems.] Sistemnyy analiz v proek- tirovanii i upravlenii : sb. nauch. tr. 16-y Mezhdunar. nauch.-prakt. konf. [System analysis in design and management: Coll.of sci.papers 16th Int. Sci.-Pract. Conf.] St. Petersburg: Polytechnic University Publ. House, 2012, pp. 112–122 (in Russian). 12. Kureichik, V.V., Zhilenkov, M.A. Pchelinyy algoritm dlya resheniya optimizatsionnykh zadach s yavno vyra- zhennoy tselevoy funktsiey. 2. Kureichik, V.V., Zaruba, D.V., Zaporozhets, D.Y. Algoritm parametricheskoy optimizatsii na osnove modeli povedeniya roya svetlyachkov. [Parametric optimization algorithm based on the model of glowworm swarm behavior] Izvesti- ya SFedU. Engineering Sciences. 2015, no. 6 (167), pp. 615 (in Russian). Сдана в редакцию 25.09.2016 Поступила в редакцию 25.09.2016 References 1. Chernyshev, Y.O., et al. Kriptograficheskie metody i geneticheskie algoritmy resheniya zadach kriptoanaliza. [Cryptographic techniques and genetic algorithms for solving cryptanalysis problems.] Krasnodar: FVAS, 2013, 138 p. (in Russian). Вестник Донского государственного технического университета 2017, №1(88), 144-159 2. Kureichik, V.V., Zaruba, D.V., Zaporozhets, D.Y. Algoritm parametricheskoy optimizatsii na osnove modeli povedeniya roya svetlyachkov. [Parametric optimization algorithm based on the model of glowworm swarm behavior] Izvesti- ya SFedU. Engineering Sciences. 2015, no. 6 (167), pp. 615 (in Russian). 3. Chernyshev, Y.O., et al. Bioinspirirovannye algoritmy resheniya zadach kriptoanaliza klassicheskikh i asimmet- richnykh kriptosistem. [Bioinspired algorithms for solving cryptanalysis problems of classic and asymmetric cryptosystems.] Krasnodar higher military school named after army general S. M. Shtemenko, 2015, 132 p. (in Russian). 4. Chernyshev, Y.O., et al. Issledovanie vozmozhnosti primeneniya geneticheskikh algoritmov dlya realizatsii kripto- analiza blochnykh kriptosistem. [Feasibility study of genetic algorithms application for implementation of block cryptosystem cryptanalysis.] Vestnik of DSTU, 2015, no. 3 (82), pp. 65–72 (in Russian). 5. Chernyshev, Y.O., et al. Issledovanie vozmozhnosti primeneniya metodov evolyutsionnoy optimizatsii dlya reali- zatsii kriptoanaliza blochnykh metodov shifrovaniya. [Research of possibility of application of evolutionary optimization methods for realization of cryptanalysis of enciphering block methods.] Izvestiya SPbGETU “LETI”, 2015, no. 10, pp. 32–40 (in Russian). 6. Lebedev, B.K., Lebedev, O.B. Modeli adaptivnogo povedeniya kolonii pchel dlya resheniya zadach na grafakh. [Modeling of an ant colony adaptive behavior by search of the decisions interpreted by trees.] Izvestiya SFedU. Engineering Sciences. 2012, no. 7, pp. 4249 (in Russian). 6. Lebedev, B.K., Lebedev, O.B. Modeli adaptivnogo povedeniya kolonii pchel dlya resheniya zadach na grafakh. [Modeling of an ant colony adaptive behavior by search of the decisions interpreted by trees.] Izvestiya SFedU. Engineering Sciences. 2012, no. 7, pp. 4249 (in Russian). 7. Lebedev, O.B. Trassirovka v kanale metodom murav'inoy kolonii. [Chanel routing bases on method of ant colony optimization.] Izvestiya SFedU. Engineering Sciences. 2009, no. 4, pp. 4652 (in Russian). 7. Lebedev, O.B. Trassirovka v kanale metodom murav'inoy kolonii. [Chanel routing bases on method of ant colony optimization.] Izvestiya SFedU. Engineering Sciences. 2009, no. 4, pp. 4652 (in Russian). 8. Chernyshev, Y.O., et al. Issledovanie vozmozhnosti primeneniya bionicheskikh metodov pchelinykh koloniy dlya realizatsii kriptoanaliza klassicheskikh shifrov perestanovok. [Research on applicability of bionic techniques of bee colonies for implementation of classical transposition cipher cryptanalysis.] Vestnik of DSTU, 2014, vol. 14, no. 1 (76), pp. 62–75 (in Russian). 9. Chernyshev, Y.O., Sergeev, A.S., Dubrov, E.O. References [Bee algorithms for solving optimization problems with the explicit objective function.] Informa- tika, vychislitel'naya tekhnika i inzhenernoe obrazovanie, 2015, no. 1 (21), pp. 1–8 (in Russian). 13. Sergeev, A.S. Parallel'noe programmirovanie. [Concurrent programming.] Rostov-on-Don: DSTU Publ. Centre, 2002,77 p. (in Russian). 14. Aho, A.V., Hopkroft, J.E., Ullman, J.D. Struktury dannykh i algoritmy. [Data Structures and Algorithms. ] Moscow: Williams, 2003, 384 p. (in Russian). 15. Chernyshev, Y.O., et al. Primenenie bioinspirirovannykh metodov optimizatsii dlya realizatsii kriptoanaliza blochnykh metodov shifrovaniya. [Application of bioinspired optimization methods for implementation of cryptanalysis block encryption methods.] Rostov-on-Don: DSTU Publ. Centre, 2016, 177 p. (in Russian). 16. Kapustin, S.A., et al. Primenenie metodov evolyutsionnoy optimizatsii dlya realizatsii kriptoanaliza blochnogo metoda shifrovaniya AES. [Application of evolutionary optimization methods for implementation of cryptanalysis of the block cryptography technique AES.] Izvestiya SPbGETU “LETI”, 2016, no. 8, pp. 25–40 (in Russian). 16. Kapustin, S.A., et al. Primenenie metodov evolyutsionnoy optimizatsii dlya realizatsii kriptoanaliza blochnogo metoda shifrovaniya AES. [Application of evolutionary optimization methods for implementation of cryptanalysis of the block cryptography technique AES.] Izvestiya SPbGETU “LETI”, 2016, no. 8, pp. 25–40 (in Russian). Поступила в редакцию 25.09.2016 Сдана в редакцию 25.09.2016 Запланирована в номер 11.01.2017 Received 25.09.2016 Submitted 25.09.2016 Scheduled in the issue 11.01.2017 Received 25.09.2016 Submitted 25.09.2016 Scheduled in the issue 11.01.2017 Received 25.09.2016 Submitted 25.09.2016 Scheduled in the issue 11.01.2017 Поступила в редакцию 25.09.2016 Сдана в редакцию 25.09.2016 Запланирована в номер 11.01.2017 Запланирована в номер 11.01.2017 Чернышев Ю. О. и др. Разработка и исследование параллельной модели алгоритмов пчелиных колоний Authors: Chernyshev, Yury O., professor of the Production Automa- tion Department, Don State Technical University (RF, Ros- tov-on-Don, Gagarin sq., 1), Dr. Sci. (Eng.), professor, ORCID: http://orcid.org/ 0000-0002-4901-1101, myvnn@list.ru Чернышев Юрий Олегович, профессор кафедры «Авто- матизация производственных процессов» Донского госу- дарственного технического университета (РФ, 344000, г. Ростов-на-Дону, пл. Гагарина, 1), доктор технических наук, профессор, ORCID: http://orcid.org/ 0000-0002-4901- 1101, myvnn@list.ru Chernyshev, Yury O., professor of the Production Automa- tion Department, Don State Technical University (RF, Ros- tov-on-Don, Gagarin sq., 1), Dr. Sci. (Eng.), professor, ORCID: http://orcid.org/ 0000-0002-4901-1101, myvnn@list.ru Чернышев Юрий Олегович, профессор кафедры «Авто- матизация производственных процессов» Донского госу- дарственного технического университета (РФ, 344000, г. Ростов-на-Дону, пл. Гагарина, 1), доктор технических наук, профессор, ORCID: http://orcid.org/ 0000-0002-4901- 1101, myvnn@list.ru Sergeev, Alexander S., postdoctoral student, Don State Technical University (RF, 344000, Rostov-on-Don, Gagarin sq., 1) Cand. Sci. (Eng.), ORCID: http://orcid.org/0000-0001- 6634-2722, sergeev00765@mail.ru Сергеев Александр Сергеевич, докторант Донского государственного технического университета (РФ, 344000, г. Ростов-на-Дону, пл. Гагарина, 1), кандидат технических наук, ORCID: http://orcid.org/0000-0001- 6634-2722, sergeev00765@mail.ru Сергеев Александр Сергеевич, докторант Донского государственного технического университета (РФ, 344000, г. Ростов-на-Дону, пл. Гагарина, 1), кандидат технических наук, ORCID: http://orcid.org/0000-0001- 6634-2722, sergeev00765@mail.ru Ryazanov, Alexander N., assistant general director, “711 Voenproekt” JSC (RF, Rostov-on-Don, M. Nagibina Prospekt, 28), ORCID: http://orcid.org/0000-0002-5471-4477, alexandr_r89@mail.ru Рязанов Александр Николаевич, помощник генераль- ного директора, Открытого акционерного общества «711 Военпроект» (РФ, 344038, г. Ростов-на-Дону, пр. М. Нагибина, 28), ORCID: http://orcid.org/0000-0002- 5471-4477, alexandr_r89@mail.ru Дубров Евгений Олегович, инженер, Федерального государственного унитарного предприятия «Ростов- ский-на-Дону научно-исследовательский институт радио- связи» (РФ, 344038, г. Ростов-на-Дону, ул. Нансена 130), ORCID: http://orcid.org/0000-0001-8866-4001, dubrov@spark-mail.ru Dubrov, Evgeny O., engineer, Rostov Scientific Research Institute for Radiocommunication (RF, Rostov-on-Don, Nan- sen St., 130), ORCID: http://orcid.org/0000-0001-8866-4001, dubrov@spark-mail.ru
https://openalex.org/W2969791206	https://sajbm.org/index.php/sajbm/article/download/967/907	English	null	Developing and implementing communications strategies: A descriptive model	South African journal of business management	1,988	cc-by	5,000	Loma A. Hill and A.A. Archer* University of Stellenbosch Business School, P.O. Box 610, Bellville, 7535 Republic of South Africa Received 2 July 1987 Received 2 July 1987 The world-vi.ide environment. but particularly the South African environment today has created _a growi_ng ne~d for businesses to communicate effectively with their stakeholders. The number of stakeholders mteractmg with companies has burgeoned. their demands have become greater and th~ need for change is ~ritical. As _t~~se pressures increase. the need for companies to communicate effectively will grow._ In So_u~h Africa the poss1~1hty for misunderstanding is compounded by communication barriers such ~s the soc10-po!1t1cal system. d1, as_ity of cultures and languages. as well as the many different levels of educatmn. In an environment such as this ~he need for effective communication is so great that a company can clearly no longer rely on ad hoc. reactive communications to facilitate the achievement of corporate goals. In order to ensure effective communication a company needs to have an o,·erall communications strategy which has its roots in corpora~e goa)s and strategies. Developing and implementing communications strategies involves many complex considerations s~ch as the identification and anal,·sis of stakeholders and their power bases. issues management. corporate image and culture. crisis communications planning. media planning and dealing with communication barriers. This article provides a suggested framework for integrating these and other considerations into the development and implementation of communications strategies. Die wereldwye omgewing. maar veral die omgewing in Suid-Afrika vandag bet ·n groeiende behoefte geskep vir ondernemings om doeltreffend met hut belangegroepe te kommunikeer. Die behoefte vir vcrandering is noodsaaklik en ondememings bevind hulself in wisselwerking met ·n toenemende aantal bdangegroepc wat al hoe groter eise aan hulle stet. Soos hierdie druk toeneem. sat die behoefte vir ondernemings om doeltreffend te kommunikeer groei. In Suid-Afrika word die moontlikheid van misverstand deur kommunikasiehindernisse soos die volgende vergroot: die sosio-politieke stelsel. verskeidenheid van kultuurgroepe en tale asook die verskillende vlakke van opvoeding. In 'n omgewing soos hierdie is die behoefte aan doeltreffende kommunikasie so groot dat ·n onderneming duidelik nie meer op ad hoc. reaktiewe kommunikasie kan staatmaak om die bereiking van maatskappydoelstellings te vergemaklik nie. Om docltreffendc kommunikasie te verseker. moet 'n ondememing 'n algemene kommunikasiestrategie he met maatskappydoelstellings en -strategiee as grondslag. Die ontwikkeling en implementering van kommunikasiestrategiee behels baie ingewikkelde oorwegings soos die identifisering en ontleding van belangegroepe en hul magsbasisse. hantering van vraagpunte. maatskappybeeld en -kultuur. beplanning van krisiskommunikasie. mediabeplanning en die bantering van kommunikasiehindernisse. Hierdie artikel. Loma A. Hill and A.A. Archer* University of Stellenbosch Business School, P.O. Box 610, Bellville, 7535 Republic of South Africa bied 'n voorgestelde raamwerk om hierdie en ander oorwegings te integreer in die ontwikkeling en implementering van kommunikasiestrategiee. • To whom COITespondence should be addressed S.-Afr.Tydskr.Bedryfsl.1988, 19( Developing and implementing communications strategies: A descriptive model S.-Afr.Tydskr.Bedryfsl.1988, 19(1) 26 Loma A. Hill and A.A. Archer* University of Stellenbosch Business School, P.O. Box 610, Bellville, 7535 Republic of South Africa The need for communications strategies Communication is the lubricant that can make it possible for these changes to take place in an atmosphere of understanding. If these non-commercial areas of business communication, i.e. communication activity not directly related to promoting the sale of a company's products or services, are neglected society will tend to react. And if the reaction is one of protest it can be seriously disruptive to the income-generating ability of a company. A positive image is important in recruiting employees, cultivating employee commitment and morale and achieving success in the marketplace. Many different messages that in sum result in the corporate image are communicated through many sources and channels. A business communicates directly and indirectly with its stakeholders on various issues. Direct (verbal) communication includes advertising, promotion campaigns, speeches, press releases, annual reports, meetings, etc. A business also, however, communicates indirectly (non-verbally) through its actions. An overall communications strategy enables the various departments of a company to develop a cohesive position and speak with one voice to its many audiences on issues that are dealt with publicly. It also ensures that communications accurately reflect corporate actions and achievements. Communicating effectively with stakeholders is a problem world-wide, but in South Africa the situation is compounded by barriers such as the socio-political system, diversity of cultures and languages, as well as the many different levels of education and cultural development. These barriers increase the possiblity for misunderstanding and conflict. Unless there is an overall communications strategy, it is likely that some of the messages (verbal and non- verbal) will not reinforce one another and others may be contradictory. All of this can result in a blurred or negative image which can seriously undermine the credibility of the l:Ompany. For instance, if a company claims to have the interests of the community at heart in its social advertising campaign, but is guilty of polluting the environment, its credibility will be jeapordized. Communicated reputation should not be a fabricated facade or superficial image. The Human Sciences Research Council reported in its Investigation into Intergroup Relations (1985) that ineffective communication and often even a total lack of communication was one of the major factors causing conflict to escalate into violence in South Africa. The need for communications strategies The human climate has become a determining factor in the future of all organizations. Businesses are having to deal to an increasing degree with stakeholder perceptions. This trend has magnified the role of communication in business enterprises. As the pressure from stakeholders increases, effective communication will become increasingly necessary for the survival and success of a business. While this is true world-wide, it is particularly pertinent in the rapidly changing South African environment of today. Little conceptual discussion or empirical research has, however, been done in South Africa on this topic. The environment in South Africa today has created a growing need for businesses to communicate effectively with their publics. The number of publics or stakeholders interacting with companies has burgeoned and their relative power and sophistication have increased dramatically. Business is having to deal to an increasing degree with issues such as black resistance to the free enterprise ethic, militance in labour relations, disinvestment. sanctions and organized consumerism and environmentalism. Business success depends to a great extent on whether stakeholders can be persuaded to support corporate goals rather than obstruct or actively oppose them. Communication can therefore play an important role in facilitating the achievement of corporate goals. This article presents a descriptive model for de- veloping and implementing communications strategies. The three-fold purpose of the model presented in this article is The three-fold purpose of the model presented in this article is Furthermore, the demands made by these various groups upon the business are many and varied. including the request to assist with social problems such as education, housing, urban renewal and development, and equal opportunity. This social role of business is closely linked with its economic mission. The more aware a company is of social forces and the more it uses its influence to push for positive change, the better the • provide management with guidelines for using communication to facilitate the achievement of corporate goals and gain a competitive advantage; • promote strategic thinking in the field of communication; • indicate how stakeholder information can be integrated into communications strategies. S.Afr.J.Bus.Mgmt.1988,19(1) 27 general business climate will be in the long run. general business climate will be in the long run. Image The demands by stakeholders for business to play an active role in initiating social and political change in South Africa have never been greater. Identity An overall communications strategy helps to give a company a clear and distinctive identity. This is a source and means of competitive differentiation in strategy development. Identity is also the source of corporate culture. Peters & Waterman ( 1982) identified a distinct corporate culture as one of the features that successful companies have in common. Reactive communication is less effective than communication that is used as a strategic tool. When a business anticipates events and communicates with its stakeholders before a crisis arises, it will have more control over its communications than when it uses communication to react under pressure, i.e. as a tactical weapon. The need for communications strategies In an environment such as this, the need for effective communication has become so great that a company can clearly no longer rely on ad hoc communications with its various stakeholders on issues affecting business. In order to ensure effective communication a company needs to have an overall communications strategy that organizes and coordinates what, when, how, and if the company communicates. These are important business decisions which necessarily tie communication with strategic management. Effective communication should have its roots in corporate goals, objectives and strategies. It is therefore essential that the verbal and non-verbal messages emanating from the business be carefully coordinated into an overall communications strategy. Stakeholders An important phase in the development of communications strategies is the identification of stakeholders. This enables the company to identify those audiences that may be important to its business in the future. By regularly monitoring the opinion of stakeholders the company will be alerted to emerging issues and therefore be in a position to respond to those issues that may affect it and to develop timely and effective strategies before the problems become crises. Effective communication is necessarily a two-way street. It involves listening, seeing and observing as well as transmitting information. The failure of a business to understand the nature of the human climate in which it operates will create a situation in which much of what it communicates is irrelevant or unintelligible to the target audiences concerned. The relative deafness and blindness of a business also means that it is out of touch with social and political realities. Scanning the stakeholder environment is therefore an important phase in the development of communications strategies. Communications network .For those crises that do occur, the company will have an organized communications network of people who are familiar with the process of developing communications strategies and who are ready to respond as a team. Advantages of communications strategies An overall communications strategy can work for a company in many ways. S.-Afr. Tydskr. Bedryfsl.1988,19(1) 28 typical stakeholders of a company are the shareholders, employees, customers, suppliers, government, the general public, the financial community and the media. Stakeholders are therefore the judges of organizational effectiveness. In order to be effective a company first has to know who will be judging its effectiveness. In other words, stakeholders must be identified. The model The various components of the model depicted in Figure 1 will now be discussed. Identifying stakeholders The first step in developing communications strategies is identifying the potential audiences with whom the company may need to communicate. This will include all individuals and groups who influence or may be influenced by the actions of the business, i.e. the stakeholders. Given that management has a clear understanding of corporate goals, objectives, strategies and their scope, identifying the individuals and groups with which a company should communicate is largely a question of determining who is in a position to significantly help or hinder the achievement of those goals - in the long as well as in the short term. The extent to which a company is able to meet the demands of stakeholders is termed effectiveness. Hofer & Schendel (1978:2) define effectiveness as the ratio of outputs produced by an organization to the societal outputs desired from the organization. Clearly the societal outputs desired from the organization can only be judged by those groups or individuals who come into contact with it, i.e. the company's stakeholders. The Stakeholder analysis The second step in the development of communications strategies requires an analysis of the stakeholder Identify potential crises .. Identify inclivi- -:J duals, groups .I., influenced Identify isue11 Corporate goals, atrategles 1111d actions ·~ ,I, AnalJle Identify atratep: lltakeholders H important to H issues stalteholdera Identify inclivi- ~ duals, groups who ~ ,I\ -1 can influence ,Ji company Formulate corpo, Formulate corpo, Formulate~ formulate func- rate communica- H rate communica- i.., communic:a- ~ tional communica- tions goals tions strategies tions goals tion1 strategies J, l J. .I. fonnul ation formulate corpo- Formulate corpo, fonnulate func- Fonnulate func- rate communica- rate communica- tional communca- tional communica· ~ tions goals for H tiona strategies for H tions Fall for H tiona 1trateaY for each stakeholder each stakeholder each stakeholder each stakeholder gn,up gn,up gn,up gn,up ,, Determine media ~ mplemen , ltrategy ' tation I Eval Figure 1 Model of communications strategies Identify potential crises Corporate goals, atrategles 1111d actions Identify inclivi- duals, groups influenced Identify isue11 important to stalteholdera Identify atratep: issues AnalJle lltakeholders Identify inclivi- duals, groups who can influence company Eval Figure 1 Model of communications strategies S.Afr.J.Bus.Mgmt.1988, 19(1) 29 environment or human climate within which the business operates. compatible with stakeholders' value systems. • Cultural factors, for example cultural composition, behaviour patterns, habits, language, etc. An awareness of the relationship between culture and communication as well as an understanding of the differences between cultures is helpful - and at times essential - in communicating successfully, especially in the multicultural South African environment. Lack of intercultural communication skills is probably one of the major reasons for ineffective communication between South African businesses and their stakeholders. In our complex cultural environment it is essential that cultural sensitivity and empathy be cultivated. Culture comprises many elements: language, semantics, religion, values and attitudes towards time, achievement, wealth, change and private enterprise all influence how a company's general actions and communication efforts are interpreted. Every time one of these cultural barriers must be crossed there is a potential communication problem. The consequences of environmental threats and opportunities are largely determined not by a situation per se , but by how people perceive a situation and how they respond to it. Although managers are continually being confronted by issues such as boycotts, product safety, personnel recruitment, retrenchment and productivity, many of these are problems of human attitude rather than material, tangible practicalities. Stakeholder analysis The fact is that all major strategic decisions have implications for stakeholders. The bigger the decision, the larger the company and the greater the number of stakeholders that are affected, the more significant the communi- cations component becomes. In formulating communications strategies, stake- holder analysis helps management to understand its audiences and to consider how they will interpret company communications. Without such analysis communications decisions will be made in a vacuum; the message may not be relevant to the interests of the stakeholders, it may not reach them or it may be misinterpreted by them. • Micropolitical environment of the stakeholder group, for example how decisions are made, the basis and location of power. Macmillan (1978) identifies the following sources of power: Stakeholders should be analysed on an on-going basis and not only when problems occur. A company should not wait until the opinions of stakeholders are suddenly crucial before undertaking attitude research. It may then be too late. Regular attitude research enables a company to detect potential threats before they become crises. The company can then engage in opinion-forming communications to prevent the crisis from occurring. (i) Possession of resources. This power arises from the fact that the stakeholders may refrain from supplying the company with much needed resources. Skilled workers who resort to strike action are in a powerful position to assert their demands for pay increases if the company cannot employ replacement workers. (ii) Dictation of alternatives (substitutes). A sole supplier, for example, only has a strong power base as long as no substitute materials exist. Formal stakeholder analysis takes the formulation of communications strategies out of the realms of assumptions and guesswork and provides results which serve as a benchmark for evaluating the effectiveness of communications strategies. It provides management with a more tangible form of data. This data will have to be gathered from a wide variety of sources - from management, independent appraisers and the stakeholders themselves. (iii) Authority. Authority is the right to enforce obedience. The power bases of government and regulatory bodies are derived from authority. (iv) Influence. An ecological body could for instance influence politicians to use their power, rooted in authority, to pass anti-polution legislation. • Goals, objectives and strategies of stakeholder groups. Some stakeholder groups are so amorphous that they do not have cohesive group goals or strategies. Identifying strategic issues Essential stakeholders of the company, i.e. groups or individuals who are irreplacable, should receive special attention. These include sole suppliers of goods or services for which there are no substitutes, key employees and major customers. Once issues that are important to stakeholders have been detected, they must be evaluated in terms of the following dimensions in order to prioritize them: • Impact on the company and industry in the short term and the long term • Impact on the company and industry in the short term and the long term A company should also pay particular attention to stakeholders who occupy judicial positions, for example government departments and regulatory hodies. Failure to do this may result in legislative action. • Probability of occurrence • Probable time of impact • Probable behaviour of stakeholders The purpose of this exercise is to narrow down the number of issues so that the company can concentrate its communications effort on those issues which, if handled correctly, can enhance the achievement of the company's strategic objectives. It is also important to identify special interest groups such as environmentalists and consumer associations who have a high degree of exposure and are capable of imposing legislation via government departments. This allocation of priorities will be influenced by the kind of business a company is in. For example, a manufacturer of stoves, refrigerators and television sets will have consumer protection groups high on his priority list, while environmental protection groups will be high on the list of a manufacturer of chemicals or fertilizers. Stakeholder analysis may also reveal a communication gap between a company and an important stakeholder group which would indicate that this group should receive special attention. This allocation of priorities will be influenced by the kind of business a company is in. For example, a manufacturer of stoves, refrigerators and television sets will have consumer protection groups high on his priority list, while environmental protection groups will be high on the list of a manufacturer of chemicals or fertilizers. These so-called strategic issues are forthcoming developments, either inside or outside the company, which are likely to have an important impact on the ability of the enterprise to meet its objectives. A strategic issue may be a welcome issue, i.e. an opportunity to be grasped in the environment or an internal strength which can be exploited to advantage. Identifying strategic issues It can also be an unwelcome external threat, or an internal weakness which imperils continuing success, and even the survival of the enterprise (Ansoff, 1980:133). Stakeholder analysis may also reveal a communication gap between a company and an important stakeholder group which would indicate that this group should receive special attention. Corporate goals, objectives and strategies clearly play a major role in the establishment of strategic issues. An effective linkage between communications on these issues and strategic planning is essential to both the success of broader corporate strategies and communications strategies. Strategic issues should be an integral part of the process of developing communications strategies to ensure that stakeholder issues are adequately addressed and that the achievement of strategic goals is not jeapordized by any form of social censure. Table 1 Impact of stakeholders on company success ~ c ... c <I) ~ ~ <I) -0 <I) ... ~ E .; ·2 0 <I) <I) ... .; ;,,-, E -~ C ·;; :I .c 0 ... ... .:,! E <I) C. B 0.. <I) <I) C ... ;,. C j:i ~ E ~ E ~ 0.. 0 <I) C .c ::I ::I 0 0 :::, l.i: 0 Cl) UJ u Cl) 0.. <.) Financial returns I 2 3 3 3 3 Truth in advertising 3 2 2 2 2 3 Environmental pollu- tion 2 2 2 3 1 3 Product service 3 2 2 3 3 3 Remuneration 2 3 3 2 2 3 Creation of jobs 3 3 3 2 3 I = Critically important to stakeholders; 2 Important to stakeholders; 3 = Unimportartant to stakeholders stakeholders, the more choices it has in responding to the issue and the greater probability it has of influencing the outcome of the issue in its favour by means of opinion-forming communication. company's resources are, however, finite and management must establish priorities in terms of those with which it will communicate. This information can be displayed in grid form. Table 1 is an example of how this grid may look. Stakeholder analysis Where group goals can be identified it is useful to analyse them in terms of (I) what the stakeholder group is trying to accomplish in the long term, (2) what the stakeholder group is trying to accomplish as regards a certain issue, and (3) what the linkage is between a certain issue and the stakeholder's long-term goals. Stakeholders should not be regarded as one homogenous mass. Stakeholder analysis will reveal that they differ in terms of the issues which are important to them and how they interpret what the company communicates. In order to better understand stakeholders they should be analysed according to the following dimensions: • Opinions and attitudes. By regularly measuring the attitudes, perceptions and opinions of stakeholders, issues important to them and to the company can be identified. If the company decides to speak out on these issues, they will form the content of the company's communications. It is important that messages transmitted by a company have relevance for the audience at which they are aimed. • Company information required by stakeholders. What do stakeholders want to know about the company? What information is relevant to their needs? What are their horizons of interests? This information is important for determining the content of corporate communi- cations. • Social values. Social values influence the attitudes and opinions of people. They direct people to selectively attend to some goals and to subordinate others. It is therefore worth the trouble to investigate whether the people with whom the company deals have different priorities. Messages transmitted by a company must be Once stakeholders have been analysed in terms of these dimensions, they must be ranked according to their potential impact on the company. Given unlimited communication resources, the audiences with whom a company could profitably communicate is extensive. A S.-Afr.Tydskr.Bedryfsl.1988,19(1) 30 Table 2 Stakeholders/issues matrix Table 2 Stakeholders/issues matrix Table 1 Impact of stakeholders on company success Table 1 Impact of stakeholders on company success Very Not Vital important Important Helpful applicable Shareholders Employees 1,2 Customers 1,2,3 Suppliers 3 Government 3 General public Financial community Media Trade unions 3 1,2 2 2 2,3 1,2 1,2 1,2,3 J = Achievement of corporate goals (objectives) in the short term 2 = Achievement of corporate goals (objectives) over the next five to ten years 3 = The climate of opinion towards private enterprise Evaluation A company should also avoid being over-selective in the information it divulges. The credibility of the company will be enhanced if it has the reputation of giving a complete account in both good times and bad, i.e. if it has an open communications climate with stakeholders. This is the final phase of the model. Periodic evaluation of communications strategies is essential as no communications strategy is permanent. As a company's strategies change and as the internal and external stakeholder environments change. so may the communications goals, objectives and strategies. It is possible that the communications strategies may remain the same, but that the communication media may need to change. Identifying issues important to stakeholders The monitoring of stakeholders described in the previous section is likely to reveal a number of emerging issues which could impact on the company. These issues will relate to such aspects as profits, product safety, service, truth in advertising and promotion. A list of key concerns or issues must be developed for each stakeholder group. This information can be displayed in a matrix of stakeholders versus issues. Table 2 is an example of how this matrix may look. The sooner a company can detect an issue that is important to A scheme which can aid in the identification of 31 Table 3 Probability/Impact grid Probability of occurrence Impact High Medium High Issue Issue Issue Issue Issue Issue Issue Issue Issue Medium Issue Issue Issue Issue Issue Issue Issue Issue Low Issue Issue Issue Issue Issue Low Issue Issue Table 3 Probability/Impact grid strategy begins with identifying potential crises. These would include existing situations or problems which have the potential to develop into crises; previous crises that may recur; and crises that have beset similar companies. Once potential crises have been identified, the next step is to determine who the target audiences of crisis communications will be. This will include employees who are directly involved in handling the crisis, employees who are affected by it, as well as external stakeholders who may be directly or indirectly affected by the crisis. The potential reactions of these stakeholders should receive attention during stakeholder analysis. strategy begins with identifying potential crises. These would include existing situations or problems which have the potential to develop into crises; previous crises that may recur; and crises that have beset similar companies. Once potential crises have been identified, the next step is to determine who the target audiences of crisis communications will be. This will include employees who are directly involved in handling the crisis, employees who are affected by it, as well as external stakeholders who may be directly or indirectly affected by the crisis. The potential reactions of these stakeholders should receive attention during stakeholder analysis. Formulation Once management has decided on which strategic issues it needs to communicate with stakeholders, specific communications goals and stategies must be formulated. This provides the framework for all corporate communications. Communications goals and strategies can be formulated at three levels, namely corporate, functional and stakeholder. Communications strategies should be consistent with each other and with the overall corporate strategy in order to enhance credibility. strategic issues is the probability/impact grid (Johnson, 1983:26). The grid, illustrated in Table 3, evaluates issues which could have a significant impact on the company according to the probability of occurrence and level of impact. The high probability/high impact issues are the strakgic issues which should be given top priority in the development of communications strategies. Once strategic issues have been identified the company must decide on which of these it needs to communicate with stakeholders. This decision is based on a number of important considerations: Communication~ goals must be written, realistic, measurable and set in a time frame so that progress can be monitored and evaluated. • What are the pros and cons of speaking out on a certain issue? Implementation Once the communications components of strategic issues have been formalized into communications goals and strategies, management must decide how to put these strategies into practice. • Do the actions and strategy of the company support what it has to say publicly • What are the long-term implications of speaking out publicly? An important aspect of implementing communications strategies is deciding on what mix of communication techniques and media to use. Factors such as the size, diversity and nature of the target audience, the message to be communicated and the funds available play a role in this regard. Although the media decisions are usually left to communications specialists, management should be aware of the relative strengths and weaknesses of the media available. • How will the company respond if it decides not to speak out publicly? A company cannot afford to speak out on too many issues. Its expertise and resources would not be sufficient. It may be unwise for a company to take a public stand on issues that are very controversial or volatile and pose a no-win situation. A company, however, communicates by default just as surely as by design, and if left to chance the wrong message may get across to stakeholders. Identifying potential crises Crises, by definition, cannot be planned. However, a company can plan its response to a crisis. The consequences of not planning crisis communications can be damaging to all aspects of a company. Many companies have had to contend with image problems years after a crisis, simply because of their lack of crisis communications planning. Research should be carried out at given intervals and measured against the benchmark research done prior to implementing the communications strategy in order to monitor the success of the strategy in terms of the communications goals. At these evaluation points new knowledge or information may shape new goals or reconfirm the original strategy. A coherent approach to crisis communications S.-Afr.Tydskr.Bedryfsl. 1988, 19(1) 32 Conclusion Ansoff. H.J. 1980. Strategic issue management. Strat. Manage.}., vol. 1, 131-148. The need for effective communication in the complex rapidly changing South African environment of today is great, and will increase as stakeholder pressure increases. Unless a company communicates effectively with its stakeholders, its success will be seriously jeapordized in the future. Hofer, C.W. & Schendel, D. 1978. Strategy formulation: analytical concepts. St. Paul: West, 219p. Human Sciences Research Council. 1985. Investigation into Intergroup Relations. Pretoria: Human Sciences Research Council. 182p. Effective communication is, however, not easy to achieve - it involves many complex considerations. The framework suggested in this article is an attempt to integrate these considerations into a model for developing and implementing communications strategies. Johnson, J. 1983. Issue management - what are the issues? Bus. Quart., vol. 48, no. 3, 22-31. Macmillan, J.C. 1978. Strategy formulation: political concepts. St. Paul: West, 167p. St. Paul: West, 167p. Peters, T.J. & Waterman, R.H. 1982. In search of excellence. Peters, T.J. & Waterman, R.H. 1982. In search of excellence. New York: Harper & Row. 360p. New York: Harper & Row. 360p.
https://openalex.org/W2016388862	https://europepmc.org/articles/pmc2267794?pdf=render	English	null	Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle	BMC structural biology	2,008	cc-by	11,662	BioMed Central BioMed Central p Research article Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle Gerhard Schenk, Tristan W Elliott, Eleanor Leung, Lyle E Carrington, Nataša Mitić, Lawrence R Gahan and Luke W Guddat Address: School of Molecular and Microbial Sciences, The University of Queensland, St. Lucia, QLD 4072, Australia Email: Gerhard Schenk* - schenk@uq.edu.au; Tristan W Elliott - stranman84@hotmail.com; Eleanor Leung - e.leung@uq.edu.au; Lyle E Carrington - l.carrington@uq.edu.au; Nataša Mitić - n.mitic@uq.edu.au; Lawrence R Gahan - gahan@uq.edu.au; Luke W Guddat* - luke.guddat@uq.edu.au * Corresponding authors Received: 10 August 2007 Accepted: 31 January 2008 This article is available from: http://www.biomedcentral.com/1472-6807/8/6 © 2008 Schenk et al; licensee BioMed Central Ltd. © 2008 Schenk 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 Background: Purple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood. Results: Red kidney bean purple acid phosphatase is a heterovalent enzyme with an Fe(III)Zn(II) center in the active site. Two new structures with bound sulfate (2.4 Å) and fluoride (2.2 Å) provide insight into the pre-catalytic phase of its reaction cycle and phosphorolysis. The sulfate-bound structure illustrates the significance of an extensive hydrogen bonding network in the second coordination sphere in initial substrate binding and orientation prior to hydrolysis. Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic μ-hydroxide present in the metal-bridging position. The fluoride-bound structure provides visual support for an activation mechanism for this μ-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity. Conclusion: In combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general. for functionality but more complex multinuclear metal clusters also occur. Amongst metalloenzymes binuclear hydrolases form a diverse family with biological functions including signal transduction and cell cycle progression, nucleotide homeostasis and bone metabolism [2-9]. BMC Structural Biology Open Access The structure of the rkbPAP-sulfate complex: a model for a pre-catalytic complex In this study rkbPAP was co-crystallized with sulfate, and diffraction data were collected at a resolution (2.4 Å) which is better than that for any previously determined plant PAP structure [18,40,41]. The Fe(III) and Zn(II) in this structure are separated by an average distance of 3.1 Å (Table 1), similar to that reported for the other rkbPAP Schematic illustration of the active site of red kidney bean purple acid phosphatase (rkbPAP), a representative binuclear metallohydrolase Figure 1 Schematic illustration of the active site of red kidney bean purple acid phosphatase (rkbPAP), a representative binuclear metallohydrolase. In most (if not all) binuclear metallohydro- lases the binding affinities of the two metal centers vary, with M1 representing the tight binding site and M2 the lower affin- ity site [8]. In rkbPAP M1 and M2 are occupied by Fe(III) and Zn(II), respectively. Combined crystallographic and spectro- scopic data for PAPs indicate the presence of a bridging (hydr)oxo group and one terminal water ligand (see text). The presence of a terminal Fe(III)-bound hydroxide is cur- rently debated with spectroscopic data suggesting its absence [8], but the crystal structure of rat PAP supporting its pres- ence [8]. PAPs are active in the pH range between 3.0 and 8.0 and have been purified and characterized from a number of mammals and plants [21-28], and PAP-like genes have been identified in a limited number of microorganisms [29]. The animal enzymes are 35 kDa monomers with redox-active Fe(III)-Fe(II/III) centers where only the heter- ovalent form is catalytically active [30,31]. Proposed bio- logical roles include iron transport, the generation of reactive oxygen species and bone resorption [32]. The lat- ter has made the enzyme a target for the development of anti-osteoporotic drugs [32,33]. Plant PAPs are 110 kDa homodimers, containing Fe(III)-Zn(II) or Fe(III)-Mn(II) centers [21-23,34,35], and a recombinant isoform from sweet potato has been shown to contain a di-iron center [36]. http://www.biomedcentral.com/1472-6807/8/6 http://www.biomedcentral.com/1472-6807/8/6 BMC Structural Biology 2008, 8:6 Members of this group of enzymes have evolved into tar- gets for the development of chemotherapeutic agents. mammalian PAPs (human, pig, rat) have been deter- mined [42-45]. Despite low overall sequence homology between PAPs from animal and plant sources their active sites are remarkably conserved, with seven invariant metal ligands (Figure 1) [8,9,18,40-47]. Binuclear metallohydrolases employ variants of the same basic mechanism to catalyze esterolysis of a large number of substrates, in some cases under inversion of stereo- chemistry, and in others without [2,8,10-13]. In the latter (e.g. alkaline phosphatase [13]) a covalently modified enzyme intermediate is formed upon nucleophilic attack by a reactive amino acid residue. In the former a metal ion-bound water ligand is the proposed nucleophile, but its precise identity has been subject to debate and may vary in different enzymes [2-9,12,14-18]. The majority of binuclear metallohydrolases require the presence of two metal ions for reactivity, although their precise roles in catalysis and/or substrate or product binding have also remained conjectural [2-9,12,14-18]. Note also that some members of this group of enzymes can operate with a sin- gle metal ion in the active site (e.g. the amino peptidase from Aeromonas proteolytica [19], the methionyl ami- nopetidase from Escherichia coli [19] or the metallo-β- lactamase from Bacillus cereus [20]. The requirement for particular metal ions and the coordination environments of the metal ions may vary significantly amongst mem- bers of this group of enzymes with di-M(II) centers (where M = Zn, Mn, Ni, Co) being most prevalent. Heterovalent centers of the Fe(III)-M(II) form have also been observed in a group of enzymes termed purple acid phosphatases (PAPs) [2,8,9]. In this study the structures of the rkbPAP-sulfate and rkb- PAP-fluoride complexes were determined, providing insight into what we propose are two crucial steps in its catalytic mechanism, (i) the pre-catalytic stage, and (ii) the chemical step. In combination with previously reported structural, kinetic and spectroscopic data a com- prehensive model for the reaction mechanism involving eight steps is proposed. The structure of the rkbPAP-sulfate complex: a model for a pre-catalytic complex Proposed biological roles for plant PAPs include phosphate metabolism and the generation of reactive oxy- gen species [37]. The characteristic purple color is due to a charge transfer transition between a tyrosine side-chain and the Fe(III) [38,39]. Background g At least one-third of enzymes characterized require metal ions to function. Roles include electron transfer reactions, oxidations and a plethora of hydrolytic processes [1]. The majority of these enzymes require one or two metal ions Page 1 of 13 (page number not for citation purposes) Page 1 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/6 http://www.biomedcentral.com/1472-6807/8/6 BMC Structural Biology 2008, 8:6 structures [40,41]. Spherical electron density is observed 2.1 Å from Fe(III) and 2.3 Å from Zn(II) and therefore in a bridging position (Figure 2a). This group is also within 3.1 Å of the carbonyl oxygen of His323, indicating the for- mation of a hydrogen bond (Figure 3). We therefore assign this density to that of a μ-hydroxo group, an inter- pretation in agreement with a previous study involving measurements of the magnetization of rkbPAP under cat- alytically relevant conditions [48]. The Fe(III) is coordi- nated by a nitrogen atom from the side chain of His325, oxygen atoms from the side chains of the chromophoric Tyr167, Asp135, Asp164 (which bridges both metal ions), and the bridging hydroxide (Figure 2a). These five ligands coordinate the Fe(III) in a trigonal bipyramidal geometry, where Asp135, the bridging hydroxide and Tyr167 are coplanar with Fe(III), and Asp164 and His325 are in axial positions. Zn(II) is also pentacoordinate, ligated by the side-chain oxygen atoms of Asp164 and Asn201, the side- chain nitrogen atoms of His286 and His323 and the μ- hydroxide. Its geometry can be described as distorted square pyramidal (Figure 2a). Mechanistically, it is impor- tant to stress that the μ-hydroxide group is the only water molecule present in the active site, and given the location of this group the fitting of a sixth ligand to either metal is implausible (Figure 2b). The structure of the rkbPAP-fluoride complex: insight into the chemical step of the hydrolytic reaction Table 1: Some interatomic distances (Å). Atoms rkbPAP-sulfate rkbPAP-fluoride Fe(III) Zn(II) 3.1 3.5 Fe(III) OH- 2.1 - Zn(II) OH- 2.3 - Fe(III) F- - 2.5 Zn(II) F- - 2.1 OH- SO42- - O1 2.7 - OH- SO42- - O3 2.5 - F- Na+ - 2.6 Na+ SO42- - O1 - 2.4 Na+ SO42- - O3 - 2.6 His323 C = O OH 3.1 - His323 C = O F- - 3.6 Asn201 Nδ2 SO42- - O3 3.2 - His202 Nε2 SO42- - O1 3.0 2.9 His202 Nε2 SO42- - O2 3.1 - His295 Nε2 SO42- - O4 3.3 - His296 Nε2 SO42- - O3 2.9 - Table 1: Some interatomic distances (Å). The pre-catalytic phase of the reaction cycle p y p f y The rate of the oxidation of reduced pig PAP by [Fe(III)(CN)6]3- displays saturation behavior as the con- centration of the oxidant is increased [56]. The model pro- posed to explain the observed saturation behavior invokes an initial rapid formation of an enzyme-[Fe(III)(CN)6]3- complex, followed by a considerably slower catalytic step. This conclusion was further augmented by measurements of the rate of phosphate binding to the Fe(III) of pig PAP using stopped-flow spectroscopy, leading to the sugges- tion that the substrate is also likely to associate initially with the enzyme, forming a catalytically non-competent complex (pre-catalytic complex) [16,56]. It was specu- lated that interactions between conserved histidine resi- dues in the active site may be responsible for the formation of this pre-catalytic complex (His92 and His195 in pig PAP, corresponding to His202 and His296 http://www.biomedcentral.com/1472-6807/8/6 Atoms rkbPAP-sulfate rkbPAP-fluoride Fe(III) Zn(II) 3.1 3.5 Fe(III) OH- 2.1 - Zn(II) OH- 2.3 - Fe(III) F- - 2.5 Zn(II) F- - 2.1 OH- SO42- - O1 2.7 - OH- SO42- - O3 2.5 - F- Na+ - 2.6 Na+ SO42- - O1 - 2.4 Na+ SO42- - O3 - 2.6 His323 C = O OH 3.1 - His323 C = O F- - 3.6 Asn201 Nδ2 SO42- - O3 3.2 - His202 Nε2 SO42- - O1 3.0 2.9 His202 Nε2 SO42- - O2 3.1 - His295 Nε2 SO42- - O4 3.3 - His296 Nε2 SO42- - O3 2.9 - Table 1: Some interatomic distances (Å). small sphere of electron density is observed between the two metal ions (Figure 4), in a position similar, but not identical, to that of the μ-hydroxo in the rkbPAP-sulfate complex. The center of the bridging electron density in the rkbPAP-F structure is 2.5 Å and 2.1 Å from Fe(III) and Zn(II), respectively (Table 1). In contrast, the bridging electron density in rkbPAP-SO4 is, respectively, 2.1 Å and 2.3 Å from Fe(III) and Zn(II). Thus, the bridging ligand is shifted 0.2 Å closer to the Zn(II) and 0.4 Å further away from the Fe(III); its distance to the carbonyl oxygen of His323 is 3.6 Å, compared to 3.1 Å observed in the rkb- PAP-SO4 structure (Figure 2). These differences indicate that fluoride rather than μ-hydroxide acts as the bridging ligand, as anticipated from kinetic and spectroscopic data collected for PAPs from pig, human and red kidney bean [15,49,50], and other binuclear metallohydrolases [51]. Additional electron densities in the second coordination sphere, close to the fluoride, are tentatively assigned to a sodium ion and a sulfate ion (see Methods section). The sodium ion may offset the charge of the metal bridging fluoride group, and possibly prevents cations (i.e. sulfate, acetate) from binding to the second coordination sphere. structures [40,41]. Spherical electron density is observed 2.1 Å from Fe(III) and 2.3 Å from Zn(II) and therefore in a bridging position (Figure 2a). This group is also within 3.1 Å of the carbonyl oxygen of His323, indicating the for- mation of a hydrogen bond (Figure 3). We therefore assign this density to that of a μ-hydroxo group, an inter- pretation in agreement with a previous study involving measurements of the magnetization of rkbPAP under cat- alytically relevant conditions [48]. http://www.biomedcentral.com/1472-6807/8/6 The Fe(III) is coordi- nated by a nitrogen atom from the side chain of His325, oxygen atoms from the side chains of the chromophoric Tyr167, Asp135, Asp164 (which bridges both metal ions), and the bridging hydroxide (Figure 2a). These five ligands coordinate the Fe(III) in a trigonal bipyramidal geometry, where Asp135, the bridging hydroxide and Tyr167 are coplanar with Fe(III), and Asp164 and His325 are in axial positions. Zn(II) is also pentacoordinate, ligated by the side-chain oxygen atoms of Asp164 and Asn201, the side- chain nitrogen atoms of His286 and His323 and the μ- hydroxide. Its geometry can be described as distorted square pyramidal (Figure 2a). Mechanistically, it is impor- tant to stress that the μ-hydroxide group is the only water molecule present in the active site, and given the location of this group the fitting of a sixth ligand to either metal is implausible (Figure 2b). Discussion h d f The study of the reaction mechanism of PAP-catalyzed hydrolysis has received considerable attention over the past decade, with much of the focus directed towards the investigation of individual steps of the catalytic cycle. Var- ious stages of the mechanism have been subject to debate, none more so than the identity of the reaction-initiating nucleophile and the mode of substrate binding [2,3,8,12,14-18,28,35,49,50,52-55]. In this study, we pro- vide insight into these two points of contention by com- bining the obtained structural data with previously reported spectroscopic, kinetic and crystallographic infor- mation, and a comprehensive eight-step model for the reaction cycle is proposed. Schematic purple acid metallohyd Figure 1 Schematic illustration of the active site of red kidney bean purple acid phosphatase (rkbPAP), a representative binuclear metallohydrolase. In most (if not all) binuclear metallohydro- lases the binding affinities of the two metal centers vary, with M1 representing the tight binding site and M2 the lower affin- ity site [8]. In rkbPAP M1 and M2 are occupied by Fe(III) and Zn(II), respectively. Combined crystallographic and spectro- scopic data for PAPs indicate the presence of a bridging (hydr)oxo group and one terminal water ligand (see text). The presence of a terminal Fe(III)-bound hydroxide is cur- rently debated with spectroscopic data suggesting its absence [8], but the crystal structure of rat PAP supporting its pres- ence [8]. The crystal structure of the free red kidney bean PAP (rkb- PAP) and complexes with phosphate (both a reaction product and substrate analogue) and tungstate (an inhib- itor) are available [40,41]. In addition, structures of phos- phate-bound sweet potato PAP [18] and several Page 2 of 13 (page number not for citation purposes) Page 2 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/6 The structure of the rkbPAP-fluoride complex: insight into the chemical step of the hydrolytic reaction rkbPAP incubated with fluoride crystallized in a different space group and with different unit cell parameters com- pared to the sulfate complex (Table 2). Two dimers in the asymmetric unit are observed yielding four copies of the active site. In these structures the metal ions are on aver- age 3.5 Å apart, a distance significantly larger than that observed in any of the other rkbPAP structures [40,41]. A Page 3 of 13 (page number not for citation purposes) Page 3 of 13 (page number not for citation purposes) BMC Structural Biology 2008, 8:6 http://www.biomedcentral.com/1472-6807/8/6 BMC Structural Biology 2008, 8:6 http://www.biomedcentral.com/1472-6807/ (a) Stereodiagram of the active site of the rkbPAP-sulfate complex Figure 2 (a) Stereodiagram of the active site of the rkbPAP-sulfate complex. Fo-Fc electron density for the sulfate group is overlayed. The sulfate group is bound in the second coordination sphere via extensive hydrogen bonding interactions. General legend Fe(III) is in tan, Zn(II) in grey, carbon in green, oxygen in red, nitrogen in blue and sulphur in orange. Hydrogen bonds and other contacts are shown as dashed lines. (b) Stereodiagram of the superimposition of the active site of the rkbPAP-sulfate complex (green) with the active site of the rat PAP-sulfate complex [8] (magenta). In the rkbPAP-sulfate complex the bridg hydroxide adopts an elevated position, precluding the binding of a terminal water to Fe(III). Both metals in the rkbPAP-sulfa complex structure are five coordinate. aValues in parentheses are for the outer resolution shells. bRsym = Σh Σi\|Ih,i - <Ih>\|/Σh Σi Ih,i where Ih,i is the intensity of the ith measurement of reflection h and <Ih> is the average value over multiple measurements. cRfactor = Σ\|\|Fobs\|-\|Fcalc\|\|/Σ\|Fobs\|, where the R-factor is calculated based on the reflections used in the refinement (90% of the total data) and R-free is calculated using the remaining 10% of the data. dRMSD = root mean square deviation. (a) Stereod Figure 2 (a) Stereodiagram of the active site of the rkbPAP-sulfate complex Figure 2 (a) Stereodiagram of the active site of the rkbPAP-sulfate complex. Fo-Fc electron density for the sulfate group is overlayed. The sulfate group is bound in the second coordination sphere via extensive hydrogen bonding interactions. General legend: Fe(III) is in tan, Zn(II) in grey, carbon in green, oxygen in red, nitrogen in blue and sulphur in orange. Hydrogen bonds and other contacts are shown as dashed lines. (b) Stereodiagram of the superimposition of the active site of the rkbPAP-sulfate complex (green) with the active site of the rat PAP-sulfate complex [8] (magenta). In the rkbPAP-sulfate complex the bridging hydroxide adopts an elevated position, precluding the binding of a terminal water to Fe(III). Both metals in the rkbPAP-sulfate complex structure are five coordinate. Page 4 of 13 (page number not for citation purposes) Page 4 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/6 http://www.biomedcentral.com/1472-6807/8/6 BMC Structural Biology 2008, 8:6 Hydrogen bond network in the active site of rkbPAP Figure 3 Hydrogen bond network in the active site of rkbPAP. The hydrogen bond pattern demonstrates how the initial, pre- catalytic enzyme-substrate complex may be stabilized. The protonation state of sulfate at the pH of crystallization (pH 4.0) is SO42-. Residue His295 (indicated by a "") is the only amino acid that is not invariant among PAPs from different sources. in rkbPAP; Figure 1), but the precise mode of binding for this complex remained obscure [16]. X-ray crystallogra- phy is an ideal method to visualize bonding interactions between a protein and a ligand. However, in order to study enzyme-substrate interactions suitable substrate analogues need to be available. A possible candidate mimic is phosphate. The crystal structure of rkbPAP with bound phosphate was solved showing that the tetraoxo anion is coordinating to both metal ions (μ-1,3 bidentate mode) [41]. However, since hydrolysis requires the coor- dination of the substrate to the metal center, followed by a nucleophilic attack by a metal-bound hydroxide [2- 12,14-18,28,35,39], the phosphate-bound rkbPAP struc- ture is likely to illustrate the product-bound state rather than providing a mimic for the pre-catalytic substrate- bound complex. The pH for crystallization was 4.5 [41], hence phosphate is likely in its monoanionic form. How- ever, substrates such as para-nitrophenolphosphate (pNPP) may interact with the enzyme as a dianion [5]. Thus, on the basis of similar charges, sulfate is a more appropriate mimic for the substrate (pKa2~2). In contrast to the phosphate-bound rkbPAP structure [41], the sulfate ion in the active site does not coordinate directly to the metal ions. Instead it is positioned in the second coordi- nation sphere, stabilized via an extensive hydrogen bond network involving His202, His295, His296 and the Zn(II) ligand Asn201 (Figures 2 and 3). Hydrogen bonds can also be formed between the μ-hydroxide and two of the Hydrogen Figure 3 y g g Hydrogen bond network in the active site of rkbPAP. The hydrogen bond pattern demonstrates how the initial, pre- catalytic enzyme-substrate complex may be stabilized. The protonation state of sulfate at the pH of crystallization (pH 4.0) is SO42-. Residue His295 (indicated by a "") is the only amino acid that is not invariant among PAPs from different sources. Table 2: Data collection and refinement statistics. Table 2: Data collection and refinement statistics. rkbPAP-sulfate rkbPAP-fluoride Crystal Parameters Unit cell lengths (Å) a = b = 148.03 c = 160.09 a = 85.72 b = 188.45 c = 192.40 Unit cell angle (°) α = β = γ = 90.0 α = β = γ = 90.0 Space group I41 P212121 Crystal dimensions (mm) 0.5 × 0.2 × 0.2 0.5 × 0.3 × 0.3 Diffraction Dataa Temperature (K) 293 100 Resolution Range (Å) 50.0 – 2.40 50.0 – 2.20 Observations (I > 0σ (I)) 152,476 425,475 Unique reflections (I > 0σ (I)) 55,092 142,147 Completeness (%) 82.0 (52.0) 89.4 (65.1) Rsymb 0.115 (0.302) 0.075 (0.34) Refinement cRfactor 0.1712 0.2237 Rfree 0.2113 0.2543 RMSDd bond lengths (Å) 0.007 0.007 RMSDd bond angles (°) 1.285 1.233 Ramachandran Plot (%) Most favoured 83.2 83.3 Additionally allowed 15.1 15.1 Generously allowed 1.2 1.2 Disallowed 0.5 0.7 aValues in parentheses are for the outer resolution shells. bRsym = Σh Σi\|Ih,i - <Ih>\|/Σh Σi Ih,i where Ih,i is the intensity of the ith measurement of reflection h and <Ih> is the average value over multiple measurements. cRfactor = Σ\|\|Fobs\|-\|Fcalc\|\|/Σ\|Fobs\|, where the R-factor is calculated based on the reflections used in the refinement (90% of the total data) and R-free is calculated using the remaining 10% of the data. dRMSD = root mean square deviation. aValues in parentheses are for the outer resolution shells. bRsym = Σh Σi\|Ih,i - <Ih>\|/Σh Σi Ih,i where Ih,i is the intensity of the ith measurement of reflection h and <Ih> is the average value over multiple measurements. cRfactor = Σ\|\|Fobs\|-\|Fcalc\|\|/Σ\|Fobs\|, where the R-factor is calculated based on the reflections used in the refinement (90% of the total data) and R-free is calculated using the remaining 10% of the data. dRMSD = root mean square deviation. Stereodiag Figure 4 Alter- natively, it is possible that due to the presence of the sul- fate group the pKa of the bridging hydroxide is sufficiently elevated so that at pH 4.0 (crystallization condition) it is protonated; in this case each of the protons may point towards one sulfate oxygen atom. At present the two pos- sibilities cannot be distinguished. The intricacies of the extended hydrogen bond network are further illustrated by bi- and trifurcated interactions involving three of the four oxygen atoms of sulfate and several conserved first and second coordination sphere residues of rkbPAP (Fig- ure 3). At pH 4.0 (crystallization condition) sulfate is expected to enter the active site in its dianionic (SO42-) form. However, due to its extensive hydrogen bonding interactions the effective charge of sulfate is lowered, reducing its electrostatic attraction for the positively charged metal cluster. Thus, the rkbPAP-sulfate complex demonstrates, for the first time, how the pre-catalytic complex predicted from stopped-flow measurements [16,52,56] may be formed and stabilized in the second coordination sphere of the enzyme. Note, however, that in addition to mimicking the pre-catalytic stage the bound sulfate may also mimic the penultimate phase of catalysis, In the absence of crystallographic data for the complex between an actual substrate and rkbPAP, in silico docking has been used to estimate the most likely binding mode of the leaving group based on the conformation of the sul- fate group in the proposed pre-catalytic state (Figure 5). Similarly, the binding mode of the leaving group in the rkbPAP-phosphate complex (which is likely to represent the product-bound state after hydrolysis, see above) was also modeled (see Methods section for details), illustrat- ing the required movement of the substrate in the active site during catalysis. A sulfate-bound structure has also been reported for rat PAP [43], with a first coordination sphere geometry simi- lar to that of rkbPAP (Figure 2b). However, in contrast to the latter in the rat enzyme SO42- is bound in a monoden- tate manner to the metal ion at the M2 site (Figure 1) and is stabilized by hydrogen bonding interactions with Asn112 (Asn201) and His113 (His202; residues num- bered in brackets indicate the corresponding residues in rkbPAP). A similar mode of sulfate coordination has been observed in the Ser/Thr protein phosphatase from bacteri- ophage λ (λPP) [57]. Stereodiag Figure 4 g p g Stereodiagram of the active site of the rkbPAP-fluoride complex. Fo-Fc electron density for the bridge is overlayed. The fluoride replaces the hydroxide in the bridging position. General legend: Fe(III) is in tan, Zn(II) in grey, sodium in purple, fluoride in cyan, carbon in green, oxygen in red, nitrogen in blue and sulphur in orange. Hydrogen bonds and other contacts are shown as dashed lines. where the reaction product phosphate is still bound to the active site (see below). sulfate oxygens (note that due to its proximity to three donor oxygens the μ-hydroxide is able to form hydrogen bond interactions with each of these atoms (Figure 3); the crystal structure thus displays the averaged hydrogen bond pattern, evident as a "trifurcated" interaction). Alter- natively, it is possible that due to the presence of the sul- fate group the pKa of the bridging hydroxide is sufficiently elevated so that at pH 4.0 (crystallization condition) it is protonated; in this case each of the protons may point towards one sulfate oxygen atom. At present the two pos- sibilities cannot be distinguished. The intricacies of the extended hydrogen bond network are further illustrated by bi- and trifurcated interactions involving three of the four oxygen atoms of sulfate and several conserved first and second coordination sphere residues of rkbPAP (Fig- ure 3). At pH 4.0 (crystallization condition) sulfate is expected to enter the active site in its dianionic (SO42-) form. However, due to its extensive hydrogen bonding interactions the effective charge of sulfate is lowered, reducing its electrostatic attraction for the positively charged metal cluster. Thus, the rkbPAP-sulfate complex demonstrates, for the first time, how the pre-catalytic complex predicted from stopped-flow measurements [16,52,56] may be formed and stabilized in the second coordination sphere of the enzyme. Note, however, that in addition to mimicking the pre-catalytic stage the bound sulfate may also mimic the penultimate phase of catalysis, sulfate oxygens (note that due to its proximity to three donor oxygens the μ-hydroxide is able to form hydrogen bond interactions with each of these atoms (Figure 3); the crystal structure thus displays the averaged hydrogen bond pattern, evident as a "trifurcated" interaction). Hydrogen Figure 3 Page 5 of 13 (page number not for citation purposes) BMC Structural Biology 2008, 8:6 http://www.biomedcentral.com/1472-6807/8/6 Stereodiagram of the active site of the rkbPAP-fluoride complex Figure 4 Stereodiagram of the active site of the rkbPAP-fluoride complex. Fo-Fc electron density for the bridge is overlayed. The fluoride replaces the hydroxide in the bridging position. General legend: Fe(III) is in tan, Zn(II) in grey, sodium in purple, fluoride in cyan, carbon in green, oxygen in red, nitrogen in blue and sulphur in orange. Hydrogen bonds and other contacts are shown as dashed lines. Stereodiag Figure 4 Page 6 of 13 (page number not for citation purposes) Stereodiag Figure 4 Since kinetic and spectroscopic experiments with mammalian PAPs indicate that a mon- odentate coordination of the substrate to site M2 precedes Page 6 of 13 (page number not for citation purposes) Page 6 of 13 (page number not for citation purposes) BMC Structural Biology 2008, 8:6 http://www.biomedcentral.com/1472-6807/8/6 Model of substrate (para-nitrophenyl phosphate) binding to rkbPAP based on the sulfate complex (this study; cyan carbons) and on the structure of the phosphate complex (magenta carbons) Figure 5 Model of substrate (para-nitrophenyl phosphate) binding to rkbPAP based on the sulfate complex (this study; cyan carbons) and on the structure of the phosphate complex (magenta carbons). The ' symbol represents a residue from the neighboring subunit. Model of substrate (para nitrophenyl phosphate) binding to rkbPAP based on the sulfate complex (this study; cyan carbons) and on the structure of the phosphate complex (magenta carbons) Figure 5 Model of substrate (para-nitrophenyl phosphate) binding to rkbPAP based on the sulfate complex (this study; cyan carbons) and on the structure of the phosphate complex (magenta carbons). The ' symbol represents a residue from the neighboring subunit. tive inhibitor with a Ki ~170 μM at pH 4.90 [50]. The above observation that the bridging hydroxide is the only nucleophile found in the active site of the rkbPAP-SO4 crystal structure (Figure 2a) leads to the suggestion that fluoride is able to displace this group, thus forming a μ- fluoro binuclear center. This interpretation is supported by the crystal structure of the rkbPAP-fluoride complex (Figure 4). Importantly, this structure lends credence to an activation mechanism for the hydrolysis-initiating nucle- ophile. Previous studies proposed a terminal M1-bound hydroxide as the most likely candidate as the nucleophile, and it was suggested that the nucleophilicity of a bridging hydroxide would be too low to assure efficient reactivity [12,14,52]. However, the rkbPAP-SO4 structure (see above) and ENDOR measurements of pig PAP [17] indi- cate that there is no terminal water ligand at the M1 site in the resting and pre-catalytic state (Figures 1 and 3). The latter study [17] suggested that the reduced nucleophilic- ity of the bridging hydroxide is compensated by an increased electrophilicity of the substrate when compar- ing the possibilities of (i) a terminally coordinated nucle- ophile and terminally coordinated substrate, with (ii) a bridging nucleophile and bridging substrate. Stereodiag Figure 4 Further- more, binding of the substrate decreases the coupling interaction between the two metal centers, indicating a lengthening of the metal-metal distance and a concomi- tant additional increase of the nucleophilicity of the bridge by effectively shifting it towards the divalent cation ("quasi-monodentate" ligation) [17]. This substrate- induced shift of the bridging ligand is also illustrated for fluoride-bound pig PAP by a combination of inhibition kinetics, resonance Raman, electron paramagnetic reso- hydrolysis [12,14,16,17], the rat PAP and λPP structures are thus likely to represent the step following the forma- tion of the pre-catalytic complex (but preceding hydroly- sis). The different binding modes of sulfate in rat and rkbPAP may be ascribed to: (1) His216 (His296) in the rat enzyme being prevented from forming a hydrogen bond with sulfate due to the presence of a Zn(II) ion (added in the crystallization buffer); (2) His295 in rkbPAP (Figure 2a) being replaced by Glu215 in rat PAP, thus altering the chemistry at this site; (3) the μ-hydroxide in the rat PAP structure being deprotonated under the conditions of crystallization (pH 7.5); and (4) the di-iron rat enzyme was likely to be crystallized in its inactive diferric form. Thus, in the rat PAP structure the combination of reduced hydrogen bonding interactions and increased positive charge in the metal cluster is likely to increase the electro- static attraction between the metal center and sulfate, leading to direct metal ion coordination. Page 7 of 13 (page number not for citation purposes) The chemical step of the catalytic cycle p y y Apart from providing insight into a possible mode of interaction between substrate and enzyme, the rkbPAP- sulfate structure also identifies the metal ion-bridging hydroxide as a candidate nucleophile (Figure 2a). A simi- lar conclusion was reached using a range of spectroscopic techniques, including electron-nuclear double resonance (ENDOR) [17] and X-ray absorption spectroscopy [15]. Fluoride has been shown to inhibit a number of metal- loenzymes, and in PAPs is able to replace nucleophilic hydroxides coordinated to the metal ions [15,49- 51,58,59]. Specifically, for rkbPAP fluoride is a competi- Page 7 of 13 (page number not for citation purposes) Page 7 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/6 BMC Structural Biology 2008, 8:6 nance and extended X-ray absorption fine structure spec- troscopies, which have demonstrated that in the ternary enzyme-fluoride-substrate complex the fluoride ion is located closer to the divalent metal ion [15]. Furthermore, studies involving absorption spectroscopy and (magnetic) circular dichroism have also indicated that the binding of substrate analogues to pig PAP decreases the exchange coupling with an accompanying red-shift of the charge transfer transition [39]. These observations were inter- preted in terms of a weakening of the Fe(III)-μ-OH bond. Thus, both μ-fluoride and μ-hydroxide in PAP are affected in a similar manner when substrate binds to the enzyme. Here, the comparison between the sulfate- and fluoride- bound rkbPAP structures corroborates this mechanism for the activation of the bridging nucleophile. The metal- metal distance in rkbPAP-F is elongated by ~0.4 Å and the metal ion-bridging ligand is shifted towards M2 and away from M1 (Table 1). In summary, the combined structural, spectroscopic and kinetic data support the proposal that substrate binding triggers the shift of the μ-hydroxide into a "quasi-terminal" position, thus increasing its nucle- ophilicity. rangements in the M1 site permit the formation of a μ-1,3 substrate complex (Figure 6c). In support of this hypoth- esis EPR measurements have indicated that, in fluoride- inhibited pig PAP, substrates and the reaction product phosphate bind in a similar metal ion-bridging μ-1,3 mode to the binuclear center [15]. The resulting μ-1,3 phosphate complex, visualized in the structure of oxi- dized (inactive) pig PAP [42], places the phosphorus atom in an ideal position for a nucleophilic attack by the μ- hydroxide moiety (Figure 6c). The proposed structural rearrangements in the M1 site are likely to be mediated via the hydrogen bond network in the second coordination sphere. A comprehensive model for the catalytic mechanism A comprehensive model for the catalytic mechanism Based on all of the available data for PAPs the following model of a comprehensive mechanism of catalysis emerges (Figure 6). Monitoring the oxidation (and thus inactivation) of Fe(III)-Fe(II) pig PAP by [Fe(CN)6]3- has provided evidence for the formation of a pre-catalytic complex in the initial phase of the reaction, where the phosphate group of the substrate does not directly coordi- nate to the metal ions [16,52,56]. The structure of rkb- PAP-SO4 demonstrates a plausible model for the pre- catalytic complex where the substrate mimic, sulfate, is bound in the second coordination sphere, an arrange- ment stabilized via an extensive hydrogen bonding net- work (Figure 3). The bridging μ-hydroxide appears to play an essential role in the initial binding and orientation of the substrate (Figure 2a). In this pre-catalytic state both metal ion sites are five-coordinate with distorted trigonal- bipyramidal geometry. The only solvent molecule in the active site is the μ-hydroxide. Since an ENDOR study pro- vided evidence for the presence of an additional water lig- and bound terminally to the metal ion in the M2 site in resting pig PAP (Figure 1) [17], the formation of the pre- catalytic complex may thus lead to the expulsion of this labile terminal water ligand (Figure 6a). Nucleophilic attack by the μ-hydroxide and esterolysis of the substrate (depending on the basicity of the leaving group these steps may occur in a concerted or sequential manner [18]) leaves the phosphate bound to the active site in a tripodal geometry (Figure 6d). This mode of coor- dination has been observed in the crystal structures of sweet potato PAP and di-Ni(II) urease, both with bound phosphate, and di-Mn(II) λPP, with bound sulfate [18,57,61]. Hydrogen bond interactions with the carbo- nyl oxygen of the metal ion ligand His323 may stabilize this tripodal arrangement, at least at low pH [18]. In Fig- ure 7 the movement required for the phosphate group of the substrate from initial binding in the pre-catalytic com- plex to the tripodal arrangement following hydrolysis is illustrated. At this point it is important to point out that evidence has recently emerged that suggests that the chemical step may be affected by both the substrates used in the reaction and the metal ion composition of the active site [53-55]. The chemical step of the catalytic cycle A related observation has been made in the mononuclear soybean lipoxygenase-1 where the degree of coordination flexibility mediated via a hydrogen bond network in the second coordination sphere directly correlates with the reactivity of this enzyme [60]. Superposition of the rkbPAP structures with bound sulfate, fluoride and phosphate demonstrates that the geometry of the protein ligands in the M2 site and the position of the M2 metal ion (Zn) are, within experimen- tal error, fixed. However, for the M1 site in the fluoride bound structure the metal ion is shifted by up to 0.4 Å when compared to rkbPAP-SO4 and rkbPAP-PO4 which then results in compensatory rotations of the side chains of Tyr167 and His325. Page 8 of 13 (page number not for citation purposes) A comprehensive model for the catalytic mechanism Spe- cifically, it could be shown that both pig and rkbPAP hydrolyze both ester bonds in the diester substrate methyl-pNPP in a processive manner [53]. This observa- tion is interpreted in terms of an initial monodentate coordination of the substrate to the M2-site, followed by a nucleophilic attack by a terminal Fe(III)-bound hydrox- ide. This process is also observed in reactions catalyzed by The formation of the pre-catalytic complex with the con- comitant expulsion of the M2 site bound terminal water ligand generates a vacant coordination position for an oxygen atom of the phosphate group (monodentate coor- dination), as exemplified in sulfate-bound structures of rat PAP and λPP (Figure 6b) [43,57]. Coordination of the substrate to the M2 site and subsequent structural rear- Page 8 of 13 (page number not for citation purposes) Page 8 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/6 BMC Structural Biology 2008, 8:6 Proposed mechanism of binuclear metallohydrolase-catalyzed esterolysis Figure 6 Proposed mechanism of binuclear metallohydrolase-catalyzed esterolysis. Following the binding of the substrate in a pre-cata- lytic complex, structural rearrangements lead to "quasi-monodentate" and bidentate coordination of the μ-hydroxide and phosphate groups, respectively (a-c). Nucleophilic attack by the μ-hydroxide is followed by the release of the leaving group, and the active site is returned to its resting state by the exchange of the bound phosphate group by two water molecules (d-h). A terminal M1-bound hydroxide is observed in the structure of rat PAP (b), which appears to be an artefact of crystallization [46] and is not supported by solution studies on resting PAP [17]. Where available crystallographic pictures of relevant active site structures are included. (a) rkbPAP-sulfate complex; (b) rat PAP-sulfate complex [43]; (c) pig PAP-phosphate complex [42]; (d) sweet potato PAP-phosphate complex [18] ; (e) rkbPAP-phosphate complex [41]; (h) rkbPAP [40] (the bridging and termi- nal M2-bound water ligands were modelled based on ENDOR studies [17]). ! A comprehensive model for the catalytic mechanism " ! ! ! ! ! A comprehensive model for the catalytic mechanism " ! ! ! ! Proposed Figure 6 Proposed mechanism of binuclear metallohydrolase catalyzed esterolysis Figure 6 Proposed mechanism of binuclear metallohydrolase-catalyzed esterolysis. Following the binding of the substrate in a pre-cata- lytic complex, structural rearrangements lead to "quasi-monodentate" and bidentate coordination of the μ-hydroxide and phosphate groups, respectively (a-c). Nucleophilic attack by the μ-hydroxide is followed by the release of the leaving group, and the active site is returned to its resting state by the exchange of the bound phosphate group by two water molecules (d-h). A terminal M1-bound hydroxide is observed in the structure of rat PAP (b), which appears to be an artefact of crystallization [46] and is not supported by solution studies on resting PAP [17]. Where available crystallographic pictures of relevant active site structures are included. (a) rkbPAP-sulfate complex; (b) rat PAP-sulfate complex [43]; (c) pig PAP-phosphate complex [42]; (d) sweet potato PAP-phosphate complex [18] ; (e) rkbPAP-phosphate complex [41]; (h) rkbPAP [40] (the bridging and termi- nal M2-bound water ligands were modelled based on ENDOR studies [17]). The regeneration of the resting form of the enzyme, which requires the removal of the phosphate from the active site, is currently not well understood. A plausible sequence involves the rearrangement of the bound phosphate group from tripodal (Figure 6d) to μ-1,3 coordination (Figure 6e) via a rotation around the axis formed by the two oxygen atoms of phosphate that are terminally coor- dinated to the metal ions in sites M1 and M2. Although we are not aware of a precedent where the simultaneous loss of two Lewis interactions is invoked as part of a cata- lytic mechanism, the resulting μ-1,3-PO4 complex has been observed in the structure of rkbPAP, where a μ- a Fe(III)Zn(II) biomimetic of PAPs, where only the termi- nal hydroxide is sufficiently nucleophilic to induce hydrolysis [62]. Subsequently, but only in the enzyme- catalyzed reaction, the bridging hydroxide initiates the cleavage of the second ester bond of the substrate in a manner similar to that described in the preceding para- graph [53]. Since no terminal Fe(III)-bound hydroxide appears to be present in the resting state of PAPs (see above) the interaction between the diester substrate and the enzyme active site may lead to the introduction of an additional water ligand. The regeneration of the resting form of the enzyme, which requires the removal of the phosphate from the active site, is currently not well understood. Page 9 of 13 (page number not for citation purposes) Conclusion hydroxide bridge was also modelled into the active site although this ligand could not be identified in the 2.7 Å electron density map [41]. It is speculated that the loss of strain is a major contributing force to drive the conversion from a tripodal to a μ-1,3 coordination mode. No experi- mental data for the subsequent steps in regeneration are yet available but a possible mechanism is depicted in Fig- ures 6f–6h. The exchange of the M2-coordinated phos- phate oxygen atom by water leads to a monodentate, M1- bound phosphate. Due to geometric constraints the M2- bound water ligand is likely to form a hydrogen bond with the phosphate group (Figure 6f), thus facilitating its deprotonation. The M2-hydroxide then interacts with the metal ion in the M1 site, thus regenerating the μ-hydrox- ide bridge, weakening the M1-phosphate bond, and pro- viding a vacant coordination position for another water molecule in the M2 site (Figure 6g). The subsequent release of the phosphate group enables the M1 site to regain resting state, trigonal-bipyramidal geometry (Fig- ure 6h). Although the precise details of the phosphate release mechanism remain unknown it is conceivable that the anion is shuttled away from the active site via the sec- ond coordination sphere, where it may interact through hydrogen bonds with histidine residues in a manner anal- ogous to that of the substrate in the pre-catalytic complex (Figure 2a). It appears thus likely that the reversible inter- actions between the phosphate group and the second coordination sphere trigger conformational changes that take place during a catalytic turnover in the active site of PAPs. By a combination of crystallographic, kinetic and spectro- scopic data an eight-step model for the reaction mecha- nism of rkbPAP-catalyzed hydrolysis has been developed. Modifications of this basic mechanism are anticipated depending on the substrates used. The mechanistic model developed here may also be relevant for several other binuclear enzymes from the binuclear metallohydrolase family. The observed binding mode for sulfate provides a new lead in the design of metallohydrolase-selective ther- apeutics. Protein purification and characterization rkbPAP was purified as described previously [21] concen- trated to 25 mg/mL and stored at 4°C in 0.5 M NaCl. Sam- ples for crystallization were > 95% pure, as judged by SDS- PAGE analysis. Metal analysis was carried out using induc- tively coupled plasma mass spectrometry and indicated the presence of 1.0 Fe, 0.9 Zn, and trace amounts of Mn and Cu per active site. Overlay of [42] and th Figure 7 y p ( g g yg ), g g y (p ) p g [ ] p p p [ ] g Overlay of the rkbPAP-sulfate complex (with the bridging oxygen in red), the bridging hydroxide (pink) as observed in pig PAP [42] and the phosphate as observed in sweet potato PAP [18]. The image demonstrates a plausible trajectory for the substrate with the bridging hydroxide as the nucleophile in catalysis. Inversion of configuration around the phosphorous atom is observed. General legend: Fe(III) is in tan, Zn(II) in grey, carbon in green, oxygen in red, nitrogen in blue, sulphur in orange, hydrogen in white and phosphorous in magenta. Methods Materials All reagents were analytical grade and from Sigma. Proposed Figure 6 A plausible sequence involves the rearrangement of the bound phosphate group from tripodal (Figure 6d) to μ-1,3 coordination (Figure 6e) via a rotation around the axis formed by the two oxygen atoms of phosphate that are terminally coor- dinated to the metal ions in sites M1 and M2. Although we are not aware of a precedent where the simultaneous loss of two Lewis interactions is invoked as part of a cata- lytic mechanism, the resulting μ-1,3-PO4 complex has been observed in the structure of rkbPAP, where a μ- Page 9 of 13 (page number not for citation purposes) Page 9 of 13 (page number not for citation purposes) BMC Structural Biology 2008, 8:6 http://www.biomedcentral.com/1472-6807/8/6 Overlay of the rkbPAP-sulfate complex (with the bridging oxygen in red), the bridging hydroxide (pink) as observed in pig PAP [42] and the phosphate as observed in sweet potato PAP [18] Figure 7 Overlay of the rkbPAP-sulfate complex (with the bridging oxygen in red), the bridging hydroxide (pink) as observed in pig PAP [42] and the phosphate as observed in sweet potato PAP [18]. The image demonstrates a plausible trajectory for the substrate with the bridging hydroxide as the nucleophile in catalysis. Inversion of configuration around the phosphorous atom is observed. General legend: Fe(III) is in tan, Zn(II) in grey, carbon in green, oxygen in red, nitrogen in blue, sulphur in orange, hydrogen in white and phosphorous in magenta. Authors' contributions All authors have read and approved the final version of the manuscript. GS was responsible for the conceptualiza- tion of experiments, analysis and interpretation of data, and drafting and critical review of the manuscript. TWE carried out protein purification, acquisition and analysis of data. EL was assisting acquisition and analysis of data. NM was involved in analysis of data and was responsible for the revision of the manuscript. LEC was involved in protein purification and acquisition of data. LRG was involved in conceptualization of experiments, interpreta- tion of data, and critical review of the manuscript. LWG was responsible for the conceptualization of experiments, acquisition, analysis and interpretation of data, and criti- cal review of the manuscript. Abbreviations ENDOR – electron-nuclear double resonance; λPP – Ser/ Thr protein phosphatase from bacteriophage λ; PAP – purple acid phosphatase; rkbPAP – red kidney bean pur- ple acid phosphatase; pNPP – para-nitrophenolphosphate The crystal structures of rkbPAP in the presence of sulfate or fluoride were determined by molecular replacement and refined to 2.4 Å with an Rfreevalue of 0.2113 and to 2.2 Å with an Rfree of 0.2543, respectively (Table 2). The sulfate complex crystallized with one dimer in the asym- metric unit while the fluoride complex has two such dim- ers. The overall structures of the polypeptides strongly resemble those of the previously determined structures of free rkbPAP and the phosphate and tungstate bound com- plexes of this enzyme [41] with r.m.s.d. values for all Cα atoms ranging from 0.2 to 0.3 Å. For the sulfate-bound structure the asymmetric unit (and final model) consisted of two polypeptide subunits of 423 residues (9–432), two Fe(III), two Zn(II), two sulfate groups, eight N-acetyl glu- cosamine residues and 399 water molecules. The first eight N-terminal amino acid residues of the polypeptide in each subunit were not visible. For the fluoride structure the asymmetric unit (and final model) consisted of four polypeptide subunits of 423 residues (9–432), four Fe(III), four Zn(II), four fluoride ions, eight sodium ions, eight sulfate groups, sixteen N-acetyl glucosamine resi- dues and 547 water molecules. Within the crystallization solution, the only other small molecules present at a detectable concentration are acetate and glycerol. The fit- ting of these molecules into the electron density occupied by the sulfate ions was tested but they did not fit the con- tours as precisely as sulfate. Furthermore, in Fo-Fc differ- ence maps the electron density at the location of the sulfate atoms was generally between 7.5–8.0 σ above the mean, compared to 3.0–4.0 σ above the mean for other Protein crystallization Crystals were grown by vapor diffusion, with hanging drops consisting of 5 μl of well solution and 5 μl of the concentrated protein solution. For the sulfate complex, the well solution for crystallization consisted of 2 M ammonium sulfate and 0.1 M sodium acetate, pH 4.0. Page 10 of 13 (page number not for citation purposes) Page 10 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/8/6 http://www.biomedcentral.com/1472-6807/8/6 BMC Structural Biology 2008, 8:6 Crystals of the fluoride complex were obtained by co-crys- tallization of rkbPAP in the presence of 50 mM NaF (an excess of ~550 fold). The well solution for these crystals consisted of 2.3 M ammonium sulfate and 0.1 M sodium acetate pH 4.0. No suitable condition for cryoprotection could be found for the sulfate crystals, therefore data were obtained at room temperature. The fluoride crystal was cryoprotected in a solution containing 30% (v/v) glycerol and 70% (v/v) well solution. nearby atoms which we assign as either oxygen, fluoride or sodium. The backbone dihedral angles (Ramachandran plot values) of His 323, Ile 340 and Ala 243 are in disal- lowed regions in all polypeptides of both complexes. Only His323 is within the active site. In all of the rkbPAP structures previously determined this residue has similar dihedral angle values [40,41]. Coordinates and structure factors have been deposited into the protein databank with accession numbers 2QFR and 2QFB for rkbPAP-sul- fate and rkbPAP-fluoride, respectively. All figures were generated with the progam PyMOL [67]. Diffraction data collection and structural refinement All X-ray data were collected using an Raxis IV++ imaging plate and an FR-E rotating anode generator operated at a current of 45 mA and a voltage of 45 kV. The program Crystalclear 1.3.6 [63] was used for the integration and scaling of the data. The sulfate and fluoride bound crystals were not isomorphous with any of the previously deter- mined structures of rkbPAP or with each other. Therefore, these structures were solved by molecular replacement in the program EPMR [64] using the polypeptide coordi- nates of the rkbPAP-phosphate complex (PDB code 4KBP [41]). Rounds of refinement and modelling were under- taken using the programs CNS [65] and O [66], respec- tively. For in silico docking studies a three dimensional model of the substrate para-nitrophenyl phosphate was constructed using Sketcher in Insight2000. The phosphorous and the oxygen atoms of the substrate were superimposed onto sulfate in the rkbPAP-sulfate complex or phosphate in the rkbPAP-phosphate [41] complex. In both dockings the aromatic ring of para-nitrophenyl phosphate was then rotated to make optimal contacts with the nearby amino acids of the enzyme. Acknowledgements g This work was funded by a grant from the Australian Research Council (DP0558652). X-ray data were measured using facilities provided by the University of Queensland Macromolecular X-ray Crystallography Facility. References 1. Lippard SJ, Berg JM: Principles of Bioinorganic Chemistry, University Science Books, Mill Valley, CA; 1994. 2. Wilcox DE: Binuclear metallohydrolases. Chem Rev 1996, 96:2435-2458. 3. Dismukes GC: Manganese enzymes with binuclear active sites. Chem Rev 1996, 96:2909-2926. References Sträter N, Lipscomb WN, Klabunde T, Krebs B: Two-metal ion catalysis in enzymatic acyl- and phosphoryl-transfer reac- tions. 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 13 of 13 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 61. Benini S, Rypniewski WR, Wilson KS, Ciurli S, Mangani S: Structure- based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism. J Biol Inorg Chem 2001, 6:778-790. 62. Neves A, Lanznaster M, Bortoluzzi AJ, Peralta RA, Casellato A, Cas- tellano EE, Herrald P, Riley MJ, Schenk G: An Unprecedented FeIII(μ-OH)ZnII Complex that Mimics the Structural and Functional Properties of Purple Acid Phosphatases. J Am Chem Soc 2007, 129:7486-7487. 63. Pflugrath JW: The finer things in X-ray diffraction data collec- tion. 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https://openalex.org/W2103543093	https://uu.diva-portal.org/smash/get/diva2:578633/FULLTEXT01	English	null	Phosphodiesterase 8B gene polymorphism in women with recurrent miscarriage: A retrospective case control study	BMC medical genetics	2,012	cc-by	5,230	© 2012 Granfors 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 Background: Recurrent miscarriage affects approximately 1% of all couples. There is a known relation between hypothyroidism and recurrent miscarriage. Phosphodiesterase 8B (PDE8B) is a regulator of cyclic adenosine monophosphate (cAMP) with important influence on human thyroid metabolism. Single nucleotide polymorphism (SNP) rs 4704397 in the PDE8B gene has been shown to be associated with variations in serum Thyroid Stimulating Hormone (TSH) and thyroxine (T4) levels. The aim of this study was to investigate whether there is an association between the SNP rs 4704397 in the PDE8B gene and recurrent miscarriage. Methods: The study was designed as a retrospective case control study. 188 cases with recurrent miscarriage were included and compared with 391 controls who had delivered at least once and with no history of miscarriage or assisted reproduction. Results: No difference between cases and controls concerning age was found. Bivariate associations between homozygous A/A (OR 1.57, 95% CI 0.98-2.52) as well as G/G carriers (OR 1.52, 95% CI 1.02-2.25) of SNP rs 4704397 in PDE8B and recurrent miscarriage were verified (test for trend across all 3 genotypes, p = 0.059). After adjustment for known confounders such as age, BMI and smoking the association between homozygous A/A (AOR 1.63, 95% CI 1.01 - 2.64, p = 0.045) and G/G (AOR 1.52, 95% CI 1.02 - 2.27, p = 0.039) carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage remained. Conclusions: Our findings suggest that there is an association between homozygous A/A as well as homozygous G/G carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage. Keywords: Phosphodiesterase 8B, Recurrent miscarriage, Single nucleotide polymorphism, Thyroid more often occurs despite normal fetal cytogenetic find- ings, and in 50% of cases the underlying cause remains unexplained [1,3-5]. Hypothyroidism is a common dis- ease, with an estimated incidence of 0.3% to 0.5% for overt and 3% for subclinical hypothyroidism in pregnant women [1,6-8]. There is a known relation between hypothyroidism and recurrent miscarriage [1]. Granfors et al. BMC Medical Genetics 2012, 13:121 http://www.biomedcentral.com/1471-2350/13/121 Granfors et al. BMC Medical Genetics 2012, 13:121 http://www.biomedcentral.com/1471-2350/13/121 Open Access Phosphodiesterase 8B gene polymorphism in women with recurrent miscarriage: A retrospective case control study Michaela Granfors1, Helena Karypidis1,2, Frida Hosseini2, Lottie Skjöldebrand-Sparre2, Anneli Stavreus-Evers1, Katarina Bremme3, Britth-Marie Landgren4, Inger Sundström-Poromaa1, Anna-Karin Wikström1 and Helena Åkerud1 Correspondence: michaela.granfors@kbh.uu.se 1Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden Full list of author information is available at the end of the article Background Recurrent miscarriage affects approximately 1% of all couples trying to conceive and is defined as three or more consecutive pregnancy losses before 20 weeks of amenorrhoea [1,2]. Predisposing factors include mater- nal age, parental chromosomal aberrations, uterine ab- normalities, antiphospholipid syndrome, immunological and thrombophilic disorders, and endocrine diseases such as hypothyroidism and diabetes mellitus [1]. Unlike sporadic spontaneous miscarriage, recurrent miscarriage The human phosphodiesterase type 8B (PDE8B) gene is located at human chromosome 5q14.1 in intron 1 and encodes a high affinity cyclic adenosine monophosphate (cAMP) specific nucleotide phosphodiesterase [9-11]. The PDE8B gene is abundantly expressed in the thyroid but has also been detected in human placenta and ovar- ies [9,10,12,13]. Based on a recent genome-wide Granfors et al. BMC Medical Genetics 2012, 13:121 http://www.biomedcentral.com/1471-2350/13/121 Page 2 of 6 women. Since May 31 2007, all women aged 18 and older attending the second trimester (16–19 weeks of gestation) routine ultrasound scan at Uppsala University Hospital have been approached for inclusion in this bio- bank, and inclusion to our study was ongoing until June 30 2010. In the control group, none had a history of miscarriage and 74.9% had at least two spontaneous pregnancies, including the ongoing pregnancy, resulting in a term (≥37 weeks) birth of a live infant. Beyond that, the same inclusion and exclusion criteria were applied for the control group as for the cases. association study, six different single nucleotide poly- morphisms (SNP) in the PDE8B gene were associated with increased serum concentrations of thyroid stimulat- ing hormone (TSH) [14]. The strongest association with increased TSH levels (although in the normal range) was reported for one specific SNP in PDE8B, rs 4704397, which is found in the promoter region of the gene [14,15]. A recent meta-analysis confirmed the associ- ation between SNP rs 4704397 in PDE8B and elevated TSH levels, but also found an association with decreased levels of free T4, although free T3 levels were normal [16]. In addition, the association between SNP rs 4704397 in PDE8B and levels of TSH has been con- firmed in pregnant women, although no association with free T3 or T4 levels was found [17]. Both cases and controls attended a brief health exam- ination including measurements of weight and height and answered standardised questions on reproductive history. Further, we reviewed the medical records to ob- tain relevant information on pregnancy outcomes, health problems and medication. Study population The study was designed as a case–control study. Cases (n = 188) were recruited from the Department of Obste- trics and Gynaecology at Uppsala University Hospital, Karolinska University Hospital, Huddinge University Hospital and Danderyd University Hospital, Sweden. Eli- gible cases with a diagnosis of recurrent miscarriage, defined as three or more verified consecutive miscar- riages in the first or second trimester of pregnancy (5–21 completed weeks of gestation), were identified in the out-patient registers of the participating clinics and invited to participate in the study. The women were included between April 29 2009 and June 30 2010. Women with known risk factors for recurrent miscar- riage, such as systemic lupus erythematosus, diabetes mellitus type 1, severe thrombophilia and major chromosomal aberrations were not included in the study. Furthermore, two women with hyperthyroidism were excluded. Blood sample collection Blood samples were collected in EDTA-containing tubes and centrifuged at 1500 g for 10 min. Plasma and buffy coat were separated and stored at −20°C. Background In SNP rs 4704397 of PDE8B an adenine (A) nucleo- tide is replaced by a guanine (G). The association be- tween the polymorphism and high levels of TSH and low free T4 levels, indicating relative hypothyroidism, is found in homozygous carriers of A/A [16]. Based on previous results it has been proposed that the SNP rs 4704397 in PDE8B and in particular the presence of A alleles might induce increased phosphodiesterase activity in PDE8B, thereby reducing the ability of the thyroid gland to generate free T4 when stimulated by TSH [14]. Related to this, we hypothesized that homozygous car- riers of A/A would be at increased risk of recurrent mis- carriage. The aim of this study was to investigate whether there is an association between the SNP rs 4704397 in the PDE8B gene and recurrent miscarriage. According to routine clinical procedures, TSH-levels were analysed in all women when diagnosed with recur- rent miscarriage. In the case-group, hypothyroidism was defined as TSH above the current defined upper limit of the reference range at the different hospitals. Pregnant controls at high risk for thyroid disease were subjected to selected TSH screening according to local guidelines based on international recommendations [6]. This case- finding procedure was applied in first or early second trimester of pregnancy, before routine ultrasound scan and inclusion in the study. The study was approved by the Regional Ethics Com- mittee of the Medical Faculty of Uppsala University Hos- pital and the Ethical Committee of the Karolinska Institutet, Stockholm. Informed consent was obtained from all women included in the study. SNP-analysis SNP-analysis Genomic DNA was extracted from blood using QIAampW DNA Blood Maxi kits (Qiagen, Venlo, the Netherlands). The samples were genotyped for the SNP rs4704397 of PDE8B, using the TaqManW SNP Genotyp- ing Assay (Applied Biosystems, Foster City, CA, USA). Briefly, PCR reactions were performed in a 96 well plate in a total volume of 25 μl for each reaction. Each reac- tion consisted of 1x TaqMan Universal PCR Master Mix (PCR buffer, ROX passive reference dye, dNTPs and AmpliTaq Gold polymerase), 1x SNP Genotyping Assay (sequence-specific forward and reverse primers to amp- lify the polymorphic sequence of interest, i.e. HRG exon 5, TaqManW MGB probes labelled with VIC Wdye to de- tect allele 1 sequence and with FAM™to detect allele 2 The control subjects (n=391) were matched for age at first planned pregnancy and were randomly chosen from the Uppsala University Hospital biobank of pregnant Granfors et al. BMC Medical Genetics 2012, 13:121 http://www.biomedcentral.com/1471-2350/13/121 Page 3 of 6 Table 2a Demographic data and clinical characteristics of the study population according to the SNP rs 4704397 genotype A/A A/G G/G (n = 110) (n = 266) (n = 203) Age, years 29.9 ± 5.1 30.5 ± 6.0 29.6 ± 5.8 BMI at first antenatal visit, kg/m2 23.6 ± 4.2 24.5 ± 4.4 24.1 ± 4.3 Smokers, n (%) 16 (14.5%) 31 (11.7%) 29 (14.3%) Values are mean ± standard deviation. No significant differences between the groups exist. Table 2a Demographic data and clinical characteristics of the study population according to the SNP rs 4704397 genotype sequence) and 10 ng of genomic DNA. Cycling conditions were initiated for 10 min at 95°C, followed by 40 cycles of 15s at 92°C and 1 minute at 60°C. Real time fluores- cence detection was performed. The Sequence Detection System (SDS) Software (Applied Biosystems) was used to plot fluorescence (Rn) values based on the signals from each well. The plotted fluorescence signals indicated which alleles were present in each sample. Results Background characteristics and genotyping of PDE8B Demographic data and clinical characteristics for cases and controls are shown in Table 1. There were no differences between cases and controls concerning age (p = 0.99). As expected, BMI was higher (p=0.033) and smoking (18.7% vs. 10.7%, p =0.015) and hypothyroidism (8.5% vs. 3.1%, p = 0.006) were more common in women with recurrent miscarriage. Genotype and risk of recurrent miscarriage A bivariate analysis was performed including all known confounders. Based on the finding (Table 2b) that homo- zygous A/A and G/G carriers seemed to be more com- mon in women with recurrent miscarriage, heterozygous A/G carriers were chosen as reference group in the logistic regression analyses. Bivariate associations be- tween homozygous A/A as well as G/G carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage were verified and a test for trend across all 3 genotypes was performed (Table 4). After adjustment for known con- founders such as age, BMI and smoking, the association between homozygous A/A (AOR 1.63, 95% CI 1.01 - 2.64, p = 0.045) and G/G (AOR 1.52, 95% CI 1.02 - 2.27, p = 0.039) carriers of SNP rs 4704397 in PDE8B and re- current miscarriage remained. Statistics Demographic and clinical characteristics were compared between cases and controls or genotype groups using t-test and Chi-square tests. ANOVA was used for conti- nous variables. Adjusted odds ratios (AOR) for recurrent miscarriage were calculated in multivariate regression analyses using the following variables: maternal age as completed years at the first pregnancy or first miscar- riage, body mass index (BMI, kg/m2) defined as BMI recorded at inclusion (cases) or BMI at first antenatal visit (controls), smoking during pregnancy (yes/no) defined as smoker during ≥1 pregnancy ending with miscarriage (cases) or smoker at first visit to the prenatal centre in gestational week 10 (controls), and genotype (A/G as reference). Only variables with possible associ- ation with each outcome (p < 0.25) in the bivariate ana- lyses were entered into the final model. All significance tests were two-tailed. P-values < 0.05 were considered as statistically significant. All statistical analyses were per- formed using SPSS 16.0 for Windows software pack (SPSS, Chicago, IL). Among the 579 genotyped women, 110 (19.0%) were homozygous carriers of A/A, 266 (45.9%) were heterozy- gous carriers (A/G) and 203 (35.1%) were homozygous carriers of G/G (Table 2a). Homozygous A/A and G/G carriers, compared to heterozygous carriers, were more common among women with recurrent miscarriage (Table 2b). Homozygous and heterozygous carriers were in accordance with the Hardy Weinberg equilibrium. Discussion PDE8B is known to be abundantly expressed in the thy- roid and is of importance for thyroid function [9,12,13,15]. Increased levels of TSH have previously been shown in homozygous A/A carriers of SNP rs 4704397 in PDE8B and we hypothesized that there might be an association between A allele carriers and recurrent miscarriage. In our study, associations between Table 1 Demographic data and clinical characteristics of the study population Table 1 Demographic data and clinical characteristics of the study population Cases (n = 188) Controls (n = 391) p-value Age, years 30.1 ± 5.8 30.1 ± 5.8 0.989 BMI, kg/m2 24.7 ± 4.8 23.9 ± 4.0 0.031 Smokers, n (%) 34 (18.1%) 42 (10.7%) 0.015 Hypothyroidism, n (%) 16 (8.5%) 12 (3.1%) 0.006 Values are mean ± standard deviation; n, numbers of women; BMI, body mass index: BMI at inclusion (cases), BMI at first antenatal visit (controls); Smokers: smoker during ≥1 pregnancy ending with miscarriage (cases), smoker at first visit to the prenatal center in gestational week 10 (controls); Hypothyroidism: elevated serum TSH-levels according to local laboratory limits. Table 2b Distribution of SNP rs 4704397 genotype among controls and cases Controls (n=391) Recurrent miscarriage (cases, n=188) All Hypothyroidism A/A 69 (17.6%) 41 (21.8%) 3 (18.8%) A/G 193 (49.4%) 73 (38.8%) 4 (25.0%) G/G 129 (33.0%) 74 (39.4%) 9 (56.2%) Table 2b Distribution of SNP rs 4704397 genotype among controls and cases Table 2b Distribution of SNP rs 4704397 genotype among controls and cases Granfors et al. BMC Medical Genetics 2012, 13:121 http://www.biomedcentral.com/1471-2350/13/121 Page 4 of 6 Page 4 of 6 homozygous A/A as well as homozygous G/G carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage were found. prevalence of hypothyroidism in G/G carriers did not differ from that of A/G carriers in our study population (Table 2a). It is very likely that another mediator is underlying this association. It has been shown that deranged levels of TSH in serum are related to inheritable factors in 65% of cases [18,19]. According to a genome-wide association scan in a Sardinian population, the specific SNP rs 4704397 in PDE8B has a strong association with increased levels of TSH and explains 2.3% of the variance in TSH. Each copy of the A allele was associated with an average in- crease of 0.13 μIU/ml in TSH serum concentrations [14]. Moreover, it is noteworthy that G/G carriers were more common among hypothyroid women with recur- rent miscarriage, compared to both controls and all cases (Table 2b). Thus, concerning recurrent miscar- riage, it seems to be a disadvantageous combination to be G/G carrier and to suffer from hypothyroidism. How- ever, it is important to remember that the number of women with hypothyroidism is quite small in this material. Limitingly, as the study population was evaluated at clinics with different reference ranges for TSH analyses, and serum was not stored at time of diagnosis, TSH levels were unavailable for further analysis. Thus, we were not able to draw conclusions about whether the increased risk of recurrent miscarriage in homozygous A/A carriers in our material was mediated through rela- tively increased TSH levels. However, the prevalence of hypothyroidism was slightly, but not enough to be statis- tically significant, greater in homozygous A/A than in G allele carriers. The importance of PDE8B in the placenta and human ovaries is unknown, but the relevance of phoshodies- terases (PDE) in human reproduction has been discussed [10,13]. The mechanisms that regulate oocyte maturation in vivo and in vitro are still not well understood but the second messenger, cyclic adenosine mono-phosphate (cAMP), plays a critical role in maintaining the oocytes at meiotic arrest in the diplotene stage of the first meiotic prophase [22]. Conclusions Our findings suggest that there is an association between homozygous A/A as well as homozygous G/G carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage. 9. Hayashi M, Matsushima K, Ohashi H, Tsunoda H, Murase S, Kawarada Y, Tanaka T: Molecular cloning and characterization of human PDE8B, a novel thyroid-specific isozyme of 3',5'-cyclic nucleotide phosphodiesterase. Biochem Biophys Res Commun 1998, 250(3):751–756. 9. Hayashi M, Matsushima K, Ohashi H, Tsunoda H, Murase S, Kawarada Y, Tanaka T: Molecular cloning and characterization of human PDE8B, a novel thyroid-specific isozyme of 3',5'-cyclic nucleotide phosphodiesterase. Biochem Biophys Res Commun 1998, 250(3):751–756. In our study, different alleles of SNP rs 4704397 in PED8B were associated with the same outcome, recur- rent miscarriage, but the explanation for this association is a subject for speculation. Genetic variability plays a role in many different diseases and has been proposed to be of relevance in screening for distinct syndromes with known genetic defects, and it might also be used for pre- diction of success related to disease treatment [29,30]. 10. Hayashi M, Shimada Y, Nishimura Y, Hama T, Tanaka T: Genomic organization, chromosomal localization, and alternative splicing of the humanphosphodiesterase 8B gene. Biochem Biophys Res Commun 2002, 297(5):1253–1258. 10. Hayashi M, Shimada Y, Nishimura Y, Hama T, Tanaka T: Genomic organization, chromosomal localization, and alternative splicing of the humanphosphodiesterase 8B gene. Biochem Biophys Res Commun 2002, 297(5):1253–1258. 11. Soderling SH, Bayuga SJ, Beavo JA: Cloning and characterization of acAMP- specific cyclic nucleotide phosphodiesterase. Proc Natl Acad Sci U S A 1998, 95(15):8991–8996. 11. Soderling SH, Bayuga SJ, Beavo JA: Cloning and characterization of acAMP- specific cyclic nucleotide phosphodiesterase. Proc Natl Acad Sci U S A 1998, 95(15):8991–8996. 12. Gamanuma M, Yuasa K, Sasaki T, Sakurai N, Kotera J, Omori K: Comparison of enzymatic characterization and gene organization of cyclic nucleotide phosphodiesterase 8 family in humans. Cell Signal 2003, 15(6):565–574. 12. Gamanuma M, Yuasa K, Sasaki T, Sakurai N, Kotera J, Omori K: Comparison of enzymatic characterization and gene organization of cyclic nucleotide phosphodiesterase 8 family in humans. Cell Signal 2003, 15(6):565–574. 13. Horvath A, Giatzakis C, Tsang K, Greene E, Osorio P, Boikos S, Libe R, Patronas Y, Robinson-White A, Remmers E, et al: A cAMP-specific phosphodiesterase (PDE8B) that is mutated in adrenal hyperplasia is expressed widely in human and mouse tissues: a novel PDE8B isoform in human adrenal cortex. Eur J Hum Genet 2008, 16(10):1245–1253. References 1. Ford HB, Schust DJ: Recurrent pregnancy loss: etiology, diagnosis, and therapy. Rev Obstet Gynecol 2009, 2(2):76–83. 1. Ford HB, Schust DJ: Recurrent pregnancy loss: etiology, diagnosis, and therapy. Rev Obstet Gynecol 2009, 2(2):76–83. 2. Jauniaux E, Farquharson RG, Christiansen OB, Exalto N: Evidence-based guidelines for the investigation and medical treatment of recurrent miscarriage. Hum Reprod 2006, 21(9):2216–2222. 2. Jauniaux E, Farquharson RG, Christiansen OB, Exalto N: Evidence-based guidelines for the investigation and medical treatment of recurrent miscarriage. Hum Reprod 2006, 21(9):2216–2222. g p 3. Sullivan AE, Silver RM, LaCoursiere DY, Porter TF, Branch DW: Recurrent fetal aneuploidy and recurrent miscarriage. Obstet Gynecol 2004, 104(4):784–788. g p 3. Sullivan AE, Silver RM, LaCoursiere DY, Porter TF, Branch DW: Recurrent fetal aneuploidy and recurrent miscarriage. Obstet Gynecol 2004, 104(4):784–788. 4. Stirrat GM: Recurrent miscarriage. Lancet 1990, 336(8716):673–675. 4. Stirrat GM: Recurrent miscarriage. Lancet 1990, 336(8716):673–675. 4. Stirrat GM: Recurrent miscarriage. Lancet 1990, 336(8716):673–675. 5. Rai R, Regan L: Recurrent miscarriage. Lancet 2006, 368(9535):601–611. 4. Stirrat GM: Recurrent miscarriage. Lancet 1990, 336(8716):673–675. 5. Rai R, Regan L: Recurrent miscarriage. Lancet 2006, 368(9535):601–611. Rai R, Regan L: Recurrent miscarriage. Lancet 2006, 368(9535):601–61 6. Abalovich M, Amino N, Barbour LA, Cobin RH, De Groot LJ, Glinoer D, Mandel SJ, Stagnaro-Green A: Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2007, 92(8 Suppl):S1–S47. 6. Abalovich M, Amino N, Barbour LA, Cobin RH, De Groot LJ, Glinoer D, Mandel SJ, Stagnaro-Green A: Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2007, 92(8 Suppl):S1–S47. Guideline. J Clin Endocrinol Metab 2007, 92(8 Suppl):S1–S47. 7. Casey BM, Leveno KJ: Thyroid disease in pregnancy. Obstet Gynecol 2006, 108(5):1283–1292. 7. Casey BM, Leveno KJ: Thyroid disease in pregnancy. Obstet Gynecol 2006, 108(5):1283–1292. 8. Casey BM, Dashe JS, Spong CY, McIntire DD, Leveno KJ, Cunningham GF: Perinatal significance of isolated maternal hypothyroxinemia identified in the first half of pregnancy. Obstet Gynecol 2007, 109(5):1129–1135. 8. Casey BM, Dashe JS, Spong CY, McIntire DD, Leveno KJ, Cunningham GF: Perinatal significance of isolated maternal hypothyroxinemia identified in the first half of pregnancy. Obstet Gynecol 2007, 109(5):1129–1135. Acknowledgements h h ld l k The authors would like to thank all the women participating in the study and Christine Sund- Lundström, Department of Women’s and Children’s Health, Uppsala University for valuable technical assistance. 18. Hansen PS, Brix TH, Sorensen TI, Kyvik KO, Hegedus L: Major genetic influence on the regulation of the pituitary-thyroid axis: a study of healthy Danish twins. J Clin Endocrinol Metab 2004, 89(3):1181–1187. Authors’ contributions HK, FH, LSS, ASE, KB BML and ISP designed the study and were responsible for data collection. HÅ designed the study, analyzed the data and drafted the manuscript. MG and AKW analyzed the data and drafted the manuscript. All authors read and approved the final manuscript. 17. Shields BM, Freathy RM, Knight BA, Hill A, Weedon MN, Frayling TM, Hattersley AT, Vaidya B: Phosphodiesterase 8B Gene Polymorphism Is Associated with Subclinical Hypothyroidism in Pregnancy. J Clin Endocrinol Metab 2009, 94(11):4608–4612. 17. Shields BM, Freathy RM, Knight BA, Hill A, Weedon MN, Frayling TM, Hattersley AT, Vaidya B: Phosphodiesterase 8B Gene Polymorphism Is Associated with Subclinical Hypothyroidism in Pregnancy. J Clin Endocrinol Metab 2009, 94(11):4608–4612. Conclusions Hypothetically, this specific SNP may be used for indi- vidual counselling and for optimising treatment to pre- vent recurrent miscarriage, based on which allele the woman is a carrier of. 14. Arnaud-Lopez L, Usala G, Ceresini G, Mitchell BD, Pilia MG, Piras MG, Sestu N, Maschio A, Busonero F, Albai G, et al: Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function. Am J Hum Genet 2008, 82(6):1270–1280. However, larger studies with independent replication are needed to confirm our results and their clinical importance. 15. Horvath A, Faucz F, Finkielstain GP, Nikita ME, Rothenbuhler A, Almeida M, Mericq V, Stratakis CA: Haplotype analysis of the promoter region of phosphodiesterase type 8B (PDE8B) in correlation with inactivating PDE8B mutation and the serum thyroid-stimulating hormone levels. Thyroid 2010, 20(4):363–367. Received: 19 April 2012 Accepted: 10 December 2012 Published: 13 December 2012 Of course, we cannot exclude our results concerning SNP associations to be false positive. Generally, there is a high false positive rate in small genetic association studies due to low prior probability [28]. However, SNP rs 4704397 in PDE8B has been shown to be associated with variations in serum TSH levels in several studies [14-17], and there is a well known association between hypothyroidism and recurrent miscarriage [1]. To our knowledge, this is the first study examining a possible association between SNP rs 4704397 in PDE8B and re- current miscarriage. To confirm our findings, it would be important to increase the sample size and replicate in an independent cohort in a further study. Competing interests The authors declare no conflict of interest. 16. Taylor P, Panicker V, Sayers A, Shields B, Iqbal A, Bremner A, Beilby J, Leedman P, Hattersley A, Vaidya B, et al: A meta-analysis of the associations between common variation in the PDE8B gene and thyroid hormone parameters; including assessment of longitudinal stability of associations over time and effect of thyroid hormone replacement. Eur J Endocrinol 2011, 10:0938. EJE. Table 2b Distribution of SNP rs 4704397 genotype among controls and cases The PDEs inactivate and degrade cAMP in oocytes as a response to the ovulatory luteinizing hor- mone pulse and have thus been proposed as factors of im- portance for regulating oocyte maturation [23-25]. Furthermore, it has been shown that levels of cAMP in the oocyte at meiotic resumption correlate with oocyte competence and embryonic development [26,27]. Based on the knowledge that PDE activity is relevant for levels of cAMP in oocytes, the importance of SNP rs 4704397 in PDE8B for regulation of oocyte maturation would be one pathway of interest for further study. Based on the fact that homozygous A/A and G/G car- riers, compared to heterozygous, were more common among women with recurrent miscarriage, we decided to use heterozygous carriers as references. This concept has been used before in other studies. When several dif- ferent folate-metabolizing genes were analyzed, hetero- zygous rather than wild-type homozygous carriers, appeared to have higher pregnancy success rate after IVF treatment [20,21]. Not only A/A, but also homozygous carriers of the G/ G SNP rs 4704397 in PDE8B were more common among women with recurrent miscarriage. This finding is novel and the mediator of the association is unknown. The association is presumably not mediated by inad- equate thyroid metabolism, since no association between homozygous G/G carriers and thyroid disease has been identified in previous studies. Consistent with this, the Another limitation with our study is that cases and controls were screened for thyroid disease in different ways. According to routine clinical procedures, TSH- levels in all women with recurrent miscarriage were ana- lyzed. Pregnant controls were subjected to selected TSH screening when considered to be at high risk for thyroid disease, in accordance with international guidelines [6]. Thus, the frequency of hypothyroidism might have been Table 3 Factors associated with recurrent miscarriage Unadjusted OR (95 % CI) Adjusted OR1 (95 % CI) p-value (Adjusted OR) Age years 1.0 (0.97-1.03) 0.99 (0.96-1.02) 0.500 BMI kg/m2 0.96 (0.92 – 1.00) 0.96 (0.92-0.96) 0.026 Smoker No 1 1 Yes 1.84 (1.12 - 3.00) 1.86 (1.12 – 3.09) 0.017 SNP rs4704397 A/G* 1 1 A/A 1.57 (0.98 - 2.52) 1.63 (1.01 - 2.64) 0.045 G/G 1.52 (1.02 - 2.25) 1.52 (1.02 - 2.27) 0.039 1adjusted for age, smoking, BMI and genotype. *Test for trend across genotypes in relation to recurrent miscarriage was p= 0.059. Table 3 Factors associated with recurrent miscarriage Granfors et al. Table 2b Distribution of SNP rs 4704397 genotype among controls and cases BMC Medical Genetics 2012, 13:121 http://www.biomedcentral.com/1471-2350/13/121 Page 5 of 6 Page 5 of 6 underestimated in the control group, which is of import- ance when the results are evaluated. Received: 19 April 2012 Accepted: 10 December 2012 Published: 13 December 2012 Received: 19 April 2012 Accepted: 10 December 2012 Published: 13 December 2012 20. Laanpere M, Altmae S, Stavreus-Evers A, Nilsson TK, Yngve A, Salumets A: Folate-mediated one-carbon metabolism and its effect on female fertility and pregnancy viability. Nutr Rev 2010, 68(2):99–113. Author details 1 1Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden. 2Department of Clinical Sciences, Division of Obstetrics and Gynaecology, Karolinska Institutet and Danderyd Hospital, Stockholm, Sweden. 3Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden. 4Department of Clinical Sciences, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. 19. Panicker V, Wilson SG, Spector TD, Brown SJ, Falchi M, Richards JB, Surdulescu GL, Lim EM, Fletcher SJ, Walsh JP: Heritability of serum TSH, free T4 and free T3 concentrations: a study of a large UK twin cohort. Clin Endocrinol (Oxf) 2008, 68(4):652–659. 20. Laanpere M, Altmae S, Stavreus-Evers A, Nilsson TK, Yngve A, Salumets A: Folate-mediated one-carbon metabolism and its effect on female fertility and pregnancy viability. Nutr Rev 2010, 68(2):99–113. Page 6 of 6 Page 6 of 6 Granfors et al. 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Conti M, Andersen CB, Richard F, Mehats C, Chun SY, Horner K, Jin C, Tsafriri A: Role of cyclic nucleotide signaling in oocyte maturation. Mol Cell Endocrinol 2002, 187(1–2):153–159. 24. Sasseville M, Albuz FK, Côté N, Guillemette C, Gilchrist RB, Richard FJ: Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle. Biol Reprod 2009, 81(2):415–425. 24. Sasseville M, Albuz FK, Côté N, Guillemette C, Gilchrist RB, Richard FJ: Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle. Biol Reprod 2009, 81(2):415–425. 25. Sela-Abramovich S, Edry I, Galiani D, Nevo N, Dekel N: Disruption of gap junctional communication within the ovarian follicle induces oocyte maturation. Endocrinology 2006, 147(5):2280–2286. 26. Luciano AM, Pocar P, Milanesi E, Modina S, Rieger D, Lauria A, Gandolfi F: Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization. Mol Reprod Dev 1999, 54(1):86–91. 26. Luciano AM, Pocar P, Milanesi E, Modina S, Rieger D, Lauria A, Gandolfi F: Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization. Mol Reprod Dev 1999, 54(1):86–91. 27. Luciano AM, Modina S, Vassena R, Milanesi E, Lauria A, Gandolfi F: Role of intracellular cyclic adenosine 3',5'-monophosphate concentration and oocyte- cumulus cells communications on the acquisition of the developmental competence during in vitro maturation of bovine oocyte. Biol Reprod 2004, 70(2):465–472. 27. Luciano AM, Modina S, Vassena R, Milanesi E, Lauria A, Gandolfi F: Role of intracellular cyclic adenosine 3',5'-monophosphate concentration and oocyte- cumulus cells communications on the acquisition of the developmental competence during in vitro maturation of bovine oocyte. Biol Reprod 2004, 70(2):465–472. p 28. Granfors et al. 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Annu Rev Genomics Hum Genet 2011, 12:217–244. doi:10.1186/1471-2350-13-121 Cite this article as: Granfors et al.: Phosphodiesterase 8B gene polymorphism in women with recurrent miscarriage: A retrospective case control study. BMC Medical Genetics 2012 13:121. 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https://openalex.org/W4282032901	https://zenodo.org/records/6683780/files/sensors-22-04131-v2.pdf	English	null	Supporting Smart Home Scenarios Using OWL and SWRL Rules	Sensors	2,022	cc-by	12,831	Citation: Reda, R.; Carbonaro, A.; de Boer, V.; Siebes, R.; van der Weerdt, R.; Nouwt, B.; Daniele, L. Supporting Smart Home Scenarios Using OWL and SWRL Rules. Sensors 2022, 22, 4131. https://doi.org/10.3390/ s22114131 Keywords: IoT; home automation; smart home; SAREF; ontology; SWRL; cognitive systems sensors sensors sensors sensors 1 Department of Computer Science and Engineering, University of Bologna, 40136 Bologna, Italy; roberto.reda@unibo.it 2 Computer Science Department, Faculty of Science, Vrije Universiteit Amsterdam, 1081 Amsterdam, The Netherlands; v.de.boer@vu.nl (V.d.B.); r.m.siebes@vu.nl (R.S.); r.p.vander.weerdt@vu.nl (R.v.d.W.) 2 Computer Science Department, Faculty of Science, Vrije Universiteit Amsterdam, 1081 Amsterdam, The Netherlands; v.de.boer@vu.nl (V.d.B.); r.m.siebes@vu.nl (R.S.); r.p.vander.weerdt@vu.nl (R.v.d.W.) 2 Computer Science Department, Faculty of Science, Vrije Universiteit Amsterdam, 1081 Amsterdam, The Netherlands; v.de.boer@vu.nl (V.d.B.); r.m.siebes@vu.nl (R.S.); r.p.vander.weerdt@vu.nl (R.v.d.W.) p ( ) 3 TNO—Netherlands Organization for Applied Scientiﬁc Research, 2597 The Hague, The Netherlands; barry.nouwt@tno.nl (B.N.); laura.daniele@tno.nl (L.D.) 3 TNO—Netherlands Organization for Applied Scientiﬁc Research, 2597 The Hague, The Netherlands; barry.nouwt@tno.nl (B.N.); laura.daniele@tno.nl (L.D.) 3 TNO—Netherlands Organization for Applied Scientiﬁc Research, 2597 The Hague, The Netherlands; barry.nouwt@tno.nl (B.N.); laura.daniele@tno.nl (L.D.) * Correspondence: antonella.carbonaro@unibo.it * Correspondence: antonella.carbonaro@unibo.it Abstract: Despite the pervasiveness of IoT domotic devices in the home automation landscape, their potential is still quite under-exploited due to the high heterogeneity and the scarce expressivity of the most commonly adopted scenario programming paradigms. The aim of this study is to show that Semantic Web technologies constitute a viable solution to tackle not only the interoperability issues, but also the overall programming complexity of modern IoT home automation scenarios. For this purpose, we developed a knowledge-based home automation system in which scenarios are the result of logical inferences over the IoT sensors data combined with formalised knowledge. In particular, we describe how the SWRL language can be employed to overcome the limitations of the well-known trigger-action paradigm. Through various experiments in three distinct scenarios, we demonstrated the feasibility of the proposed approach and its applicability in a standardised and validated context such as SAREF Roberto Reda 1 , Antonella Carbonaro 1,* , Victor de Boer 2 , Ronald Siebes 2 , Roderick van der Weerdt 2 , Barry Nouwt 3 and Laura Daniele 3 1 Department of Computer Science and Engineering, University of Bologna, 40136 Bologna, Italy; roberto.reda@unibo.it Article Roberto Reda 1 , Antonella Carbonaro 1,* , Victor de Boer 2 , Ronald Siebes 2 , Roderick van der Weerdt 2 , Barry Nouwt 3 and Laura Daniele 3 1. Introduction For instance, by combining data from internal thermometers and online weather services it can decide to just open the window and let the fresh air in instead of simply turning on the air conditioner thus saving energy. Additionally, the same system could automatically close the window if it starts raining outside or when the residents leave the house thus guaranteeing safety. Achieving such complex behaviour requires the smart home system to be able to com- bine and analyse data coming from heterogeneous sources according to some formalised knowledge [5,6]. Consequently, automated reasoning capabilities and the adoption of a higher abstraction programming paradigm becomes essential as well [7]. g p g g p g Semantic web (SW) technologies consist of a set of open recommendations for associat- ing data to their formal meaning and have been shown to constitute an appropriate means for achieving data interoperability in IoT systems [8,9]. Additionally, SW naturally enable inference capabilities over semantically annotated data that cannot be obtained using other traditional programming languages. Numerous studies in the literature have shown that SW technologies can be a suitable approach to tackle the complexity of many speciﬁc tasks in speciﬁc areas of modern IoT home automation [10–12]. The use of SW technologies, in particular OWL ontologies, as a means to overcome the poor interoperability in the IoT ﬁeld has been recently largely investigated [5,13,14]. One notable example in the smart home ﬁeld is the Smart Appliance REFerence ontology (SAREF) [15]. SAREF is an OWL-DL ontology, created in close interaction with the industry, that aims at formally describing the core concepts in the smart appliances domain. The ontology deﬁnes concepts for modelling the devices, their tasks and the functions they perform to accomplish the tasks. It also enables the description of the device energy proﬁle and power proﬁle. The ontology is designed to be easily extendable and can be used as basis for creating more specialised ontologies such as SAREF4Health, an extension for IoT- based healthcare systems [16]. Moreover, SAREF has been standardised by the European Telecommunications Standards Institute (ETSI) and experimentally validated [17]. y Rule-based programming approaches such as the trigger-action paradigm are widely employed in actual IoT home automation systems for deﬁning domotic scenarios due to their simplicity and intuitive use [18]. For example, IFTTT is one of the most popular tool for programming IoT scenarios using trigger-action rules [19]. 1. Introduction Academic Editor: Mehmet Rasit Yuce Academic Editor: Mehmet Rasit Yuce Over the last few years, the home automation sector has seen significant adoption of IoT devices performing the most various sensing and actuating capabilities for a plethora of different domotic tasks. Basically, IoT domotic devices are size-contained wireless systems which can be accessed and programmed through the internet to monitor or control home attributes such as air conditioning and heating, lighting, surveillance and home appliances. Received: 31 March 2022 Accepted: 23 May 2022 Published: 29 May 2022 IoT home automation, besides increasing entertainment and user comfort, can provide several potential beneﬁts in other crucial areas such as home safety [1], energy efﬁciency and preservation [2], and elderly care [3]. However, current home automation systems do not fully exploit the intrinsic potential of IoT devices due to two critical challenges they face: the high heterogeneity of the devices and protocols which results in limited interoperability (a common issue that plague the IoT landscape in general); and the limited expressivity of the paradigms adopted to program domotic scenarios [4]. In fact, the majority of commercially available IoT programming environments are proprietary technologies based essentially on the low level abstraction trigger-action model which is limited to processing only a small number of different input data sources (normally, only sensor data gathered by devices from the same vendor) and lacks reasoning features. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 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/). g For example, a typical current domotic scenario for room temperature regulation consists of turning on the air conditioner set to the desired temperature and letting the internal thermostat do the rest. However, a smart IoT system could be embedded with https://www.mdpi.com/journal/sensors Sensors 2022, 22, 4131. https://doi.org/10.3390/s22114131 Sensors 2022, 22, 4131 2 of 19 the knowledge to make it be able to autonomously ﬁnd the best way to cool down the room temperature. 1. Introduction However, even though IFTTT partially extends the expressivity of the simple trigger-action model through the integration of web services, it suffers from several limitations such as a low-level abstraction and low generalisation due to the lack of actual semantics support and reasoning capabilities [4,7]. Indeed, besides providing semantics to IoT data, SW technologies also provide support for dealing with the collected information, that is they naturally enable reasoning capabilities over the semantically annotated data, especially by means of rule languages [20]. In [21], Corno et al. employed SW technologies to overcome the low level abstraction of IFTTT rules in end-user development (EUD) environments. The authors created EUPont, a high-level OWL ontology that provides abstract representations for EUD programming environments for the IoT. The applicability of SW ontologies and rule languages in home automation has been investigated by Bonino et al. [22]. In their work, the authors employed an OWL ontology (i.e., DogOnt ontology [23]) to provide a common semantics and features description for the devices involved, and two rule languages (i.e., SWRL and Jena rules) to perform reasoning. In their system, rules are deﬁned to evaluate structural and state properties of the home environment. Fensel et al. in [24] describe the SESAME-S project (SEmantic SmArt Metering Services for Energy Efficient Houses) which makes use of linked data to assist home tenants in making informed decisions and controlling home energy consumption. Owl ontologies are used to semantically annotate data regarding automation devices, consuming measurement and energy pricing. Rules are employed to implement policy-based decision-making mechanisms. Sensors 2022, 22, 4131 3 of 19 3 of 19 More recently, in [25] Saba et al. have proposed a system for energy management in smart home environments. The OWL ontology on which the system builds up pro- vides a formal representation of the energy aspects of the appliances and other domotic environment elements such as the extent by which they positively or negatively inﬂuence the consumption of electrical energy. SWRL rules are used as reasoning mechanism to implement energy saving scenarios without compromising tenants’ comfort. Previous works in the domotics ﬁeld that have adopted SW technologies focus only on speciﬁc home automation tasks (e.g., energy management). Accordingly, automated reasoning through OWL and SWRL is performed over solely ad hoc written ontologies. 1. Introduction g g p y g Indeed, the necessity of an ontology-agnostic approach in SW solutions have been clearly highlighted in [26] for building-level automation systems. In this work, the authors propose BRICK (Building’s Reasoning for Intelligent Control Knowledge-based System) as an integrated system for intelligent building energy and security management. Stemming from these results, the aim of our work is to suggest a unifying approach in home automation systems by demonstrating how the Semantic Web Rule Language (SWRL) can be employed as a general purpose programming paradigm to implement advanced domotic scenarios which are not limited to work only in a speciﬁc domotic area or rely only on ad hoc ontologies. For this purpose, we developed the Semantic Smart Home System (SSHS), a knowledge-based system which is capable of performing home automation facilities by executing SW rules over in situ collected IoT sensor data potentially combined with external information sources (e.g., curated ontologies or third party web services). Our work builds upon the existing standardised ETSI Smart Applications REFerence (SAREF) ontology (the SAREF ontology is available at https://saref.etsi.org/ (accessed on 30 March 2022)), which, as mentioned above is speciﬁcally designed to enable semantic interoperability in IoT systems including domotics [15,17]. It is important to note that SAREF is a general purpose ontology in the IoT domain, it has not been developed for a single speciﬁc project, therefore it cannot be considered an ad hoc solution. g p p j The experiments that we have carried out showed the feasibility of our approach and the efﬁciency of the SSHS in realistic real-life settings. The remainder of this paper is organised as follows. In Section 2, a detailed description of the architecture and the functioning of the SSHS is provided and illustrates how the SWRL language can be used as a home automation scenario programming paradigm. The experiments that have been carried out using the proposed approach are described in Section 3. Finally, in Section 4 concluding remarks and future research directions close the paper. 2. Materials and Methods The main objective of this study is to demonstrate that SW technologies can be em- ployed to realise home automation scenarios that can fully exploit the potential offered by modern IoT devices. Indeed, in order to achieve an overall higher level of automation, the heterogeneous amount of information collected by IoT devices crucially requires to be integrated and analysed accordingly to some sort of formalised knowledge, such as OWL ontologies. To achieve this goal we developed the SSHS which is a knowledge-based framework that can execute complex home automation scenarios. In SSHS, automatic actions are the result of SWRL rules executed over semantically annotated IoT data potentially combined with external information sources such as curated ontologies or web services. In SSHS, semantics also provides the expressiveness and abstractions which facilitates the task of tackling the programming complexity. That is, in SSHS, rules are expressed in terms of high level entities rather than the respective lower level of the sensor raw values or device speciﬁc actuator commands. p In our study, the SSHS operates within a SAREF-based environment. A SAREF en- vironment, in this context, is essentially composed of two elements: a set of knowledge bases and a knowledge engine. The term knowledge base refers to a general entity which Sensors 2022, 22, 4131 4 of 19 can communicate and exchange data semantically annotated according to the SAREF ontology or one of its extensions. For instance, any IoT device, such as a door switch or a thermometer, in a SAREF environment can be seen as a knowledge base. All the knowledge bases are connected to the knowledge engine through a smart connector (i.e., a generic component that is being developed by the InterConnect consortium (InterConnect consortium: https://interconnectproject.eu/consortium/ (accessed on 30 March 2022))). Knowledge bases conﬁgure their smart connector with their speciﬁc capabilities and RDF graph patterns. The knowledge engine uses these to function as the coordinator sys- tem that allows the knowledge bases to exchange information to each other. The RDF graph patterns are used by the knowledge engine to route the data. Further informa- tion about the Knowledge Engine can be found in the InterConnect public git reposi- tory (InterConnect public git repository: https://gitlab.inesctec.pt/interconnect-public/ knowledge-engine/ (accessed on 30 March 2022)) and deliverable 5.2 (InterConnect Deliv- erable 5.2: https://interconnectproject.eu/wp-content/uploads/2021/02/InterConnect_ WP5_D5.2-DataFlowManagement_draftVersion.pdf (accessed on 30 March 2022)). 2. Materials and Methods g p The SSHS we developed can be seen as a SAREF knowledge base that once connected to the knowledge engine periodically receives sensor data gathered from the IoT devices, executes semantic rules over them, and sends commands back to control the actuators. Since the SAREF ontology provides a common semantics, rules can be written device vendor independently. An example of an SAREF setup is shown in Figure 1, in which four IoT devices collect data from the environment and exchange them with the SSHS that can eventually send commands back to them. IoT Device IoT Device IoT Device IoT Device Semantic Smart Home System Knowledge Engine Figure 1. The schema depicts an example of SAREF environment setup, as intended in this study, composed of four IoT devices that exchange data in RDF format, through the knowledge engine, with the Semantic Smart Home System. Figure 1. The schema depicts an example of SAREF environment setup, as intended in this study, composed of four IoT devices that exchange data in RDF format, through the knowledge engine, with the Semantic Smart Home System. Figure 2 depicts the main architecture of the SSHS. The SSHS is made up of three main components: the update module, the core system and the actuate module. These components realise three phases which are executed in order and cyclically. The ﬁrst phase is the update phase which is performed by the update module. During the update phase, the system gathers all the sensor data measured at that moment by the IoT devices installed in the house. Optionally, the incoming data can be arbitrarily augmented through a software procedure. For example, if a physical device does not Sensors 2022, 22, 4131 5 of 19 5 of 19 provide in its output a timestamp, it can be added at this stage. Data from virtual devices (i.e., web services or external data sources) and conﬁguration information are collected as well. The output of the update phase is an RDF graph which represents the current status of the house combined with all the other information gathered. SWRL RULES SAREF ONTOLOGY EXTERNAL ONTOLOGIES SENSORS DATA DATA AUGMENTATION VIRTUAL DEVICES CORE UPDATE GRAPH PATTERNS INSTANTIATION ACTUATE INTERNAL PROCEDURES SSHS Architecture SAREF Environment SAREF Environment CONFIG Figure 2. The Semantic Smart Home System architecture. 2. Materials and Methods The system is made up of three main components: the update component which collects data from the SAREF environment and constructs h RDF h i h h h t i h h SWRL SWRL RULES SAREF ONTOLOGY EXTERNAL ONTOLOGIES SENSORS DATA DATA AUGMENTATION VIRTUAL DEVICES CORE UPDATE GRAPH PATTERNS INSTANTIATION ACTUATE INTERNAL PROCEDURES SSHS Architecture SAREF Environment SAREF Environment CONFIG SWRL RULES SAREF ONTOLOGY EXTERNAL ONTOLOGIES SENSORS DATA DATA AUGMENTATION VIRTUAL DEVICES CORE UPDATE GRAPH PATTERNS INSTANTIATION ACTUATE INTERNAL PROCEDURES SSHS Architecture SAREF Environment SAREF Environment CONFIG Figure 2. The Semantic Smart Home System architecture. The system is made up of three main components: the update component which collects data from the SAREF environment and constructs the RDF graph representing the current house status, the core component in that executes the SWRL rules over the content of the main graph, and the actuate component which inspects the reasoning result and sends commands back to the actuators. These tasks are executed sequentially in a loop. The second phase is the rules execution phase that is performed by the core system. The core component represents the most important part of the SSHS. It executes the semantic web rules over the RDF graph produced during the previous phase. The semantic web rules are expressed using the SWRL language. The SAREF ontology and optionally other external OWL ontologies, takes part in the inference process as well. In our SSHS implementation, the OWL classiﬁcation and the rule execution are performed by the reasoner Pellet which is an open source OWL-DL reasoner that also features an embedded SWRL inference engine [27]. The third and the last phase is the actuate phase that is performed by the actuate module. The actuate module ﬁrst analyses the result of the reasoning process and then accordingly it generates the graph pattern instantiations (i.e., the device commands) to be sent to the knowledge engine in order to operate the actuators such as turning on a lamp or closing a shutter. Along with graph pattern instantiations, during the actuate phase internal procedures are executed to modify the status of the virtual devices. 2.2. Implementation Details We implemented a prototype of the SSHS using the Python language. The Owl- ready2 [28] framework was used to connect the update and the actuate component to the core component which is enabled by the Pellet reasoner [27]. Pellet is an open source OWL2-DL reasoner that natively supports SWRL rules execution. Some demo code is available at https://github.com/robertoReda/sshs (accessed on 30 March 2022). 2.1. IoT as Data Sources Reasoning over IoT sensor data combined with external information sources is the key feature of the SSHS. Within the SSHS, every data source can be modelled as an IoT device. In SSHS an IoT device can be of three kinds: physical device, augmented device and virtual device. Sensors 2022, 22, 4131 6 of 19 Physical devices correspond to the actual IoT devices materially installed inside the house. For instance, a physical device could be a door switch sensor mounted on a door frame or a thermometer which gauges the room temperature. For each physical sensor in the house, the update module collects its status information as an RDF graph as it comes as output from the device itself. An augmented device is basically a physical device to whose output graph one or more pieces of extra information are added through a software procedure during the update phase. This is necessary because unfortunately IoT devices do not always provide by default all the essential information that might be needed to write rules, such as their status history. For instance, most of the colour changing light bulbs available on the market provide in their status only the current colour which is actually being displayed while sometimes it could be necessary to keep track of the previous conﬁgurations to restore a previous state after temporarily changing it. A virtual device is a generic IoT device whose functionalities are fully software emu- lated. Virtual devices do not have a corresponding physical device installed in the house. For example, the information that an IoT weather station provides (e.g., temperature, humidity, wind speed, rainfall and solar radiation) can be also obtained from an online weather web service thus making unnecessary (at least in some cases) the presence of an actual physical device in the house. Virtual devices can also be used to provide function- alities that are not natively available in SWRL or OWL run-time environments, such as functions to retrieve the current date. However, in SSHS, a clock can be easily implemented as a virtual IoT that provides as output the actual date time. Virtual devices can either be implemented as software procedures invoked by the update module or as separate SAREF knowledge bases connected directly to the knowledge engine. 2.3. Rules Writing and Scenarios A home automation scenario refers to a set of actions that are performed when certain conditions are met. Most of the current home automation tools adopt the trigger-action model as a home automation scenario programming paradigm [4]. In trigger-action systems the desired behaviour is speciﬁed by means of rules in the form “if-then” where the “if” part of the rule checks whether a particular event (i.e., the trigger) has occurred and the “then” part speciﬁes the action that should be executed in response. For instance, a typical scenario rule could be: “If the leak sensor detects some water, then turn off the washing machine”. Rule-based languages provide an intuitive way to program home automation scenarios especially when IoT devices are involved [4]. However, IoT home automation tools that do not adopt semantics suffer from several important limitations. First, the impossibility to deﬁne generic sets of rules for devices which have similar functionalities instead of vendor speciﬁc rules. Second, triggers and actions can be speciﬁed only in terms of device output values and device commands which implies a low level of abstraction. Finally, actions are determined on the sole base of data input sources due to the lack of reasoning capabilities. The Semantic Web Rule Language (SWRL) [29,30] allows the deﬁnition of rules in terms of OWL entities, that is, it combines the ease of rule-languages with the capability to perform automated reasoning. SWRL is the means by which domotic scenarios are programmed in the SSHS. In SSHS automatic actions are determined by analysing the IoT input data according to knowledge expressed in OWL ontologies or in the rules themselves. Sensors 2022, 22, 4131 7 of 19 7 of 19 In other words, in SSHS automatic actions are the result of logical inferences drawn over the RDF graph that represents the current status of the house. In other words, in SSHS automatic actions are the result of logical inferences drawn over the RDF graph that represents the current status of the house. Technically, SWRL provides a high-level abstract syntax for horn-like rules fully compliant with OWL semantics. A SWRL rule is composed of two parts, an antecedent and a consequent where both the antecedent and the consequent consist of a positive conjunction of atoms. Since SWRL rules are expressed in terms of OWL concepts, atoms can be either individuals, properties or classes deﬁned within the knowledge base. 2.3. Rules Writing and Scenarios This feature of SWRL is particularly important in SSHS because, in rule deﬁnition, IoT sensor data and knowledge can be composed together in a seamless way, thus allowing to achieve a higher level of abstraction. For example, a rule to automatically turn on a lamp when the natural light drops could be written as shown in Listing 1 regardless of how the low light condition of the room has been actually determined. For instance, it could be either obtained by analysing the output of a physical photocell (this would be the only possible way with a trigger-action system) or inferred using the information contained in an ontology given the period of the year and the current time. Alternatively, the same information could be achieved by consulting a web service. Listing 1: A SWRL rule tha can be used to switch on a lamp in case of low light condition inside a room. lowLightLevel (Room) =⇒ switchOn (Lamp) lowLightLevel (Room) =⇒ switchOn (Lamp) This approach overcomes the limitations of the simple trigger-action model; the higher level of abstraction in rule deﬁnition helps signiﬁcantly to tackle the complexity of deﬁning advanced home automation scenarios. Most importantly, automated reasoning capabilities provided by OWL and SWRL dramatically increase the overall degree of automation that can be potentially achieved by the system. In trigger-action programming, to check whether scenario conditions are met two types of triggers can be employed: event triggers and state triggers [31]. Event triggers refer to when an asynchronous event occurs, that is when a certain condition becomes true at a particular instant in time. For example, when a button is pressed or when a presence sensor detects a person entering a room. State triggers occur when a condition is true over a period of time. For instance, the condition that it is raining outside or the temperature is above a certain threshold. In the SSHS, the only way to determine where scenario conditions are met is to analyse the current status of the house represented by the RDF graph that is constructed during the update phase. Since the home status RDF graph is sampled at regular intervals, state triggers are naturally supported, but asynchronous events can not activate any immediate response. However, in SSHS, event triggers can be easily simulated by checking whether the event condition has occurred in the near instant using the timestamp provided by the IoT devices. For example, the rule in Listing 2 can be used to automatically turn on the garden lights when a car passing through the gate is detected by a photocell. Listing 2: This SWRL rule switches on the garden lights if the photocell has detected the passage of a car within the last ten seconds. Listing 2: This SWRL rule switches on the garden lights if the photocell has detected the passage of a car within the last ten seconds. Listing 2: This SWRL rule switches on the garden lights if the photocell has detected the passage of a car within the last ten seconds. hasClosedState ( photocell ) ∧hasTimeStamp ( photocell , ? ts ) ∧currentTime (? ct ) ∧swrlb : subtract (? ct , ? ts , ? delta ) ∧swrlb : greaterThan (? delta , 0) ∧swrlb : lessThan (? delta , 10 ,000) =⇒ switchOn ( gardenLight ) car within the last ten seconds. lowLightLevel (Room) =⇒ switchOn (Lamp) hasClosedState ( photocell ) ∧hasTimeStamp ( photocell , ? ts ) ∧currentTime (? ct ) ∧swrlb : subtract (? ct , ? ts , ? delta ) ∧swrlb : greaterThan (? delta , 0) ∧swrlb : lessThan (? delta , 10 ,000) =⇒ switchOn ( gardenLight ) hasClosedState ( photocell ) ∧hasTimeStamp ( photocell , ? ts ) ∧currentTime (? ct ) ∧swrlb : subtract (? ct , ? ts , ? delta ) ∧swrlb : greaterThan (? delta , 0) ∧swrlb : lessThan (? delta , 10 ,000) =⇒ switchOn ( gardenLight ) Sensors 2022, 22, 4131 8 of 19 8 of 19 The above rule checks whether the difference between the current time and the instant reported in the time stamp is below a certain threshold. The rule can be simpliﬁed by deﬁning another rule which generalises this concept as shown in Listing 3. Listing 3: The ﬁrst rule is used to detect that a generic event has happened within the last ten seconds while the second rule makes use of the ﬁrst one to control the garden lights. saref : hasTimeStamp (? device , ? ts ) ∧currentTime (? ct ) ∧swrlb : subtract (? ct , ? ts , ? delta ) ∧swrlb : greaterThan (? delta , 0) ∧swrlb : lessThan (? delta , 10 ,000) =⇒ tenSecondsEvent (? device ) tenSecondsEvent ( gardenLight ) =⇒ switchOn ( gardenLight ) saref : hasTimeStamp (? device , ? ts ) ∧currentTime (? ct ) ∧swrlb : subtract (? ct , ? ts , ? delta ) ∧swrlb : greaterThan (? delta , 0) ∧swrlb : lessThan (? delta , 10 ,000) =⇒ tenSecondsEvent (? device ) tenSecondsEvent ( gardenLight ) =⇒ switchOn ( gardenLight ) Generally, time stamps enable time reasoning capabilities that are particularly useful for dealing with ordered events. For example, an action is performed only if a person has pressed a button after entering a room and not vice versa. It is worth to note that neither SWRL nor OWL natively provide current time function- ality which is essential to simulate event triggers in the SSHS. However, time information can be easily introduced into the system by using virtual IoT devices (i.e., in this case a simulated clock device). Similarly to triggers, actions can be distinguished into instant actions, sustained actions, and extended actions [31]. lowLightLevel (Room) =⇒ switchOn (Lamp) Instant actions occur at a particular instant of time such as “turn on the light”. Sustained actions are performed as long as a condition holds, for example, “light is on as long as there is someone in the room”. Extended actions are performed for a speciﬁc time interval, therefore time stamps are needed to accomplish this task. For example, “change the light colour to red for 10 s”. After a sustained or extended action it is often necessary to resume the previous state of the device. Since it is not possible to store indeﬁnitely new knowledge within the knowledge base (i.e., the house state graph is rebuilt at each update phase), augmented IoT devices can be employed when the devices do not natively provide a status history or when it is not possible to infer the previous state from the current graph. OWL and consequently SWRL knowledge bases are monotonic; new knowledge can be added, but existing knowledge can not be retracted or modiﬁed. In the SSHS, rules can not directly change the state of the IoT devices. For example, if a lamp has to be turned on, rules classify the respective OWL individual into a desired state which represents the action that should be taken. Only during the update phase actual commands are sent to devices according to the classiﬁcation results in order to reﬂect changes. Actual changes to the environment are visible within the knowledge base only after the next update phase has been completed. 3. Results An IoT home automation system is essentially composed of a variable number of devices that include both sensors to perceive the domestic environment and actuators to perform actions on it, connected to a programmable unit that implements the control logic (i.e., the domotic scenarios). The majority of traditional IoT home automation systems adopt the trigger-action model as a programming paradigm for deﬁning scenarios, according to which every time certain conditions are met, a speciﬁc action is executed. The SSHS that we developed extends the trigger-action model by introducing auto- mated reasoning capabilities in the process. In our system, domotic scenarios are expressed by means of SWRL rules that combine knowledge with trigger-action deﬁnitions. Therefore, the automatic actions are not only determined based on sensor data, but are the result of logical inferences enabled by the underlying semantics provided by the SAREF ontology. Sensors 2022, 22, 4131 9 of 19 9 of 19 The aim of this approach is to achieve greater expressivity and a higher level of abstraction needed to build knowledge-enabled and reasoning-capable home automation systems, so that the potential offered by IoT devices can be fully exploited. The aim of this approach is to achieve greater expressivity and a higher level of abstraction needed to build knowledge-enabled and reasoning-capable home automation systems, so that the potential offered by IoT devices can be fully exploited. p y y p In order to show the feasibility of the proposed approach, we have designed and tested on our prototype several different knowledge-involving domotic scenarios that operate under different conditions with different kinds of IoT devices. These use cases also are meant to demonstrate that scenarios in SSHS can be based on web standards and public ontologies and implement well-deﬁned reasoning without the necessity of ad hoc control programs or even ad hoc ontologies. Moreover, we evaluated the system performance by measuring the processing time taken by the reasoner to evaluate rules with different numbers of devices involved in order to demonstrate the efﬁciency of the SSHS in realistic settings. y g Experiments were conducted using a simulated house environment. IoT devices output data were synthetically generated using a Python script. This testing method is particularly convenient since it is economical, signiﬁcantly speeds up the process of recreating the desired experimental conditions and simpliﬁes the analysis of the resulting system behaviour. 3. Results Moreover, it is worth to note that since the semantics provided by the SAREF ontology makes data device-independent, synthetic generated IoT output graphs do not lack any relevant information that might be acquired in a real life setting. y g q g An example of a simulated domestic environment that we used as a testbed to carry out experiments is shown in Figure 3. In this case, the environment consists of a 6 room ﬂat plus a terrace, equipped with 29 IoT devices including light switches, door/windows switches, thermometers and a presence detection sensor. An excerpt of the corresponding RDF graph model of the house is shown in Listing 4. As it can be seen, rooms are modelled as OWL individuals that belong to speciﬁc classes according to their functions within the house. SAREF4BLDG (SAREF4BLDG ontology: https://saref.etsi.org/saref4bldg/ (accessed on 30 March 2022)), an extension of the SAREF core ontology, has been employed for this purpose. For example, the terrace is represented by means of an OWL individual that belongs to the class s4bldg:BuildingSpace to indicate that it is an outdoor ambient. Static knowledge about the environment and any other relevant information that cannot be inferred from the IoT data sources, such as the aforementioned spatial features of the house, can be introduced into the SSHS as conﬁguration data in RDF format. There are no constraints on the quantity and the type of information that can vary signiﬁcantly and strictly depends on the speciﬁc scenarios that make use of it. An example of a simulated domestic environment that we used as a testbed to carry out experiments is shown in Figure 3. In this case, the environment consists of a 6 room ﬂat plus a terrace, equipped with 29 IoT devices including light switches, door/windows switches, thermometers and a presence detection sensor. An excerpt of the corresponding RDF graph model of the house is shown in Listing 4. As it can be seen, rooms are modelled as OWL individuals that belong to speciﬁc classes according to their functions within the house. SAREF4BLDG (SAREF4BLDG ontology: https://saref.etsi.org/saref4bldg/ (accessed on 30 March 2022)), an extension of the SAREF core ontology, has been employed for this purpose. For example, the terrace is represented by means of an OWL individual that belongs to the class s4bldg:BuildingSpace to indicate that it is an outdoor ambient. 3. Results ex : Thermometer rdf : type saref : TemperatureSensor . ex : LivingRoom s4bldg : contains ex : PresenceDetector . ex : PresenceDetector rdf : type saref : Device . ex : PresenceDetector rdf : type saref : Sensor . Listing 4: An excerpt of the RDF graph that represents the spatial features of the domestic environ- ment shown in Figure 3. Listing 4: An excerpt of the RDF graph that represents the spatial features of the domestic environ- ment shown in Figure 3. Listing 4: An excerpt of the RDF graph that represents the spatial features of the domestic environ- ment shown in Figure 3. Listing 4: An excerpt of the RDF graph that represents the spatial features of the domestic environ- ment shown in Figure 3. ex : Building rdf : type s4bldg : Building . ex : Building s4bldg : hasSpace ex : House . ex : House rdf : type s4bldg : BuildingSpace . ex : House rdf : type saref : FeatureOfInterest . ex : House s4bldg : hasSpace ex : LivingRoom . ex : LivingRoom rdf : type s4bldg : BuildingSpace . ex : LivingRoom rdf : type saref : FeatureOfInterest . ex : House s4bldg : hasSpace ex : Terrace . ex : Terrace rdf : type s4bldg : BuildingSpace . ex : Terrace rdf : type saref : FeatureOfInterest . ex : LivingRoom s4bldg : contains ex : Door . ex : Door rdf : type s4bldg : BuildingObject . ex : LivingRoom s4bldg : contains ex :Window . ex :Window rdf : type s4bldg : BuildingObject . s4bldg : BuildingObject rdfs : subClassOf s4bldg : PhysicalObject . ex : LivingRoom s4bldg : contains ex : LightSwitch . ex : LightSwitch rdf : type saref : Device . saref : Device rdfs : subClassOf s4bldg : PhysicalObject . ex : LightSwitch rdf : type saref : Actuator . ex : LivingRoom s4bldg : contains ex : Thermometer . ex : Thermometer rdf : type saref : Device . ex : Thermometer rdf : type saref : TemperatureSensor . ex : LivingRoom s4bldg : contains ex : PresenceDetector . ex : PresenceDetector rdf : type saref : Device . ex : PresenceDetector rdf : type saref : Sensor . ment shown in Figure 3. ex : Building rdf : type s4bldg : Building . 3. Results g Static knowledge about the environment and any other relevant information that cannot be inferred from the IoT data sources, such as the aforementioned spatial features of the house, can be introduced into the SSHS as conﬁguration data in RDF format. There are no constraints on the quantity and the type of information that can vary signiﬁcantly and strictly depends on the speciﬁc scenarios that make use of it. Terrace Living room Bedroom Kitchen Entrance Utility Room W1L W2L W1B D1B D1L W1K D1K D1E DOOR/WINDOW D1U T1L T1B T1E T1K T1T THERMOMETER LIGHT SWITCH L1B L1L L1T L1K L1E L1U BUTTON B1E PRESENCE P1B P1T P1L P1U P1E P1K P2E PRESENCE P1B P1T P1L P1U P1E P1K P2E LK1E LOCK Figure 3. The schema represents the ﬂoor-plan of typical domestic environment equipped with various domotic IoT devices. Figure 3. The schema represents the ﬂoor-plan of typical domestic environment equipped with various domotic IoT devices. Sensors 2022, 22, 4131 10 of 19 Listing 4: An excerpt of the RDF graph that represents the spatial features of the domestic environ- ment shown in Figure 3. ex : Building rdf : type s4bldg : Building . ex : Building s4bldg : hasSpace ex : House . ex : House rdf : type s4bldg : BuildingSpace . ex : House rdf : type saref : FeatureOfInterest . ex : House s4bldg : hasSpace ex : LivingRoom . ex : LivingRoom rdf : type s4bldg : BuildingSpace . ex : LivingRoom rdf : type saref : FeatureOfInterest . ex : House s4bldg : hasSpace ex : Terrace . ex : Terrace rdf : type s4bldg : BuildingSpace . ex : Terrace rdf : type saref : FeatureOfInterest . ex : LivingRoom s4bldg : contains ex : Door . ex : Door rdf : type s4bldg : BuildingObject . ex : LivingRoom s4bldg : contains ex :Window . ex :Window rdf : type s4bldg : BuildingObject . s4bldg : BuildingObject rdfs : subClassOf s4bldg : PhysicalObject . ex : LivingRoom s4bldg : contains ex : LightSwitch . ex : LightSwitch rdf : type saref : Device . saref : Device rdfs : subClassOf s4bldg : PhysicalObject . ex : LightSwitch rdf : type saref : Actuator . ex : LivingRoom s4bldg : contains ex : Thermometer . ex : Thermometer rdf : type saref : Device . 3. Results ex : Building s4bldg : hasSpace ex : House . ex : House rdf : type s4bldg : BuildingSpace . ex : House rdf : type saref : FeatureOfInterest . ex : House s4bldg : hasSpace ex : LivingRoom . ex : LivingRoom rdf : type s4bldg : BuildingSpace . ex : LivingRoom rdf : type saref : FeatureOfInterest . ex : House s4bldg : hasSpace ex : Terrace . ex : Terrace rdf : type s4bldg : BuildingSpace . ex : Terrace rdf : type saref : FeatureOfInterest . g yp saref : Device rdfs : subClassOf s4bldg : PhysicalObject . ex : LivingRoom s4bldg : contains ex : Thermometer An example of an RDF graph representing an IoT data source and its associated measurement is shown in Listing 5. The graph regards a thermometer that is physically located on the terrace. The measurement value is reported along with the unit and the timestamp of the gauging instant in ISO-8601 format and Unix time. Listing 5: An excerpt of RDF graph that represents the output of an IoT thermometer annotated according to the SAREF ontology. The graph contains information about the physical collocation of the device within the house as well as the measurement in degree Celsius and the timestamp. ex : T1T rdf : type saref : TemperatureSensor ; saref : makesMeasurement ex : T1T_m1 ; s4bldg : isContainedIn ex : Terrace . ex : T1T_m1 rdf : type saref : Measurement ; saref : hasValue ‘ ‘25.5 ’ ’^^ xsd : f l o a t ; saref : isMeasuredIn om: degree_Celsius ; saref : hasTimestamp ‘ ‘2020 −12 −02T14 :30:00 ’ ’^^ xsd : dateTime ; smart : hasUnixTimestamp ‘ ‘1606919400 ’ ’^^ xsd : integer . During normal operation, the SSHS cyclically aggregates the various IoT data sources to construct an RDF graph that represents the current status of the domestic environment. Rules are then executed over this graph and the inferred knowledge is added back to it. Lastly, the system inspects the resulting graph to send, if necessary, commands to the Sensors 2022, 22, 4131 11 of 19 11 of 19 actuator devices. For our tests, the last phase was omitted and the resulting graph was directly analysed using the Protege tool. actuator devices. For our tests, the last phase was omitted and the resulting graph was directly analysed using the Protege tool. 3.1. Energy Conservation Monitoring Scenario Often, homeowners are unaware of the costs of some domestic energy wasting be- haviours they involuntarily adopt, such as forgetting the windows open or leaving them open for too long a period of time. Therefore, monitoring in-home energy wasting in order to provide tenants immediate feedback can be a crucial feature for actuating an efﬁcient energy wasting reduction in an IoT home automation system [32]. We implemented a domotic scenario that assesses the energy wasting level of a room according to the state of the windows (i.e., either open or closed). Top-down proceeding, the monitoring system consists of two main rules, shown in Listing 6, that classify the energy wasting level into two states: GreenEnergyState if there is no energy wasting (i.e., windows in the room are closed) or RedEnergyState if there is energy wasting (i.e., windows in the room are open). Listing 6: SWRL rules for classifying the energy wasting level in a room. smarthouse : ClosedWindow (?window) ∧s4bldg : isContainedIn (?window, ?room) =⇒ smarthouse : GreenEnergyState (?room) smarthouse :OpenWindow(?window) ∧s4bldg : isContainedIn (?window, ?room) =⇒ smarthouse : RedEnergyState (?room) Listing 6: SWRL rules for classifying the energy wasting level in a room. Listing 6: SWRL rules for classifying the energy wasting level in a room. Listing 6: SWRL rules for classifying the energy wasting level in a room. smarthouse : ClosedWindow (?window) ∧s4bldg : isContainedIn (?window, ?room) =⇒ smarthouse : GreenEnergyState (?room) smarthouse :OpenWindow(?window) ∧s4bldg : isContainedIn (?window, ?room) =⇒ smarthouse : RedEnergyState (?room) For each window of the house, ﬁrst its state is detected. Then, the location of the win- dow within the house is retrieved, and the corresponding room is ﬁnally classiﬁed accordingly. A possible way to detect whether a window is open or closed could be achieved by installing a door switch IoT sensor on the window frame. For example, the closed window and the open window classiﬁcation can be determined by retrieving the device state using the rules in Listing 7. Listing 7: The SWRL rules are used to detect whether a window is open using a switch sensor state. Listing 7: The SWRL rules are used to detect whether a window is open using a switch sensor state. smart :Window(?window) ∧saref : hasState (?window, ? s t a t e ) ∧saref : CloseState (? s t a t e ) =⇒ smarthouse : ClosedWindow (?window) smart :Window(?window) ∧saref : hasState (?window, ? 3.1. Energy Conservation Monitoring Scenario s t a t e ) ∧saref : OpenState (? s t a t e ) =⇒ smarthouse :OpenWindow(?window) Listing 7: The SWRL rules are used to detect whether a window is open using a switch sensor state. smart :Window(?window) ∧saref : hasState (?window, ? s t a t e ) ∧saref : CloseState (? s t a t e ) =⇒ smarthouse : ClosedWindow (?window) smart :Window(?window) ∧saref : hasState (?window, ? s t a t e ) ∧saref : OpenState (? s t a t e ) =⇒ smarthouse :OpenWindow(?window) Since it cannot be inferred that a sensor is either installed on a window or a door, it is necessary to explicitly specify it as conﬁguration data. This can be done either through the device settings or using rules. For example, the rule in Listing 8 states that the OWL individual W1L is a door switch installed on a window. Sensors 2022, 22, 4131 12 of 19 12 of 19 Listing 8: The SWRL rule asserts that a DoorSwitch sensor W1L is installed on a window. Listing 8: The SWRL rule asserts that a DoorSwitch sensor W1L is installed on a window. saref : DoorSwitch (W1L) =⇒ Window(W1L) The location of the window can be inferred through the location of the sensor (if it has been indicated in the device settings). However, not always an open window causes heat loss, for example when the differ- ence between the indoor and outdoor temperature is negligible. Therefore, to achieve more precision, rules can be modify so that the temperature is taken into account as in Listing 9. Listing 9: SWRL rules for classifying the energy wasting level in a room by taking into account several factors. smarthouse :OpenWindow(?window) ∧s4bldg : isContainedIn (?window, ?room) ∧smarthouse : hasIntExtTempDifference (?room , delta ) ∧swrlb : greaterThan (? delta , 0 . 5 ) =⇒ smarthouse : RedEnergyState (?room) smarthouse :OpenWindow(?window) ∧s4bldg : isContainedIn (?window, ?room) ∧smarthouse : hasIntExtTempDifference (?room , delta ) ∧swrlb : greaterThan (? delta , 0 . 5 ) =⇒ smarthouse : RedEnergyState (?room) The internal temperature can be acquired by installing a thermometer inside the room, while the external temperature can be acquired by either installing a thermometer outside (e.g., on the terrace) or using an IoT virtual device that wraps an online weather web service. 3.1. Energy Conservation Monitoring Scenario When the two measurements are available the difference can be easily obtained as shown in Listing 10: Listing 10: SWRL rule for calculating the difference between the internal temperature and the external temperature. smarthouse : hasIndoorTemperature (?room , ? internalTemp ) ∧smarthouse : hasOutdoorTemperature (? house , ? externalTemp ) ∧swrlb : subtract (? internalTemp , ? externalTemp , ? delta ) =⇒ smarthouse : hasIntExtTempDifference (?room , delta ) smarthouse : hasIndoorTemperature (?room , ? internalTemp ) ∧smarthouse : hasOutdoorTemperature (? house , ? externalTemp ) ∧swrlb : subtract (? internalTemp , ? externalTemp , ? delta ) =⇒ smarthouse : hasIntExtTempDifference (?room , delta ) Eventually, once an energy wasting situation has been detected, remedial actions could be performed such as warning the tenants or automatically turning off the heating system in the room. 3.2. Visual Cueing System Scenario saref : PresenceSensor ( P1E ) ∧saref : hasState (P1E , OnState ) =⇒ smarthouse : VisitorAtTheDoor ( Entrance ) Listing 12: This SWRL rule is used to detect the presence of a visitor at the door using a volumet- ric sensor. saref : PresenceSensor ( P1E ) ∧saref : hasState (P1E , OnState ) =⇒ smarthouse : VisitorAtTheDoor ( Entrance ) Similarly, the same operation has to be performed when a doorbell button (i.e., a smart button) is employed. However, since a button is kept pressed only for a few instants of time, a timing mechanism is needed since the entity Entrance should persist in the VisitorAtTheDoor state for a certain number of seconds after the button has been released. This can be achieved by checking the button timestamp, that is, the visitor presence is detected until 10 s have passed since the button press occurred. To retrieve the current time, a virtual IoT device MainClock is used. An example of such a rule is provided in Listing 13. Listing 13: SWRL rule to detect the pressure of the doorbell button using the device timestamp. Listing 13: SWRL rule to detect the pressure of the doorbell button using the device timestamp. smarthouse : hasUnixTimestamp ( MainClock , ? clockTime ) ∧smarthouse : SmartButton ( B1E ) ∧smarthouse : hasUnixTimestamp (B1E , ? doorbellTime ) ∧swrlb : subtract (? elapsedTime , ? clockTime , ? doorbellTime ) ∧swrlb : lessThan (? elapsedTime , 10,000) =⇒ smarthouse : VisitorAtTheDoor ( Entrance ) g p g p smarthouse : hasUnixTimestamp ( MainClock , ? clockTime ) ∧smarthouse : SmartButton ( B1E ) ∧smarthouse : hasUnixTimestamp (B1E , ? doorbellTime ) ∧swrlb : subtract (? elapsedTime , ? clockTime , ? doorbellTime ) ∧swrlb : lessThan (? elapsedTime , 10,000) =⇒ smarthouse : VisitorAtTheDoor ( Entrance ) The proposed scenario can be easily extended or modiﬁed. For example, tenants could receive an alert on their mobile phones if they are not inside and the visual signalling can be automatically disabled during sleep time. 3.2. Visual Cueing System Scenario Largely widespread in the modern domotic landscape, IoT smart light devices are inexpensive colour-changing LED light bulbs remotely controllable. Initially intended for ambient lighting enhancement, smart bulbs can be efﬁciently used as means to provide visual cues in IoT home automation systems. For example, if tenants are watching the TV or speaking on the phone, to signal the end of the washing machine cycle, the room light could be slightly turned to a blue-like colour instead of relying on an annoying buzzer sound. Most importantly, such a pervasive visual cueing system can potentially result in an efﬁcient low-cost assistive technology in case of hearing impaired tenants [33]. gy g p We designed a domotic scenario that exploits the colour-changing features of the IoT house lighting system to signal the presence of a visitor at the entrance door. The rule in Listing 11 turns the lights colour to red when someone has pressed the doorbell or is standing close to the entrance door. Tenant localisation within the house is performed in order to avoid unnecessary light switching (i.e., only lights in occupied rooms are involved). Alternatively, if indoor tenant localisation is not possible (e.g., due to the lack of presence sensors in every room or it cannot be inferred in another way), a restricted set of light bulbs can be speciﬁcally designated for this purpose. Sensors 2022, 22, 4131 13 of 19 Listing 11: SWRL rule that signals the presence of a visitor by turning the colour of indoor light to red. smarthouse : VisitorAtTheDoor ( Entrance ) ∧smarthouse : Tenant (? tenant ) ∧smarthouse : isLocatedIn (? tenant , ?room) ∧saref : LightSwitch (? l i g h t ) ∧smarthouse : isLocatedIn (? light , ?room) =⇒ smarthouse : TemporaryRedLightColourState (? l i g h t ) To detect a visitor at the entrance door, either a presence/motion sensor mounted on the door frame or a smart button can be used. When a presence sensor is used, the rule shown in Listing 12 classiﬁes the entity Entrance into the state VisitorAtTheDoor according to sensor state. Listing 12: This SWRL rule is used to detect the presence of a visitor at the door using a volumet- ric sensor. 3.3. Weather Based Domotic Scenario IoT weather stations are devices that provide information about the local external environment, including temperature, humidity, wind speed, and rain conditions. In home automation, local climate data can be exploited in several ways, for example, to automati- cally regulate the curtain position according to natural light and retract it in case of strong wind. One disadvantage of IoT weather stations is that they are expensive equipment, especially compared to other common IoT domotic devices. However, in the SSHS, a physical IoT weather station can be easily replaced by a virtual IoT device that retrieves the same information from an online weather web service. We designed a domotic scenario that, based on current weather conditions, automat- ically disables the garden irrigation system in case of rain, thus reducing unnecessary water consumption. Sensors 2022, 22, 4131 14 of 19 14 of 19 For this scenario, we also implemented a virtual IoT device that acts as a weather station. All the information that the virtual device provides is retrieved from the Open- Weather web service [34]. OpenWeather offers access to current weather data for any location speciﬁed by geographic coordinates or city name. Data include information about the weather conditions (e.g., rain, snow, etc.), temperature, humidity, wind speed, and in particular the rain volume for the last 3 h. p Basically, the virtual device consists in a Python script that queries the OpenWeather web service and translates the JSON result into RDF. An excerpt of the output graph is shown in Listing 14. Listing 14: An excerpt of the output RDF graph of the weather base station implemented through a virtual IoT device. Listing 14: An excerpt of the output RDF graph of the weather base station implemented through a virtual IoT device. Listing 14: An excerpt of the output RDF graph of the weather base station implemented through a virtual IoT device. ex :WS1 a smart : SmartWeatherStation ; saref : hasTimestamp ‘ ‘2020 −12 −02T14 :30:00 ’ ’^^ xsd : dateTime ; smart : hasUnixTimestamp ‘ ‘1606919400 ’ ’^^ xsd : integer ; smart : hasSunriseTime 1634018420 ; smart : hasSunsetTime 1634057589 ; smart : hasTemperature 9.29 ; smart : hasWeatherCondition smart : WeatherConditionRain ; smart : hasRain3h 3. 3.3. Weather Based Domotic Scenario ex :WS1 a smart : SmartWeatherStation ; saref : hasTimestamp ‘ ‘2020 −12 −02T14 :30:00 ’ ’^^ xsd : dateTime ; smart : hasUnixTimestamp ‘ ‘1606919400 ’ ’^^ xsd : integer ; smart : hasSunriseTime 1634018420 ; smart : hasSunsetTime 1634057589 ; smart : hasTemperature 9.29 ; smart : hasWeatherCondition smart : WeatherConditionRain ; smart : hasRain3h 3. We suppose that the irrigation system is activated every day at a ﬁxed time. The rules in Listing 15 prevent the electromechanical valve from opening if the rainfall amount in the last 3 h is greater than 2 mm. Listing 15: This SWRL rule prevent the irrigation system to water the garden if the amount of rain in the last 3 h exceeds 2 mm. Listing 15: This SWRL rule prevent the irrigation system to water the garden if the amount of rain in the last 3 h exceeds 2 mm. smart : SmartWeatherStation (WS1) ∧smart : hasRain3h : ( WS1, ? r a i n f a l l ) ∧swrlb : greaterThan (? r a i n f a l l , 2) =⇒ smart : keepClosedState (VALVE1) smart : SmartWeatherStation (WS1) ∧smart : hasRain3h : ( WS1, ? r a i n f a l l ) ∧swrlb : greaterThan (? r a i n f a l l , 2) =⇒ smart : keepClosedState (VALVE1) Similarly, the irrigation system should be disabled if it has started raining during its functioning. This can be obtained by adding another rule as shown in Listing 16. Listing 16: This SWRL rule prevents the irrigation system to water the garden while it is raining. Listing 16: This SWRL rule prevents the irrigation system to water the garden while it is raining. smart : SmartWeatherStation (WS1) ∧smart : hasWeatherCondition (WS1, WeatherConditionRain ) =⇒ smart : keepClosedState (VALVE1) It has to be noted that two separate rules are necessary since SWRL does not support atom disjunction. 3.4. Performance Evaluation The main advantage of the SSHS over traditional systems based on the trigger-action model is the capability of performing automated reasoning over sensor data and formalised knowledge provided by SWRL rules and OWL ontologies. Knowledge-based systems enabled by DL-OWL reasoners necessitate higher computational capacity compared to systems implemented using general purpose languages such as C++ or Java. Nevertheless, home automation systems are real-time applications that require logic instructions to be exe- cuted in a minimal amount of time so as not to cause unwanted delays in automatic actions. Therefore, it is essential that the SSHS completes the inferential process in appropriate time to make it suitable to operate in a real-setting environment. Sensors 2022, 22, 4131 15 of 19 15 of 19 In order to asses the time performance of the SSHS, similarly to what has been done by Zhai et al. in [35], we executed a set of rules varying the number of devices involved in each experiment repetition, and we measured the average processing time (i.e., the time that the reasoner takes to draw all the inferences) for each conﬁguration. The employed rule-set comprehends: 2 rules that determine whether a room is occupied or unoccupied according to a sensor state (i.e., classiﬁcation task); 5 rules that classify the air quality into ﬁve levels based on the concentration of the CO2 in the room (Wisconsin Department of Health Services. Health effects produced by exposure to CO2. https://www.dhs.wisconsin. gov/chemical/carbondioxide.htm (accessed on 30 March 2022) (i.e., a task that require signiﬁcant numerical comparisons); 2 rules that make use of the preceding results and an OWL ontology to automatically open the windows in the room in case of high concentration of CO2 if people are present (i.e., a task than involve a chain of inferences and additional OWL axioms). An excerpt of the rule-set is provided in Listing 17. Listing 17: An excerpt of the set of rules employed to measure the average processing time. smart : CO2Meter (? device ) ∧smart : isLocatedIn (? device , ?room) ∧saref : makesMeasurement (? device , ?measurement ) ∧saref : hasValue (? measurement , ? value ) ∧swrlb : greaterThan (? value , 400) ∧swrlb : lessThanOrEqual (? value , 1000) =⇒ smart : CO2GreenLevel (?room) smart : PresenceSensor (? device ) ∧saref : hasState (? device , example : OnState ) ∧smart : isLocatedIn (? 3.4. Performance Evaluation device , ?room) =⇒ smart : OccupiedRoom (?room) smart : CO2UnsafeLevel (?room) ∧smart : OccupiedRoom (?room) =⇒ smart : WindowsOpen(?room) Listing 17: An excerpt of the set of rules employed to measure the average processing time. smart : CO2Meter (? device ) ∧smart : isLocatedIn (? device , ?room) ∧saref : makesMeasurement (? device , ?measurement ) ∧saref : hasValue (? measurement , ? value ) ∧swrlb : greaterThan (? value , 400) ∧swrlb : lessThanOrEqual (? value , 1000) =⇒ smart : CO2GreenLevel (?room) smart : PresenceSensor (? device ) ∧saref : hasState (? device , example : OnState ) ∧smart : isLocatedIn (? device , ?room) =⇒ smart : OccupiedRoom (?room) smart : CO2UnsafeLevel (?room) ∧smart : OccupiedRoom (?room) =⇒ smart : WindowsOpen(?room) Listing 17: An excerpt of the set of rules employed to measure the average processing time. Listing 17: An excerpt of the set of rules employed to measure the average processing time. smart : CO2Meter (? device ) ∧smart : isLocatedIn (? device , ?room) ∧saref : makesMeasurement (? device , ?measurement ) ∧saref : hasValue (? measurement , ? value ) ∧swrlb : greaterThan (? value , 400) ∧swrlb : lessThanOrEqual (? value , 1000) =⇒ smart : CO2GreenLevel (?room) smart : PresenceSensor (? device ) ∧saref : hasState (? device , example : OnState ) ∧smart : isLocatedIn (? device , ?room) =⇒ smart : OccupiedRoom (?room) smart : CO2UnsafeLevel (?room) ∧smart : OccupiedRoom (?room) =⇒ smart : WindowsOpen(?room) The status graph, over which the inferences are drawn, contains the measurements collected by a presence detection sensor and a CO2 meter. Both the sensors are installed in the same room. The presence sensor indicate whether the room is occupied through an OffState-Onstate indication, while the CO2 meter measures the CO2 concentration in the room in ppm. Measurement values and presence states were randomly generated using a Python script. We increased the number of the rooms to vary the number of devices involved in each run of the experiment. Listing 18 provides an example of the status graph generated to test the system time performance. Listing 18: An excerpt of the the status graph generated to test the system time performance. Listing 18: An excerpt of the the status graph generated to test the system time performance. Listing 18: An excerpt of the the status graph generated to test the system time performance. 3.4. Performance Evaluation ex : CO2_1 rdf : type smart :CO2 ; saref : makesMeasurement ex : CO2_1_m1 ; smart : isLocatedIn ex : R1 . ex : CO2_1_m1 rdf : type saref : Measurement ; saref : hasValue ‘ ‘2186 ’ ’^^ xsd : f l o a t ; saref : isMeasuredIn om: partsPerMillion ; saref : hasTimestamp ‘ ‘2020 −12 −02T14 :30:00 ’ ’^^ xsd : dateTime . ex : PRESENCE_1 rdf : type smart : SmartPresence ; saref : hasState ex : OnState ; smart : isLocatedIn ex : R1 . Listing 18: An excerpt of the the status graph generated to test the system time performance. ex : CO2_1 rdf : type smart :CO2 ; saref : makesMeasurement ex : CO2_1_m1 ; smart : isLocatedIn ex : R1 . ex : CO2_1_m1 rdf : type saref : Measurement ; saref : hasValue ‘ ‘2186 ’ ’^^ xsd : f l o a t ; saref : isMeasuredIn om: partsPerMillion ; saref : hasTimestamp ‘ ‘2020 −12 −02T14 :30:00 ’ ’^^ xsd : dateTime . ex : PRESENCE_1 rdf : type smart : SmartPresence ; saref : hasState ex : OnState ; smart : isLocatedIn ex : R1 . ex : CO2_1 rdf : type smart :CO2 ; saref : makesMeasurement ex : CO2_1_m1 ; smart : isLocatedIn ex : R1 . Sensors 2022, 22, 4131 16 of 19 16 of 19 For each repetition of the experiment we increased the number of devices involved. The inference process has been executed three times for each conﬁguration and the average time calculated. The tests were performed using Protege 5.5.0 on a MacBook Pro equipped with an Intel Core i7 (2.2 GHz, 6-Core) processor and 16 GB (2400 MHz DDR4) of RAM. It is worth to note that existing semantic reasoners are too resource-intensive to be directly ported on resource-constrained devices (such as Raspberry or Arduino) without further engineering efforts. However, promising solutions are currently under investigation [36]. g g p g y g Figure 4 shows the results of the experiments. The execution time is speciﬁed in milliseconds. 3.4. Performance Evaluation As it can be seen from the graph, for a number of devices between 2 and 200 the execution time does not exceed 300 ms which can be considered optimum for real-time operation in a domotic environment where the number of devices of a typical deployment is expected to be in the range of 50 to 100 [37]. This result is signiﬁcant when compared to the high latency and variability of the popular IFTTT trigger-action system that has been experimentally estimated in the order of seconds [38]. Number of Devices Avg Processing Time (in ms) 0 200 400 600 0 250 500 750 1000 Figure 4. The graph displays the average processing time (in milliseconds) for a number of devices that ranges from 2 to 1200. Figure 4. The graph displays the average processing time (in milliseconds) for a number of devices that ranges from 2 to 1200. 4. Discussions The ubiquity and pervasiveness of the IoT devices in ordinary households offers important opportunities for the home automation sector in many crucial areas such as energy preservation, home safety and living assistance. Notwithstanding, the potential of these devices is still largely under-exploited due to the poor data interoperability and the limited expressivity of the most commonly adopted paradigms for programming domotic scenarios. The aim of this study was to show that SW technologies can constitute a viable solution not only for tackling the problem of data and device heterogeneity, but also for deﬁning more complex home automation scenarios that can better exploit the potential of IoT technology. To this purpose, we developed a knowledge-based IoT home automation system that can aggregate and combine IoT sensor data with external heterogeneous data sources and analyse them according to formal knowledge expressed by means of OWL ontologies and SWRL rules. In particular, we demonstrated how in such a system the SWRL language can extend and overcome the limitations of the popular trigger-action model by introducing inferential reasoning capabilities directly in domotic scenario deﬁnition. The variety of the experiments that we carried out using SAREF as the main reference ontology proved the feasibility of the proposed approach and applicability of it to a standard and well validated context. Sensors 2022, 22, 4131 17 of 19 17 of 19 Our system opens up to a plethora of possible domotic scenarios that can be imple- mented ranging from simple actions to more complex automation tasks that make use of common sense knowledge. g Other beneﬁts of the proposed approach include the high degree of explainability of the processes involved. This feature assumes particular importance when home automation is employed to realise in-home healthcare facilities, for example, for elderly care or assisted living. Moreover, due to the high degree of customisation and the overall ﬂexibility, the system could be easily re-adapted for use in other IoT domains where complex automation logic is needed. g However, it is worth to note that at present, even though reuse is partially possible, scenarios require rules to be manually written. Manual rule-writing is not a trivial task but a time consuming and error prone process especially when scenario complexity signiﬁcantly increases. Therefore, alternative ways to facilitate and potentially automatise the rule creation should be explored. 4. Discussions Future research will focus on how to apply machine learning techniques to ease the process of creating rule-based home automation scenarios. Other interesting research directions should address the problems of diagnosis and fault tolerance. Author Contributions: Conceptualisation, R.R.; Methodology, R.R.; Software, R.R.; Validation, V.d.B., R.S., B.N. and L.D.; Investigation, R.R.; Resources, R.v.d.W.; Data Curation, R.v.d.W.; Writing—Original Draft Preparation, R.R.; Writing—Review And Editing, V.d.B., R.S. and A.C.; Visualisation, R.R.; Supervi- sion, V.d.B. and A.C.; Project Administration, V.d.B.; Funding Acquisition, V.d.B. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by the European Union’s Horizon 2020 project Interconnect under grant agreement 857237. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Abbreviations API Application Programming Interface DL Description Logic ETSI European Telecommunications Standards Institute EUD End-User Development IoT Internet of Things JSON JavaScript Object Notation KGs Knowledge Graphs OWL Web Ontology Language RDF Resource Description Framework SAREF Smart Applications REFerence (Ontology) SSHS Semantic Smart Home System SW Semantic Web SWRL Semantic Web Rule Language API Application Programming Interface DL Description Logic ETSI European Telecommunications Standards Institute EUD End-User Development IoT Internet of Things JSON JavaScript Object Notation KGs Knowledge Graphs OWL Web Ontology Language RDF Resource Description Framework SAREF Smart Applications REFerence (Ontology) SSHS Semantic Smart Home System SW Semantic Web SWRL Semantic Web Rule Language References Available online: https://ifttt.org (accessed on 30 March 2022). 20. Chen, G.; Jiang, T.; Wang, M.; Tang, X.; Ji, W. 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https://openalex.org/W4213214980	https://www.researchsquare.com/article/rs-3319/v1.pdf?c=1631840578000	English	null	Hyperhomocysteinemia in association with male gender, advanced age, uric acid and metabolic syndrome: A retrospective cross-sectional study	Research Square (Research Square)	2,019	cc-by	4,624	Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/16 Page 1/16 Abstract Background Elevated homocysteine level has been proposed as a risk factor for cardiovascular disease. The aim of this study is to evaluate the connection between hyperhomocysteinemia with other factors for early detection of possible cardiovascular disease. Methods The data in this retrospectively designed cross-sectional study was retrieved from the health examination database in a medical center located in southern Taiwan in 2016. The correlation of hyperhomocysteinemia with sex, age, body mass index, waist circumference, lipid profile, blood pressure, uric acid, high sensitivity C-reactive protein and lipoprotein were investigated. Both simple and multiple stepwise logistic regression models were used for the assessment of their connection. Results A total of 878 subjects with mean age of 55.4 ± 10.8 years were included in the current study and 73 (9.1%) participants had elevated homocysteine levels. Simple logistic regression analysis showed elevated Hcy was significantly associated with sex (OR 0.11, 95% CI 0.04-0.31, P < 0.001), age (OR 1.03, 95% CI 1.00-1.05, P < 0.035), waist circumference (OR 1.04, 95% CI 1.02-1.06, P = 0.001), HDL-C (OR 0.97, CI 0.95-0.99, P = 0.011), triglyceride (OR 1.00, CI 1.00-1.01, P = 0.002), SBP (OR 1.02, CI 1.01-1.03, P = 0.004), DBP (OR 1.04, CI 1.02-1.06, P = 0.001), sugar level (OR 1.01, CI 1.00-1.02, P = 0.021), uric acid (OR 1.39, CI 1.20-1.60, P < 0.001) and the presence of metabolic syndrome (OR 1.81, CI 1.12-2.93, P = 0.016). Multiple stepwise logistic regression analysis was applied and the result showed that male gender (OR 0.15, 95% CI 0.05-0.43, P < 0.001), advanced age (OR 1.03, 95% CI 1.00-1.05, P < 0.036), triglycerides (OR 1.002, 95% CI 1.001-1.004, P = 0.022), systolic blood pressure (OR 1.02, 95% CI 1.00-1.03, P = 0.023) and uric acid (OR 1.26, 95% CI 1.07-1.47, P = 0.005) were significantly associated with the elevation of plasma homocysteine. Conclusions Several factors are associated with hyperhomocysteinemia in asymptomatic subjects including sex, age, uric acid and metabolic syndrome components. Among these factors, male, advanced age, systolic blood pressure, plasma level of triglyceride and uric acid were independently associated with hyperhomocysteinemia. Definition of hyperhomocysteinemia and metabolic syndrome The normal concentration of Hcy in this study was defined as ≤ 15 µmol/L and hyperhomocysteinemia was considered > 15 µmol/L [6]. The definition of metabolic syndrome (MetS) was adopted from the National Cholesterol Education Program-Third Adult Treatment Panel (NCEP-ATP III) [7]. Subjects were diagnosed with MetS when they met any three or more of the following five components: 1) abdominal obesity (waist circumference > 90 cm in men or > 80 cm in women, the recommended criteria for Asian Americans), 2) high fasting blood glucose level (≥100 mg/dL), 3) high triglyceride level (≥150 mg/dL), 4) high SBP (≥ 130 mmHg) or DBP (≥ 85 mmHg), or 5) low HDL-C level (≤ 40 mg/dL in men or ≤ 50 mg/dL in women). Subjects This retrospective, cross-sectional study was conducted by applying data from a health examination database in a medical center located in southern Taiwan from January 2016 to December 2016. A total of 4858 subjects had undergone health examinations during this period of time. Subjects who had missing anthropometric, metabolic factors, plasma hs-CRP, plasma lipoprotein or plasma Hcy data (n = 3980) were excluded, leaving 878 patients retained for the present study. The research was approved by the Chang Gung Medical Foundation Institutional Review Board (IRB No.201900130B0). Information from the subjects including sex, age, height (cm), weight (kg), waist circumference (cm), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were obtained by trained nurses. Body mass index (BMI) was calculated by using the formula as weight in kilograms divided by the height in meters squared. Blood samples were collected for measuring fasting plasma glucose, uric acid, hs-CRP, lipoprotein, Hcy, total cholesterol, triglyceride, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). Information from the subjects including sex, age, height (cm), weight (kg), waist circumference (cm), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were obtained by trained nurses. Body mass index (BMI) was calculated by using the formula as weight in kilograms divided by the height in meters squared. Blood samples were collected for measuring fasting plasma glucose, uric acid, hs-CRP, lipoprotein, Hcy, total cholesterol, triglyceride, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). Background Cardiovascular disease is one of the most common leading causes of mortality and morbidity globally. In Taiwan, cardiac disease was the second major cause of death following by malignant neoplasm in 2016. Well-established risk factors for cardiovascular disease include hypertension, dyslipidemia, diabetes mellitus and obesity. Homocysteine (Hcy), an amino acid produced during the metabolism of methionine, is considered as an independent factor for cardiovascular disease [1]. Previous studies concluded that Hcy exerts adverse effects on endothelium and smooth muscle cells [2], resulting in endothelial dysfunction, vascular smooth muscle cells proliferation, oxidative stress, increased collagen synthesis and arterial stiffness [2]. Besides Hcy, inflammatory markers such as high-sensitivity C-reactive protein (hs-CRP) are proposed as novel predictors for cardiovascular disease based on the concept that inflammation serves as a major role in the progression of atherothrombosis. The predictive role of hs-CRP for cardiovascular disease has been established by previous research [3]. Furthermore, oxidative stress is regarded as a contributor to cardiac and vascular abnormalities [4]. Xanthine oxidase, an enzyme participating in the uric acid synthesis, is suggested to be one of the sources of reactive oxygen species. Page 2/16 Page 2/16 The upregulation of xanthine oxidase and consequent elevation of uric acid is considered as a potential biomarker for cardiovascular disease [5]. We conducted a cross-sectional study to investigate the association between hyperhomocysteinemia and other cardiovascular risk factors mentioned above. The aim of this article is to provide evidence for early identification of asymptomatic populations at high risk of cardiovascular disease with Hcy and other serum biomarkers. Results The baseline characteristics of the participants enrolled in this study are presented in Table 1. The average age of 878 participants was 55.4 ± 10.8 years, and for gender, there were 535 females (60.9%) and 343 males (39.1%), while mean plasma Hcy level was 10.4 ± 3.64 µmol/L. The average level of metabolic factors of the 878 adults such as waist circumference (84.9 ± 10.9 cm), HDL-C (50.0 ± 13.4 mg/dL), triglyceride (136.6 ± 98.1 mg/dL), SBP (120.1 ± 20.4 mmHg), DBP (85.2 ± 11.7 mmHg) and sugar AC (104.9 ± 25.3 mg/dL) are also listed in the table. Data analysis The data were expressed as mean ± standard deviation for continuous variables and percentages for categorical variables. The 𝜒2-test was used for comparing categorical characteristics of subjects and the Student`s t-test was used for the comparison of continuous variables of subjects. Univariate and Page 3/16 multivariate logistic regression were applied for analyzing the odds ratio (OR) of significant factors associated with hyperhomocysteinemia. A P value < 0.05 was considered as statistically significant. The SPSS software version 22.0 (IBM Corp., Armonk, NY, USA) was used for all analyses. multivariate logistic regression were applied for analyzing the odds ratio (OR) of significant factors associated with hyperhomocysteinemia. A P value < 0.05 was considered as statistically significant. The SPSS software version 22.0 (IBM Corp., Armonk, NY, USA) was used for all analyses. Table 1. Baseline characteristics Subjects were categorized into two groups by Hcy level (Table 2). Of the total 878, Subjects were categorized into two groups by Hcy level (Table 2). Of the total 878, hyperhomocysteinemia was observed in 73 subjects. In participants with elevated Hcy level, the proportion of males was significantly higher than in a normal sample of Hcy level participants (94.5% versus 34.0%, P < 0.001). Besides, participants with high plasma Hcy had significantly higher waist circumference, triglyceride, SBP, DBP, sugar AC and significantly lower HDL-C as compared with those with normal plasma Hcy. The percentage of those with metabolic syndrome in the high Hcy group was also significantly higher than in the normal Hcy group (46.0% versus 32.5%, P = 0.015). Participants with elevated Hcy were more likely to be older and have higher uric acid level than control group subjects. BMI, total cholesterol, LDL-C, hs-CRP, lipoprotein showed no significant difference between the two groups. Table 2. Differences between high homocysteine and normal homocysteine groups Simple logistic regression analysis in Table 3 shows elevated Hcy was significantly associated with sex (OR 0.11, 95% CI 0.04-0.31, P < 0.001), age (OR 1.03, 95% CI 1.00-1.05, P < 0.035), waist circumference (OR 1.04, 95% CI 1.02-1.06, P = 0.001), HDL-C (OR 0.97, CI 0.95-0.99, P = 0.011), triglyceride (OR 1.00, CI 1.00-1.01, P = 0.002), SBP (OR 1.02, CI 1.01-1.03, P = 0.004), DBP (OR 1.04, CI 1.02-1.06, P = 0.001), sugar level (OR 1.01, CI 1.00-1.02, P = 0.021), uric acid (OR 1.39, CI 1.20-1.60, P < 0.001) and the presence of metabolic syndrome (OR 1.81, CI 1.12-2.93, P = 0.016). The multiple stepwise logistic regression analysis conducted with these factors showed that only sex (OR 0.15, CI 0.05-0.43, P < 0.001), age (OR 1.03, CI 1.00-1.05, P = 0.036), triglyceride (OR 1.002, CI 1.001-1.004, P = 0.022), SBP (OR 1.02, CI 1.00-1.03, P = 0.023) and uric acid (OR 1.26, CI 1.07-1.47, P = 0.005) were independently significantly associated with hyperhomocysteinemia. Discussion Page 4/16 Page 4/16 This retrospective cross-sectional study revealed positive correlations between hyperhomocysteinemia and advanced age, triglyceride, blood pressure and uric acid levels. The subjects included in this current research were sampled from the Taiwanese population. Compared with a previous study based on Taiwanese people, the levels of average Hcy level were similar [8]. The positive relation between Hcy and triglyceride has been observed in several other studies as well. In research conducted in Japan with 40 patients diagnosed with type 2 diabetes mellitus, Hcy was associated with triglyceride but not with total cholesterol and LDL-C [9]. Momin et al. reported similar results based on a sample of 4660 Chinese in an urban community, with data that showed hyper- homocysteine state was independently related to elevated triglyceride [10]. The mechanism behind the relation between homocysteinemia and triglyceride has been proposed by several animal research studies. Experimental studies have suggested that Hcy-related endoplasmic reticulum stress increases the expression of sterol regulatory element-binding proteins (SREBPs), which further promote cholesterol and triglyceride synthesis [11]. The result was reproduced in an animal experiment by using methionine diet-fed mice, revealing elevated plasma triglyceride in increased plasma Hcy mice [12]. However, the relation between Hcy and triglyceride is not so consistent. Yadav et al. conducted a study involving 60 ischemic heart disease patients and failed to find a significant relationship between Hcy level and lipid profiles [13]. Another study enrolling 155 type 2 diabetes mellitus subjects also reported no significant association regarding hyperhomocysteinemia and high plasma triglyceride level [14]. Despite the inconsistent findings on the effect of Hcy, we provide a strong relation between elevated Hcy and triglyceride level in the current research. Some cross-sectional studies discussing the association between Hcy and lipid profiles other than triglyceride showed a negative relationship with HDL-C [9, 10, 15-19] and apolipoprotein [15-17, 20] but positive relationship with LDL-C [18]. The mechanism of Hcy related to decreased HDL-C and apolipoprotein has been demonstrated in animal models. Hcy down- regulated peroxisome proliferator-activated receptor (PPAR𝛂) and apolipoprotein AI protein level [21]. The result caused the suppression of lecithin-cholesterol acyltransferase substrate, which led to HDL maturation inhibition [22]. Significant relationship was noted between hyperhomocysteinemia and metabolic syndrome in our simple logistic regression analysis; however, the studies discussing the connection between Hcy and metabolic syndrome showed conflicting results. Seven hundred and twenty- two patients undergoing medical checkup in Korea revealed lack of association [23]. Discussion On the contrary, Esteghamati et al. found that homocysteine was correlated with metabolic syndrome based on a prospective study composed of 5893 subjects in Iran [24]. In our multiple stepwise logistic regression analysis, only some of the components of metabolic syndrome such as triglycerides and systolic blood pressure were independently significantly associated with elevated plasma homocysteine level. The differences between our analysis and prior studies were likely because of the adjusting variables we used, as our study also incorporated other cardiovascular disease risk factors including uric acid and CRP. Despite the inconsistent findings on the effect of Hcy, we provide a strong relation between elevated Hcy and triglyceride level in the current research. Some cross-sectional studies discussing the association between Hcy and lipid profiles other than triglyceride showed a negative relationship with HDL-C [9, 10, 15-19] and apolipoprotein [15-17, 20] but positive relationship with LDL-C [18]. The mechanism of Hcy related to decreased HDL-C and apolipoprotein has been demonstrated in animal models. Hcy down- regulated peroxisome proliferator-activated receptor (PPAR𝛂) and apolipoprotein AI protein level [21]. The l d h i f l i hi h l l l f b hi h l d HDL The connection between hyperhomocysteinemia and uric acid is explained by the methionine- homocysteine cycle [25]. In this cycle, methionine is converted to S-adenosyl-homocysteine (SAH), which becomes Hcy and adenosine, with adenosine then being degraded to uric acid. Therefore, an increase in Page 5/16 Page 5/16 Hcy leads to an increase in uric acid level; this result is consistent with our analysis. Similar findings were also observed in other research studies [26, 27]. On the other hand, sex difference was also found to be an independent factor for elevated Hcy level in the current study, the mechanism of which might be related to the sex difference in Hcy metabolism. Normally, Hcy is metabolized by 2 different pathways: remethylation and transsulfuration [26]. A study involving 11 healthy young men and premenopausal women without cardiac risk factors conducted by Fukagawa et al. reported a significant higher rate of remethylation and a tendency toward higher rates of transmethylation in women than in men [28]. Therefore, it is reasonable to anticipate that Hcy level is higher in men than women. An Indian study composed of 1243 healthy subjects confirmed the sex difference by showing a significantly higher level of Hcy over women [29]. Conclusions Elevated serum homocysteine is associated with sex, age, uric acid and metabolic syndrome components. Among these factors, male gender, advanced age, triglyceride, systolic blood pressure and uric acid were profoundly associated with hyperhomocysteinemia. Further prospective cohort studies are needed for validating the influence on cardiovascular event prediction with homocysteine and its associates. Discussion In addition, creatine is synthesized from guanidinoacetate during the formation of SAH in the methionine-Hcy cycle. Creatine and subsequent creatinine synthesis is related to muscle mass, which provides an explanation for higher Hcy levels in men than women [30]. Hormonal difference has also been proposed as a reason contributing to the different Hcy levels in males and females. A clinical study with transsexuals showed that male-to-female subjects who were treated with ethinyl estradiol and antiandrogen had significantly decreasing levels of Hcy. On the contrary, female-to-male transsexuals who underwent testosterone treatment had increased plasma Hcy [31]. The age-related elevation of Hcy could be explained by the physiologically-associated renal function decline. A previous study concluded that glomerular filtration rates served as an important role in Hcy and creatinine clearance [32]. Possible mechanisms regarding the relationship between Hcy and blood pressure is that Hcy induces endothelial injury and oxidative stress, which leads to impaired nitric oxide release [33], and the consequences of these effects contribute to the raise of blood pressure. There are several limitations in the current study. Firstly, the study is retrospectively designed, so further studies are needed to determine any causation. Secondly, the subjects were recruited from a single medical center, which might not represent the general population in Taiwan. Thirdly, participants who received medications for hypertension or hyperlipidemia were not excluded, so medication use might affect the result of our analysis. Acknowledgements Not applicable. Abbreviations BMI, body mass index; DBP, diastolic blood pressure; Hcy, homocysteine; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; MetS, Page 6/16 Page 6/16 metabolic syndrome; SBP, systolic blood pressure; TC, total cholesterol. metabolic syndrome; SBP, systolic blood pressure; TC, total cholesterol. Funding No financial support was received for this project. Authors’ contribution Study concept and design: PJC, YCL, PMW and SSL. Sampling and laboratory analysis: PJC, YCL and PMW. Statistical analysis and interpretation of data: SSL. Preparing the manuscript: PJC and SSL. Critical revision of the manuscript and corresponding author: SSL. All authors read and approved the final version of the manuscript. Ethics approval and consent to participate The study was approved by the Chang Gung Medical Foundation Institutional Review Board. In this retrospective study, informed consent was unnecessary because the information of all participants was anonymized and de-identified. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. References 1. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. Jama. 2002;288(16):2015-22. 1. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. Jama. 2002;288(16):2015-22. 2. Ganguly P, Alam SF. Role of homocysteine in the development of cardiovascular disease. 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Giltay EJ, Hoogeveen EK, Elbers JM, Gooren LJ, Asscheman H, Stehouwer CD. Effects of sex steroids on plasma total homocysteine levels: a study in transsexual males and females. The Journal of Page 9/16 clinical endocrinology and metabolism. 1998;83(2):550-3. clinical endocrinology and metabolism. 1998;83(2):550-3. clinical endocrinology and metabolism. 1998;83(2):550-3. 32. Arnadottir M, Hultberg B, Nilsson-Ehle P, Thysell H. The effect of reduced glomerular filtration rate on plasma total homocysteine concentration. Scandinavian journal of clinical and laboratory investigation. 1996;56(1):41-6. 32. Arnadottir M, Hultberg B, Nilsson-Ehle P, Thysell H. The effect of reduced glomerular filtration rate on plasma total homocysteine concentration. Scandinavian journal of clinical and laboratory investigation. 1996;56(1):41-6. 33. Pushpakumar S, Kundu S, Sen U. Endothelial dysfunction: the link between homocysteine and hydrogen sulfide. Current medicinal chemistry. 2014;21(32):3662-72. 33. Pushpakumar S, Kundu S, Sen U. Endothelial dysfunction: the link between homocysteine and hydrogen sulfide. Current medicinal chemistry. 2014;21(32):3662-72. Tables Table 1. Baseline characteristics Page 10/16 Participants (n = 878) Sex Female, n, % 535 (60.9%) Male, n, % 343 (39.1%) Age (years) 55.4±10.8 BMI (kg/m2) 25.2±3.8 Waist circumference (cm) 84.9±10.9 Total cholesterol (mg/dl) 203.0±37.0 HDL-C (mg/dl) 50.0±13.4 Triglycerides (mg/dl) 136.6±98.1 LDL-C (mg/dl) 123.3±32.6 SBP (mmHg) 129.1±20.4 DBP (mmHg) 85.2±11.7 Sugar AC (mg/dl) 104.9±25.3 TC/HDL-C (mg/dl) 4.28±1.18 LDL-C/HDL-C (mg/dl) 2.62±0.92 Uric acid (mg/dl) 6.34±1.56 Plasma hs-CRP (ug/dl) 2.33±4.09 Plasma lipoprotein (mg/dl) 16.7±19.9 Plasma homocysteine (umol/l) 10.4±3.64 Plasma lipoprotein (mg/dl) 16.7±19.9 Plasma homocysteine (umol/l) 10.4±3.64 Abbreviations: BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; hs-CRP, high-sensitivity C-reactive protein. Table 2. Differences between high homocysteine and normal homocysteine groups Page 12/16 High homocysteine (n=73) Normal homocysteine (n= 805) P value Sex <0.001* Male, n, % 69 (94.5%) 274 (34.0%) Female, n, % 4 (5.5%) 531 (66.0%) Age (years) 58.2±9.9 55.2±10.9 0.034* BMI (Kg/m2) 25.9±4.1 25.2±3.8 0.140 Waist circumference (cm) 88.9±11.7 84.6±10.7 0.001* Total cholesterol (mg/dl) 202±42.2 203.1±36.5 0.806 HDL-C (mg/dl) 46.2±12.0 50.4±13.5 0.011* Triglycerides (mg/dl) 176.6±76.8 133.0±86.8 <0.001* LDL-C (mg/dl) 122.2±37.8 123.4±32.1 0.764 SBP (mmHg) 135.7±19.7 128.5±20.4 0.004* DBP (mmHg) 89.8±12.4 84.8±11.5 <0.001* Sugar AC (mg/dl) 111.6±30.7 104.3±24.7 0.018* TC/HDL-C 4.63±1.46 4.25±1.15 0.009* LDL-C/HDL-C 2.81±1.09 2.60±0.91 0.062 Uric acid (mg/dl) 7.12±1.87 6.26±1.51 <0.001* Hs-CRP (ug/dl) 3.15±6.73 2.25±3.76 0.076 Lipoprotein (mg/dl) 13.3±17.7 17.0±20.1 0.135 MetS, n, % 0.015* yes 34(46.6%) 262(32.5%) no 39(53 4%) 543(67 5%) : Indicates a significant difference, p < 0.05 : Indicates a significant difference, p < 0.05 Abbreviations: BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; hs-CRP, high-sensitivity C-reactive protein; MetS, metabolic syndrome. Table 3. Regression analysis for association of high homocysteine with different variables Page 14/16 Variables Simple logistic regression OR (95% CI) P value Multiple stepwise logistic regression OR (95% CI) P value Sex 0.11(0.04-0.31) <0.001* 0.15(0.05-0.43) <0.001* Age 1.03(1.00-1.05) <0.035* 1.03(1.00-1.05) 0.036* BMI 1.05(0.98-1.11) 0.149 Waist circumference 1.04(1.02-1.06) 0.001* Total cholesterol 1.00(1.00-1.01) 1.000 HDL-C 0.97(0.95-0.99) 0.011* Triglyceride 1.00(1.00-1.01) 0.002* 1.002(1.001-1.004) 0.022* LDL-C 1.00(0.99-1.01) 0.764 SBP 1.02(1.01-1.03) 0.004* 1.02(1.00-1.03) 0.023* DBP 1.04(1.02-1.06) 0.001* Sugar AC 1.01(1.00-1.02) 0.021* Uric acid 1.39(1.20-1.60) <0.001* 1.26(1.07-1.47) 0.005* Hs-CRP 1.04(0.99-1.08) 0.090 Lipoprotein 0.99(0.97-1.00) 0.137 MetS 1.81(1.12-2.93) 0.016* : Indicates a significant difference, p < 0.05 Abbreviations: BMI body mass index; HDL high-density lipoprotein; LDL low-density lipoprotein; SBP systolic : Indicates a significant difference, p < 0.05 *: Indicates a significant difference, p < 0.05 Abbreviations: BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; hs-CRP, high-sensitivity C-reactive protein; MetS, metabolic syndrome. Page 15/16 Page 16/16
https://openalex.org/W4224329869	https://zenodo.org/record/6410386/files/Yuriane%20Canabisfa_1910841019_Artikel%20Pelayanan%20Publik.pdf	Indonesian	null	Inovasi Sistem Pelayanan PA Tanjung Pati Keliling (SIMPATIK) dalam Upaya Meningkatkan Kualitas Pelayanan Publik	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	4,822	Inovasi Sistem Pelayanan PA Tanjung Pati Keliling (SIMPATIK) dalam Upaya Meningkatkan Kualitas Pelayanan Publik Pelayanan Publik Yuriane Canabisfa Yuriane Canabisfa Program Studi Ilmu Administrasi Publik, Universitas Andalas, Indonesia yurianecanabisfa123@gmail.com Abstract: Facing a sophisticated era, the government as a leader who regulates social life must be able to adapt to these conditions. The government is expected to take advantage of developments in technology, information and communication in terms of meeting the needs of the community through public services. Like most other regions, Kabupaten Fifty Cities have started implementing digital-based public services. One of the digitalization of public services is carried out by the Tanjung Pati Religious Court Institution through an innovation called SIMPATIK. SIMPATIK is a service innovation provided with a ball pick-up system by Religious Court officers. With this innovation, it is expected to be able to answer the demands of the community in receiving services effectively and efficiently. Research related to service innovation by the Religious Courts has not been widely carried out, therefore researchers are interested in deepening this SIMPATIK innovation. The research method chosen is a qualitative approach with a descriptive analysis method. This study uses Rogers' theory in Mona Melinda (2020) which measures the innovation attribute variable with 5 things, namely relative advantage; suitability (compatibility); complexity (complexity); the possibility of being tried (triability); and easy to observe (observability). Based on the results of the study, it is known that the services provided by the Tanjung Pati Religious Court through SIMPATIK Innovation are good. However, there are still obstacles, such as the use of the new website, which is limited to registration of interested parties, and has not been added to other activities. Keywords: Innovation, Services, SIMPATIK, Tanjung Pati Abstrak: Menghadapi era yang serba canggih menjadikan pemerintah sebagai pemimpin yang mengatur kehidupan bermasyarakat harus bisa menyesuaikan dengan keadaan tersebut. Pemerintah diharapkan dapat memanfaatkan perkembangan teknologi, informasi dan komunikasi dalam hal memenuhi kebutuhan masyarakat melalui pelayanan publik. Seperti kebanyakan daerah lainnya, Kabupaten lima Puluh Kota sudah mulai menerapkan pelayanan publik berbasis digital. Digitalisasi pelayanan public tersebut salah satunya dilakukan oleh Lembaga Pengadilan Agama Tanjung Pati melalui inovasi yang Bernama SIMPATIK. SIMPATIK merupakan inovasi layanan yang diberikan dengan system jemput bola oleh petugas Pengadilan Agama. Dengan adanya inovasi ini diharapkan dapat menjawab tuntutan masyarakat dalam menerima pelayanan secara efektif dan efesien. Penelitian terkait inovasi pelayanan oleh Pengadilan Agama belum banyak dilakukan oleh karena itu peneliti tertarik untuk memperdalam inovasi SIMPATIK ini. Metode penelitian yang dipilih adalah pendekatan kualitatif dengan metode deskriptif analisis.. Yuriane Canabisfa Penelitian ini menggunakan teori Rogers dalam Mona Melinda (2020) yang mengukur variabel atribut inovasi dengan 5 hal yakni keuntungan relatif (relatif advantage); kesesuaian (compatibility); kerumitan (complexity); kemungkinan dicoba (triability); dan kemudahan diamati (observability). Berdasarkan hasil penelitian, diketahui bahwa pelayanan yang diberikan Pengadilan Agama Tanjung Pati melalui Inovasi SIMPATIK sudah baik. Namun masih terdapat kendala seperti pemanfaatan website baru sebatas pendaftaran pihak yang berkepentingan, belum bertambah pada kegiatan yang lain. Kata Kunci: Inovasi, Pelayanan, SIMPATIK, Tanjung Pati Kata Kunci: Inovasi, Pelayanan, SIMPATIK, Tanjung Pati A. PENDAHULUAN Dalam kehidupan berbangsa dan bernegara, pemerintah sebagai aparatur birokrasi memiliki tujuan untuk selalu memenuhi kebutuhan masyarakatnya. Pemenuhan kebutuhan tersebut dilakukan melalui pelayanan yang diberikan kepada setiap individu sesuai keperluannya masing-masing. Semakin berkembangnya system kehidupan, kebutuhan masyarakat yang diperlukan juga semakin banyak. Pemerintah dituntut untuk bisa memenuhi kebutuhan masyarakat yang beragam tersebut, mulai dari Pendidikan, Kesehatan, Keamanan, hingga kebutuhan social seperti urusan Perkawinan, Perceraian dan sebagainya. Dalam penanganan permasalahan dan kebutuhan masyarakat tersebut, pemerintah harus dapat memberikan pelayanan yang efektif dan efisien kepada masyarakat sebagai negara yang berdaulat (Maisy Triwahyuni dkk, 2020). Menurut Siagian dalam Hardiyansyah (2018) menyatakan teori ilmu administrasi mengajarkan pemerintahan negara pada hakikatnya menyelenggarakan dua jenis fungsi utama, yaitu fungsi pengaturan dan fungsi pelayanan. Fungsi pengaturan biasanya dikaitkan dengan hakikat negara modern sebagai suatu negara hukum (legal state), sedangkan fungsi pelayanan dikaitkan dengan hakikat negara sebagai suatu negara kesejahteraan (welfarestate). Kedua fungsi tersebut dipercayakan kepada aparatur pemerintah sesuai bidangnya masing- masing. Pengertian pelayanan (service) menurut American Marketing Association, seperti dikutip oleh Cowell dalam Hardiyansyah (2018) bahwa pelayanan merupakan kegiatan atau manfaat yang ditawarkan oleh suatu pihak kepada pihak lain dan pada hakekatnya tidak berwujud serta tidak menghasilkan kepemilikan sesuatu, proses produksinya mungkin juga tidak dikaitkan dengan suatu produk fisik. Jadi, bisa dikatakan bahwa pelayanan merupakan kegiatan yang dilakukan oleh pihak tertentu untuk membantu seseorang untuk mendapatkan kebutuhannya dalam hal berupa barang atau jasa, aktivitasnya tidak bisa dilihat namun dapat dirasakan. Menurut Undang-Undang Nomor 25 Tahun 2009 tentang Pelayanan Publik, pelayanan publik adalah kegiatan atau kebutuhan pelayanan bagi setiap warga negara dan penduduk atas barang, jasa, dan atau pelayanan administratif yang disediakan oleh penyelenggara pelayanan publik. Pelayanan public merupakan sesuatu kewajiban yang harus diberikan pemerintah kepada masyarakat sebagai pihak yang harus dilayani. Sedangkan menurut Wasistiono dalam Hardiyansyah (2018), Pelayanan umum atau pelayanan publik adalah pemberian jasa baik oleh pemerintah, pihak swasta atas nama pemerintah ataupun pihak swasta kepada masyarakat, dengan atau tanpa pembayaran guna memenuhi kebutuhan dan atau kepentingan masyarakat. Pemberian pelayanan public oleh aparatur pemerintah kepada masyarakat sebenarnya merupakan implikasi dari fungsi aparat negara sebagai pelayan masyarakat (Hamirul, 2018). Disimpulkan bahwa pelayanan public adalah aktivitas yang dilakukan oleh pemerintah kepada masyarakat sesuai dengan peraturan perundang-undangan yang ada dalam rangka memenuhi kebutuhan setiap penduduknya melalui instansi-instansi yang dikelola oleh pemerintah sendiri dan swasta. A. PENDAHULUAN Dari pengertian pelayanan public diatas, menurut Hardiyansyah (2018) terdapat 3 unsur penting dalam pelayanan public, yaitu unsur pertama, adalah organisasi pemberi (penyelenggara) pelayanan yaitu Pemerintah/Pemerintah Daerah, unsur kedua, adalah penerima layanan (pelanggan) yaitu orang atau masyarakat atau organisasi yang berkepentingan, dan unsur ketiga, adalah kepuasan yang diberikan dan/atau diterima oleh penerima layanan (pelanggan). Pelayanan public tidak hanya menjadi urusan pemerintah pusat saja namun mengikutsertakan pemerintah daerah. Pemerintah di daerah memiliki kewenangan yang luas untuk mengatur daerahnya sesuai kebutuhan masyarakat tersebut dikarenakan adanya otonomi daerah, otonomi daerah ini mencakup segala aspek kehidupan masyarakatnya termasuk untuk urusan pelayanan public yang ada diharapkan pemerintah daerah juga dapat menyesuaikannya. Pemerintah pusat memiliki kewenangan untuk merumuskan peraturan dan untuk pelaksanaan diserahkan kepada masing-masing daerah. Mona Melinda dkk (2020) menyatakan Kewajiban pemda dalam mewujudkan pelayanan yang terbaik tertuang dalam UU Nomor 23 Tahun 2014 tentang Pemerintahan Daerah. Dalam ketentuan Bab XXI UU Nomor 23 Tahun 2014 disebutkan bahwa pemda dapat melakukan inovasi dalam upaya peningkatan kinerja penyelenggaraan pemerintahan daerahnya. Hal ini diperkuat dengan adanya desentralisasi kewenangan yang dimiliki pemda sebagai konsekuensi otonomi daerah. Menurut Brinkerhoff & Wetterberg yang dikutip oleh Mona Melinda dkk (2020) Studi desentralisasi di Indonesia menunjukkan otonomi daerah berpengaruh signifikan bagi pemda termasuk dalam penyelenggaraan layanan publik. Dengan begitu bisa dikatakan bahwa, dengan adanya otonomi daerah telah membawa perubahan yang besar bagi struktur pemerintahan daerah dalam melayani masyarakatnya, mereka tidak lagi terpaku dengan keputusan pemerintah pusat namun dapat mengembangkannya berdasarkan kebutuhan demi mewujudkan masyarakat yang sejahtera. Ketika dihadapkan pada proses penyelenggaraannya pelayanan publik dengan orientasi pada kekuasaan yang amat kuat, selama ini telah membuat birokrasi menjadi semakin jauh dari misinya untuk memberikan sebuah pelayanan (Roni Ekha Putera, 2009) Dilihat dari pelaksanaan kegiatan administrative oleh pemerintah, Indonesia masih jauh berada dalam kategori baik dalam memberikan pelayanan yang layak kepada masyarakat hal ini dikarenakan masih banyak ditemukan pelayanan yang disediakan membutuhkan biaya, waktu yang lama serta system pengurusan berbelit-belit. Pelaksanaan pelayanan yang masih dirasa belum optimal ini disebabkan atas dasar kinerja birokrat yang belum memenuhi peraturan yang berlaku. Ditinjau dari sisi kelembagaan masih banyak terjadi duplikasi, dimana bentuk organisasi masih berbentuk piramida terbalik. Ditinjau dari ketatalaksanaan dan pelayanan publik terjadi sistem prosedur pelayanan yang belum transparan, berbelit-belit dan terjadi praktik KKN (Korupsi, Kolusi, Napotisme) (Bobi Rizki Ananda, 2019). Menurut Bobi Rizki Ananda dkk (2019), Masyarakat cenderung diposisikan sebagai pihak yang ingin mendapatkan pelayanan bukan sebagai pihak yang dilayani. A. PENDAHULUAN Untuk itu, masyarakat sebagai pihak yang menginginkan keadilan dan kesejahteraan melakukan tuntutan kepada pemerintah terkait perbaikan kinerja birokrasi public. Tuntutan-tuntutan itu menjadi masukan bagi pemerintah atas apa yang perlu dilakukan kedepannya. Survey telah banyak dilakukan mengenai kinerja organisasi pemerintah Indonesia dalam memberikan pelayanan public, salah satunya seperti yang dilakukan Dwiyanto dkk yang dikutip oleh Ria Ariany (2010), dimana Dwiyanto melakukan survey GDS 2002 di 20 Provinsi di Indonesia yang salah satu hasilnya adalah organisasi pemerintah dalam menyelenggarakan pelayanan public, waktu yang diperlukan untuk menyelesaikan sebuah pelayanan seringkali tidak diatur dengan jelas, akibatnya terjadi ketidakpastian dalam hal waktu pelayanan. Tidak hanya itu, menurut Ananda dkk yang dikutip Aranzi Eriza dkk (2021) Penyelenggaraan pelayanan publik saat ini masih dihadapkan pada kondisi yang belum selesai dengan kebutuhan dan perubahan di berbagai bidang kehidupan bermasyarakat, berbangsa dan bernegara. Birokrasi pemerintahan ditinjau dari ketatalaksanaan dan pelayanan publik masih terjadi sistem pelayanan yang belum transparan, berbelit-belit dan adanya praktik KKN. Ditambah lagi ketika dihubungkan dengan unsur yang terdapat dalam pelayanan public dimana masih terdapat kendala antara lain seperti unsur pertama dalam pelayanan public yaitu pemerintah sebagai pihak pembuat aturan dan pelaksana kebijakan, kadangkala ini membuat kebingungan antara pemerintah untuk menjalankan urusan sebagai pihak yang memiliki kepentingan pembuat keputusan atau yang menjalankannya. Yang kedua itu terkait masyarakat, dimana masyarakat tidak dalam posisi untuk menawar sehingga tidak bisa memiliki kekuatan untuk mendapatkan layanan yang baik. Ketiga adalah kepuasan masyarakat menerima pelayanan yang perlu diperhatikan oleh pemerintah sebagai acuan dalam pengambilan keputusan, dengan adanya pelayanan yang belum optimal maka masyarakat tidak merasa puas atas apa yang diberikan pemerintah sehingga harus dilakukan pembenahan. Dalam hal menjawab tuntutan masyarakat dibutuhkan upaya untuk memperbaiki pelayanan yang membebani masyarakat tersebut, ini dapat dilakukan dengan reformasi birokrasi terkait dengan pelayanan. Pelayanan yang sudah ada dilakukan pembaharuan serta dilakukan inovasi. Berdasarkan PERMENPAN-RB NO 30 Tahun 2014, Inovasi pelayanan publik adalah terobosan pelayanan publik yang merupakan gagasan ide kreatif original dan adaptasi/modifikasi yang memberikan manfaat bagi masyarakat baik secara langsung maupun tidak langsung. Dengan kata lain, inovasi pelayanan publik sendiri tidak mengharuskan suatu penemuan baru, tetapi dapat merupakan suatu pendekatan baru yang bersifat kontekstual dalam arti inovasi tidak terbatas dari tidak ada kemudian muncul gagasan dan praktik inovasi, tetapi dapat berupa inovasi hasil dari perluasan maupun peningkatan kualitas pada inovasi yang ada. sedangkan menurut Evert M. A. PENDAHULUAN Roger yang dikutip Suwarno dalam Nugi Alindro dkk (2021) menyatakan inovasi adalah ide, gagasan, praktik atau/benda yang didasari dan diterima sebagai suatu hal yang baru oleh seseorang atau kelompok untuk diadopsi. Pemerintah dalam melakukan inovasi pelayanan public disesuaikan dengan kebutuhan masyarakat serta melihat perkembangan zaman. Saat ini dunia sedang dihadapkan dengan pengaruh globalisasi yang sangat kuat. Tidak ada negara yang mampu menolak arus globalisasi ini dikarenakan globalisasi telah mencakup sendi-sendi kehidupan mulai dari kehidupan ekonomi, politik, social budaya hingga pertahanan dan keamanan. Globalisasi sebagai akibat dari pertukaran ilmu pengetahuan para intelligent antar negara sehingga membawa ke arah yang serba maju dan modern. Kemajuan ini terlihat dengan berkembang pesatnya teknologi, informasi dan komunikasi yang pada akhirnya membawa pengaruh signifikan dalam kehidupan. Kemajuan-kemajuan ini dalam pelaksanaannya didorong dengan kemunculan internet. Internet menjadikan segalanya berada dalam jaringan. Dengan adanya internet, masyarakat mulai dimanjakan dengan system yang cepat dan mudah, contohnya saja ketika berbelanja, masyarakat tidak perlu lagi datang ke pasar yang ramai dan kumuh untuk mendapatkan keperluan sehari-hari cukup dengan menyediakan paket data maka masyarakat sudah dapat memesan kebutuhan tersebut melalui telepon genggam dengan hanya duduk di rumah atau di coffeshop. Begitu mudah, cepat dan murah fasilitas yang disediakan melalui internet untuk menunjang aktivitas masyarakat. Dengan demikian, pemerintah harus menyesuaikan pelayanan yang akan diberikan ditengah-tengah masyarakat modern. Berdasarkan data pada Asosiasi Penyelenggara Jasa Internet Indonesia (APJII) yang terdapat dalam Hazid Jalma dkk (2019) menggambarkan terjadinya peningkatan pengguna internet di Indonesia setiap tahunnya. Internet menjadi alat yang menghubungkan seseorang dengan yang lainnya sehingga ini terus mengalami peningkatan. Pemerintah mulai memanfaatkan keadaan untuk memperbaharui kualitas pelayanan public melalui internet. Penggunaan internet dan teknologi dalam pelayanan public merubah pelayanan konvesional menjadi serba digital. Digitalisasi pelayanan public disambut baik oleh masyarakat maupun aparat pelaksana kebijakan, hal ini dikarenakan digitalisasi pelayanan yang diberikan membawa banyak kemudahan, seperti antrian yang tidak lama, pelayanan yang cepat, dan tidak perlu mengeluarkan biaya. Pelayanan public yang digital akan meningkatkan kepercayaan masyarakat terhadap pemerintah dengan adanya transparansi dan akuntabel dari aktivitas yang dilaksanakan. Salah satu Lembaga/instansi yang melakukan digitalisasi dalam pelayanan public adalah Pengadilan Agama Tanjung Pati. Pengadilan ini berinovasi dengan Program SIMPATIK yang dirancang untuk memberikan pelayanan kepada masyarakat dengan system jemput bola. Dengan adanya inovasi ini diharapkan pelayanan keadilan yang disediakan dapat menjangkau seluruh masyarakat hingga ke daerah terpencil. A. PENDAHULUAN SIMPATIK merupakan salah satu bentuk pelayanan prima yang diberikan oleh PA Tanjung Pati yang dilaksanakan setiap hari Kamis pekan pertama dan ketiga setiap bulannya, berlokasi di Kantor Wali Nagari Limbanang, Kabupaten Limapuluh Kota. Lembaga Pengadilan Agama juga berupaya untuk meningkatkan kualitas pelayanan public melalui pengembangan inovasi pelayanan dan peningkatan kualitas SDM. Studi relevan yang memiliki lokus Pengadilan Agama sendiri telah dilakukan oleh ahli. Studi yang dilakukan oleh Annisa Trijultian (2022) melihat Pengaruh Kualitas Pelayanan Administrasi Pos Bantuan Hukum Terhadap Kepuasan Masyarakat di Pengadilan Agama Kota Sukabumi, dimana penelitian lebih ke arah melihat pelaksanaan pelayanan yang telah ada. Berbeda dari studi yang telah dilakukan sebelumnya, inovasi SIMPATIK yang dilakukan sebagai wujud meningkatkan kualitas layanan agar masyarakat dapat menerima keadilan dengan mudah memiliki karakteristik pelayanan yang berbeda dengan pengadilan agama dimana layanan dilakukan secara daring dan luring. Untuk studi terkait inovasi pelayanan oleh pengadilan agama belum ada dilakukan, Dengan demikian ada keterbaruan dari inovasi yang dilakukan oleh Pengadilan Agama Tanjung Pati. Ini menjadi dasar peneliti tertarik untuk melakukan penelitian ini. B. METODE PENELITIAN Artikel ini menggunakan pendekatan kualitatif dengan metode deskriptif analisis. Pendekatan kualitatif dipilih karena kemampuan peneliti untuk mendapatkan pemahaman yang mendalam, autentik, dan mendasar mengenai fenomena yang tengah diamati. Metode deskriptif analisis digunakan dengan alasan data dan informasi yang dihimpun memerlukan perhatian pada fenomena atau masalah aktual melalui proses pengumpulan data, penyusunan, pengolahan, dan penarikan kesimpulan. Hasil dari proses tersebut kemudian digunakan untuk mendeskripsikan suatu keadaan empiris yang objektif atas fenomena atau masalah yang sedang dikaji. Sedangkan teknik pengumpulan data yang digunakan adalah studi kepustakaan. Hal ini dilakukan karena keterbatasan peneliti untuk melakukan wawancara secara langsung kepada narasumber-narasumber terkait pada saat tulisan ini dibuat. Studi kepustakaan yang dimaksud dalam penelitian ini adalah upaya yang dilakukan oleh peneliti dalam mencari, mengumpulkan dan mempelajari bahan tertulis berupa ebook, artikel, berita online (seperti minangkabaunews.com) dan website Lembaga terkait (seperti https://badilag.mahkamahagung.go.id/) yang menjadi sumber terhadap masalah dan fenomena yang dikaji. Teknik Analisis Data dalam penelitian ini merujuk pada penjelasan Creswell yang dikutip Leo Agustino (2020) yang menjabarkan tekniknya pada pengorganisasian data, pembacaan dan memoing (pembuatan catatan), serta pendeskripsian, pengklarifikasian, serta penafsiran data menjadi kode dan tema. C. KERANGKA TEORI Inovasi yang ada harus diuji dan dicoba terlebih dahulu untuk dilihat keuntungan dan nilai lebih yang dimilikinya. 5. Observability atau kemudahan diamati. Observability adalah sejauh mana hasil inovasi dapat dilihat dan diamati oleh orang lain. Beberapa inovasi dapat dengan mudah untuk diamati, dipahami dan dikomunikasikan dengan orang lain (Rogers, 1995). Sebuah inovasi perlu untuk dilihat dan diamati bagaimana ia bekerja untuk dapat menghasilkan sesuatu yang lebih. 4. Triability atau kemungkinan dicoba. Triability atau kemungkinan dicoba adalah sejauh mana inovasi dapat diujicobakan secara terbatas (Rogers, 1995). Inovasi yang ada harus diuji dan dicoba terlebih dahulu untuk dilihat keuntungan dan nilai lebih yang dimilikinya. 5. Observability atau kemudahan diamati. Observability adalah sejauh mana hasil inovasi d dilih d di i l h l i B b i i d d d h k 4. Triability atau kemungkinan dicoba. Triability atau kemungkinan dicoba adalah sejauh mana inovasi dapat diujicobakan secara terbatas (Rogers, 1995). Inovasi yang ada harus diuji dan dicoba terlebih dahulu untuk dilihat keuntungan dan nilai lebih yang dimilikinya. 5. Observability atau kemudahan diamati. Observability adalah sejauh mana hasil inovasi dapat dilihat dan diamati oleh orang lain. Beberapa inovasi dapat dengan mudah untuk diamati, dipahami dan dikomunikasikan dengan orang lain (Rogers, 1995). Sebuah inovasi perlu untuk dilihat dan diamati bagaimana ia bekerja untuk dapat menghasilkan sesuatu yang lebih. Dalam artikel ini, penulis memilih menggunakan teori atribut inovasi oleh Everett M. Rogers dikarenakan ingin melihat dan mendeskripsikan bagaimana atribut inovasi pelayanan dari SIMPATIK sehingga dapat dilihat bagaimana keuntungan, kendala, dan kerumitan yang dialami dalam pelaksanaannya. C. KERANGKA TEORI Inovasi merupakan sarana untuk memupuk kepercayaan masyarakat kepada pemerintah dalam hal melaksanakan aktivitas pemerintahan. Menurut Wike Yuliana (2020) menyatakan inovasi adalah suatu kegiatan yang menciptakan sebuah ide baru yang tujuannya mengubah sesuatu baik berupa produk atau jasa menjadi lebih baik dari sebelumnya. Mulgan & Alburry dikutip dalam Muluk yang dikutip Wati (2021) membagi inovasi pelayanan kedalam lima tipe dengan kriteria masing-masing. Pertama Inovasi Produk atau Layanan, yaitu inovasi yang dilakukan dengan merubah bentuk dan desain produk layanan. Kedua Inovasi Proses Pelayanan, yaitu inovasi yang dilakukan dengan merubah organisasi, prosedur, dan kebijakan yang dibutuhkan untuk berinovasi. Ketiga Inovasi Metode Pelayanan, yaitu inovasi yang dilakukan dengan merubah cara berinteraksi dengan masyarakat atau memberikan pelayanan pada masyarakat. Keempat Inovasi Kebijakan yaitu inovasi yang mengacu pada kebaharuan visi, misi, tujuan dan strategi serta alasan berdasarkan realitas yang ada. Kelima Inovasi Sistem yaitu inovasi yang dilakukan dalam berinteraksi dengan aktoraktor lain atau dengan kata lain adanya perubahan dalam tata kelola pemerintahan. Rogers dalam Mona Melinda (2020) menilai perlunya standar universal untuk menggambarkan atribut yang diklaim menjadi inovasi pelayanan publik, sehingga ia menyatakan bahwa terdapat lima atribut inovasi yang berbeda yang saling berhubungan secara empiris namun secara konseptual berbeda, yaitu: 1. Relative advantages atau keuntungan relatif. Relative advantages atau keuntungan relatif adalah sejauh mana inovasi yang ada dianggap lebih baik daripada ide yang digantikannya (Rogers, 1995). Sebuah inovasi harus memiliki nilai lebih dan keunggulan dibandingkan dengan inovasi sebelumnya. 2. Compatibility atau kesesuaian. Compatibility atau kesesuaian adalah sejauh mana inovasi dianggap konsisten dengan nilai-nilai yang ada, pengalaman masa lalu atau ide-ide dan keadaan sebelumnya, dan dengan kebutuhan yang ada (Rogers, 1995). Dalam hal ini inovasi yang ada seharusnya tidak terlepas langsung dari inovasi dan keadaan yang sebelumnya agar inovasi yang telah ada sebelumnya tidak hilang begitu saja namun juga menjadi bagian dari perubahan dan proses inovasi. 3. Complexity atau kerumitan. Complexity atau kerumitan adalah tingkat di mana inovasi dianggap relatif sulit untuk dapat dipahami dan digunakan (Rogers, 1995). Inovasi yang baru memungkinkan memiliki tingkat kerumitan yang lebih tinggi dari inovasi sebelumnya. 3. Complexity atau kerumitan. Complexity atau kerumitan adalah tingkat di mana inovasi dianggap relatif sulit untuk dapat dipahami dan digunakan (Rogers, 1995). Inovasi yang baru memungkinkan memiliki tingkat kerumitan yang lebih tinggi dari inovasi sebelumnya. 4. Triability atau kemungkinan dicoba. Triability atau kemungkinan dicoba adalah sejauh mana inovasi dapat diujicobakan secara terbatas (Rogers, 1995). a. Relative advantage atau keuntungan relatif Sebuah inovasi harus memiliki nilai lebih dan keuntungan dibandingkan dengan keadaan yang ada sebelum adanya inovasi. Menurut Everett M. Rogers yang dikutip Mona Melinda dkk (2020), sebuah inovasi dapat dilihat dari sejauh mana inovasi dianggap lebih baik daripada ide yang digantikannya. Nilai lebih atau keunggulan dalam inovasi memperlihatkan sejauh mana suatu inovasi yang ada dianggap lebih baik dari keadaan sebelum adanya inovasi atau lebih baik dari inovasi yang ada sebelumnya. Inovasi SIMPATIK merupakan inovasi Pengadilan Agama Tanjung Pati yang berupa layanan pengurusan dokumen atau produk pengadilan tanpa harus dating ke kantor yang bertujuan untuk menghemat waktu dan tenaga masyarakat. Inovasi SIMPATIK dibuat dalam rangka memberikan pelayanan terbaik kepada masyarakat sehingga dibuat pelayanan yang dapat dilakukan meski tidak datang ke kantor pengadilan agama. Adanya inovasi SIMPATIK dapat menciptakan pelayanan yang efektif dan efisien karena masyarakat dapat melakukan tindakan mencari keadilan kapan saja dan dimana saja melalui mobil keliling yang dijadikan untuk memberikan pelayanan ke daerah yang jauh dari kantor pengadilan agama. Mobil Pelayanan Keliling tersebut beroperasi secara terjadwal setiap hari Kamis pekan pertama dan ke tiga di Nagari Limbanang, Kecamatan Suliki. Jadwal program Simpatik dapat di akses melalui https://pa-tanjungpati.go.id/ dan Instagram pengadilan_agama_tanjung_pati. Dalam komponen layanan simpatik terdiri dari, layanan informasi dan pengaduan. Layanan Pos bantuan Huhum, Layanan pendaftaran perkara secara online melalui aplikasi E-court dan layanan pengambilan dokumen berupa akte cerai,salinan putusan atau penetapan. Inovasi ini memudahkan masyarakat untuk mengakses hukum khususnya pelayanan pengadilan agama Tanjung Pati. Sehingga masyarakat dapat dilayani dengan cepat dan mudah. b. Compatibility atau Kesesuaian Menurut Rogers yang dikutip Mona Melinda dkk (2020), kesesuaian dilihat dari sejauh mana inovasi dianggap konsisten dan sesuai dengan nilai-nilai yang ada, pengalaman masa lalu, serta sesuai dengan kebutuhan yang ada. Nilai merupakan konsep yang dianggap berharga yang dianggap baik, layak, pantas, benar dan penting dalam kehidupan masyarakat. Dalam suatu organisasi, nilai merupakan sesuatu yang didukung oleh organisasi. Jika dikaitkan dengan teori Everett M. Rogers, dijelaskan bahwa suatu inovasi akan lebih mudah diterima jika sesuai dan tidak bertentangan dengan nilai dan gagasan yang didukung dan diterapkan. Karena, jika inovasi bertentangan dengan nilai yang ada maka inovasi akan relatif sulit untuk dapat diterima. Berdasarkan kajian yang dilakukan peneliti belum menemukan adanya penolakan terhadap pelaksanaan SIMPATIK ini, sejak mulai diberlakukan hingga sekarang, masyarakat mendukung penuh pelaksanaan ini yang berarti inovasi SIMPATIK tidak melanggar nilai- nilai yang ada dalam kehidupan bermasyarakat. Selain itu, dalam pelaksanaannya SIMPATIK ini bekerja sama dengan kantor wali nagari Limbanang untuk menentukan jadwal pelayanan, kerja sama yang dilakukan sejauh ini telah berjalan dengan lancar dan tidak menganggu ketertiban. Inovasi ini mudah diterima masyarakat karena masyarakat memerlukan pelayanan ini dalam kegiatan sehari-harinya. Hal ini berpedoman pada PERMA Nomor 01 Tahun 2014 Tentang Pedoman Pemberian Layanan Hukum Bagi Masyarakat Tidak Mampu di Pengadilan. Konsistensi Pengadilan Agama dalam melaksanakan SIMPATIK ini terlihat dengan jadwal mingguan yang sudah diterapkan serta adanya update kegiatan yang telah dilakukan pada website resmi pengadilan agama Tanjung Pati itu sendiri. Inovasi SIMPATIK dilaksanakan secara daring dan luring. Daring digunakan ketika pendaftaran online sesuai kebutuhan oleh masyarakat dan menentukan jadwal pelayanan lanjutan. Sedangkan luring dilakukan ketika jadwal telah tiba, masyarakat juga dapat menunggu di daerah masing-masing karena petugas datang dengan mobil keliling. c. Complexity atau Kerumitan Sebuah inovasi baru cenderung dianggap memiliki kerumitan yang lebih tinggi dibandingkan dengan inovasi sebelumnya. Menurut Rogers yang dikutip Mona Melinda dkk (2020), kerumitan adalah tingkat di mana inovasi dianggap relatif sulit untuk dapat dipahami dan digunakan. Dalam hal ini, dilihat juga seberapa besar kendala yang dihadapi. Dalam pelaksanaannya, inovasi SIMPATIK tidak memiliki kendala yang berarti karena dilakukan dengan luring dan daring dengan system jemput bola dimana petugas yang datang ke tempat masyarakat yang akan melakukan pelayanan sehingga mengurangi kendala penerapan inovasi ini. Hal yang mungkin menghambat pelaksanaan inovasi ini adalah kemampuan masyarakat untuk melakukan pendaftaran secara online dikarenakan masih banyak masyarakat di daerah Tanjong Pati dan sekitarnya yang gagap teknologi dan juga untuk daerah limbanang sendiri jaringan seluler masih susah sehingga akan mengalami gangguan ketika masyarakat membuka websitenya. Namun, untuk meningkatkan pemahaman masyarakat, pihak pengadilan agama Tanjung Pati masih sering mensosialisasikan inovasi SIMPATIK ini sehingga masyarakat bisa terbiasa dan tidak canggung lagi. e. Observability atau kemudahan diamati. Menurut Rogers yang dikutip Mona Melinda dkk (2020), observability adalah sejauh mana hasil inovasi dapat dilihat dan diamati oleh orang lain. Sebuah inovasi perlu untuk dilihat dan diamati bagaimana ia bekerja dan dapat menghasilkan sesuatu yang lebih baik. Sebuah inovasi yang mudah diamati akan lebih mudah untuk diterima dan berkembang di kalangan masyarakat. Agar inovasi SIMPATIK mudah diamati, dipahami, dan dikenal oleh masyarakat Kecamatan Harau, Pengadilan Agama Tanjung Pati melakukan berbagai upaya salah satunya dengan melakukan sosialisasi. Salah satu bentuknya yaitu melalui mobil keliling dengan membuka stan di lokasi yang strategis. Inovasi SIMPATIK merupakan inovasi yang dapat digunakan oleh masyarakat kapan saja dan di mana saja. Dalam penggunaannya masyarakat dapat mengakses SIMPATIK dengan masuk ke website https://pn-tanjungpati.go.id/. Website tersebut menyediakan berbagai informasi dan layanan yang dibutuhkan masyarakat. Setelah layanan dipilih, masyarakat harus mengunggah berkas dan persyaratan yang dibutuhkan, kemudian masyarakat dapat melihat jadwal yang telah disediakan pada website tersebut untuk pelaksanaan layanan tadi. d. Triability atau kemungkinan dicoba Sebuah inovasi harus diuji dan dicoba terlebih dahulu agar dapat diterima dan diterapkan di masyarakat. Inovasi yang akan dilakukan harus diujicobakan agar dapat dilihat nilai lebih dan keuntungannya dibandingkan dengan sebelumnya. Sebelum dilaunching dan diperkenalkan kepada masyarakat, inovasi SIMPATIK tidak melalui uji coba terlebih dahulu, namun karena inovasi SIMPATIK pada acara perdananya diadakan secara luring yang bertempat di kantor wali nagari Limbanang, maka sudah terlihat inovasi ini diterima di masyarakat dengan kedatangan mereka untuk melakukan pelayanan pengadilan. Selain itu, inovasi SIMPATIK telah disosialisasikan terlebih dahulu sebelum diberikan pelayanannya kepada masyarakat. Kelebihan dibandingkan sebelum adanya inovasi SIMPATIK ini sangat jelas, karena masyarakat tidak perlu jauh-jauh menempuh perjalanan dating ke kantor pengadilan agama, serta adanya system yang terjadwal juga sangat membantu kejelasan waktu yang diluangkan masyarakat untuk mendapatkan pelayanan. KESIMPULAN Berdasarkan uraian yang telah dijelaskan sebelumnya dapat diambil kesimpulan yaitu inovasi SIMPATIK oleh Pengadilan Agama Tanjung Pati sudah terlaksana dengan baik dan diterima oleh masyarakat setempat. Lima atribut inovasi yang dijabarkan oleh Everett M. Rogers, yaitu relative advantages, compatibility, complexity, triability, dan observability dimana empat diantaranya sudah terdapat dalam inovasi SIMPATIK. Hal ini terlihat dari adanya manfaat yang telah diterima masyarakat berupa efisiensi waktu, biaya dan tenaga serta telah sesuai dengan kebutuhan masyarakat setempat. Meski tidak dilakukan uji coba terlebih dahulu terhadap inovasi SIMPATIK, namun inovasi ini tetap berjalan baik sudah mendekati waktu satu tahun dan tentu membawa perubahan sebelum adanya inovasi ini. Rekomendasi untuk Pengadilan Agama Tanjung Pati selaku Lembaga yang melaksanakan inovasi SIMPATIK, yaitu: 1. Perlunya sosialisasi tata cara pendaftaran pengguna secara online agar masyarakat tidak kebingungan dan harus bertanya kepada petugas ketika memerlukan pelayanan. 2. Pengadilan Agama Tanjung Pati sebaiknya memanfaatkan media social dan juga internet untuk memperkenalkan SIMPATIK ini, karena sumber yang peneliti dapat sangat terbatas untuk penulisan artikel, ini tentu berpengaruh juga kepada pengetahuan masyarakat terkait SIMPATIK ini sendiri jika tidak disediakan pemahaman lebih lanjut mengenai inovasi ini. 2. Pengadilan Agama Tanjung Pati sebaiknya memanfaatkan media social dan juga internet untuk memperkenalkan SIMPATIK ini, karena sumber yang peneliti dapat sangat terbatas untuk penulisan artikel, ini tentu berpengaruh juga kepada pengetahuan masyarakat terkait SIMPATIK ini sendiri jika tidak disediakan pemahaman lebih lanjut mengenai inovasi ini. 3. Pengadilan Agama Tanjung Pati sebaiknya melakukan survei kepuasan masyarakat terkait inovasi SIMPATIK agar dapat dilihat dengan jelas bagaimana respons masyarakat terhadap adanya inovasi SIMPATIK. 3. Pengadilan Agama Tanjung Pati sebaiknya melakukan survei kepuasan masyarakat terkait inovasi SIMPATIK agar dapat dilihat dengan jelas bagaimana respons masyarakat terhadap adanya inovasi SIMPATIK. 4. Pengadilan Agama Tanjung Pati sebaiknya melakukan pemanfaatan pelayanan online tidak hanya pada sesi pendaftaran aja melainkan sesi lain seperti diskusi melalui wa dan sebagainya. DAFTAR PUSTAKA Hardiyansyah. 2018. Kualitas Pelayanan Publik. Yogyakarta: Gava Media. Melinda, M., Syamsurizaldi, & Kabullah, M. I. 2020. Inovasi Pelayanan Administrasi Kependudukan Online (PADUKO) oleh Dinas Kependudukan dan Pencatatan Sipil Kota Padang Panjang. Nakhoda: Jurnal Ilmu Pemerintahan. Vol. 19, No. 2: 202-216. Ariany, R., & Putera, R. E. 2013. Analisis Kinerja Organisasi Pemerintah dalam Memberikan Pelayanan Publik di Kota Pariaman. MIMBAR. Vol. 29, No. 1: 33-40. Eriza, A,. Yoserizal, & Putera, R. E. 2021. Inovasi Pelayanan Publik Berbasis Android ‘Paga Nagari’ Di Polres Solok Kota. JMPKP: Jurnal Manajemen Publik & Kebijakan Publik. Vol. 3, No. 2: 12-22. Jalma, H., Putera, R. E., & Kusdarini. 2019. E-Government dengan Pemanfaatan Web OpenSID dalam Pelayanan Publik di Nagari Tanjung Haro Sikabu-kabu Padang Panjang. Jurnal Ilmu Administrasi. Vol. 8, No. 1: 24-37. Ananda, B. R., Putera, R. E., & Ariany, R. 2019. Reformasi Birokrasi Pelayanan Publik di Polres Solok Kota. Transparansi : Jurnal Ilmiah Ilmu Administrasi. Vol. 2, No. 2: 217- 223. Alindro, N., Kusdarini, & Putera, R. E. 2021. Inovasi Pelayanan Administrasi Nikah Terintegrasi di Kantor Urusan Agama Kecamatan Enam Lingkung Kabupaten Padang Pariaman. Jurnal Ilmu Administrsi Negara (AsIAN). Vol. 9 No. 1: 218-227. Triwahyuni, M., Putera, R. E., & Rahayu, W. K. 2020. Inovasi Pelayanan Kesehatan Kelas Imud di Puskesmas Padang Pasir, Kecamatan Padang Barat, Kota Padang, Sumatera Barat. Jurnal Public Policy. Vol. 6, No. 1. Ananda, B. R., Putera, R. E., & Ariany, R. 2019. Inovasi Pelayanan Kesehatan Di Rumah Sakit Umum Daerah Kota Pariaman. Jurnal Ilmu Administrasi. Vol. 8, No. 2: 167-179. Putera, R. E. 2009. E-Government dan Reformasi Birokrasi dalam Rangka Peningkatan Pelayanan Publik di Daerah. DEMOKRASI. Vol. VIII, No. 1. Yuliana, W., Putera, R. E., Yoserizal. 2020. Inovasi Pelayanan Kesehatan Public Safety Center 119 (PSC 119) Smash Care’s di Kota Solok. JIAP: Jurnal Ilmu Administrasi Publik. Vol. 8, No. 1: 265-271. Permana, L., & Izzati, U. 2020. Inovasi Pelayanan Kesehatan Publik Berbasis E-government (Studi Kasus: Inovasi Ayo Ceting di Puskesmas Andalas). JESS (Journal of Education on Social Science). Vol. 4, No. 1: 25-37. Hamirul. 2018. Patologi Pelayanan Publik Di Era Revolusi Industri 4.0. Jurnal Tata Sejuta. Vol. 4, No. 2: 82-96. Wati, N. K. Tipologi Inovasi Pelayanan Publik Dinas Kependudukan Dan Pencatatan Sipil (Disdukcapil) Kota Tangerang Selatan Di Masa Pandemi. JURNAL OF GOVERNMENT - JOG (Kajian Manajemen Pemerintahan & Otonomi Daerah). Vol. 6, No. 2: 89-110. Trijultian, A., Basori, Y. F., & Amirulloh, M. R. 2022. DAFTAR PUSTAKA Pengaruh Kualitas Pelayanan Administrasi Pos Bantuan Hukum Terhadap Kepuasan Masyarakat Di Pengadilan Agama Kota Sukabumi. JIP: Jurnal Inovasi Penelitian. Vol. 2, No. 10: 3399-3406. AM, Fordilag. 2021. PA Tanjung Pati Layani Masyarakat dengan Inovasi SIMPATIK. AM, Fordilag. 2021. PA Tanjung Pati Layani Masyarakat dengan Inovasi SIMPATIK. Tersedia https://badilag.mahkamahagung.go.id/ diakses 3 April 2022. Pengadilan Agama Tanjung Pati. 2022. Sosialisasi "Inovasi" Simpatik (Sistem Pelayanan Pa Tanjung Pati Keliling) Pa Tanjung Pati. Tersedia https://www.pa-tanjungpati.go.id/ diakses 3 April 2022. MinangkabauNews. 2021. Inovasi Produk layanan PA Tanjung Pati, Bupati: Manfaatkan dan Lihat Jadwalnya. Tersedia https://minangkabaunews.com/ diakses 3 April 2022. MinangkabauNews. 2021. Inovasi Produk layanan PA Tanjung Pati, Bupati: Manfaatkan dan Lihat Jadwalnya. Tersedia https://minangkabaunews.com/ diakses 3 April 2022.
https://openalex.org/W4290635951	https://www.journals.vu.lt/kriminologijos-studijos/article/download/28827/28019	English	null	Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics	Kriminologijos studijos	2,022	cc-by	12,532	Kriminologijos studijos ISSN 2351-6097 eISSN 2538-8754 2021, vol. 9, pp. 100–128 DOI: https://doi.org/10.15388/CrimLithuan.2021.9.4 Contents lists available at Vilnius University Press Kriminologijos studijos ISSN 2351-6097 eISSN 2538-8754 2021, vol. 9, pp. 100–128 DOI: https://doi.org/10.15388/CrimLithuan.2021.9.4 Contents lists available at Vilnius University Press ISSN 2351-6097 eISSN 2538-8754 DOI: https://doi.org/10.15388/CrimLithuan.2021.9.4 Kriminologijos studijos 2021, vol. 9, pp. 100–128 Received: 29/04/2022. Accepted: 03/08/2022 Copyright © 2021 Sigita Černevičiūtė. Published by Vilnius University Press. This is an Open Access article distributed un- der the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics Sigita Černevičiūtė Sigita Černevičiūtė Sigita Černevičiūtė Vilniaus universitetas, Filosofijos fakultetas Sociologijos ir socialinio darbo institutas Kriminologijos katedra Vilnius University, Faculty of Philosophy Institute of Sociology and Social Work Department of Criminology Universiteto g. 9, LT-01513 Vilnius tel. (8 5) 266 7600 sigita.cerneviciute@fsf.vu.lt ORCID iD https://orcid.org/0000-0003-4409-4961 Abstract. This article calls into question the reliability of the official historical statistical sources of homicides in 1920s-1930s Lithuania and aims to evaluate the limitations of the mentioned sources. With this in mind, we will attempt to make some assumptions about homicide types and trends in interwar Lithuania. The Central Statistical Bureau had been publishing statistical data relevant to the investigation of homicides in Statistical Bulletins and the Statistical Yearbooks of Lithuania since 1924. These statistical sources in the homicide study were problematic due to an unclear data collection methodology, the use of different homicide terms, unrealistic definitions of urban and rural areas and changes in Lithuania’s territory and population. We cannot determine the exact rates of the 1930s homicides due to the change in the homicide terminology and its content. Between 1924-1931 the term violent deaths, except suicides, was used, and homicides were not singled out. The analysis also shows that until 1931 violent deaths included homicides and accidental deaths too. From 1932 to 1939 violent deaths were divided into 4 groups: suicide, homicide, accidents and other violence. The more detailed data of the 1930s have revealed that the most frequent victims of homicide were in the 100 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics 15-29 and 29-44 age groups for men, while the 15-29 age group stands out for females. The most common method of killing was shooting. The police-published homicide statistics also reveal a problem of terminology. According to the Penal Statute of Lithuania, deprivation of life was distinguished into the crimes of homicide; infanticide; abortion; preparation to murder; attempted murder and persuasion and help to commit suicide. However, this terminology was only partially reflected in the police statistics, as between 1927-1930 the police used the term homicides and classified them into the ones committed: for-profit; during brawls; for other purposes. Infanticide was separated from homicides. Leaving out others, infanticide was the most common murder type in interwar Lithuania. Sigita Černevičiūtė Since 1931 the statistics of the police had been using the term of deprivation of life and distinguished it into the 7 types according to the motive: for-profit; during brawls; defending one’s own life or the lives of others; involuntary; infanticide; abortion and for other purposes. It did not include dead bodies found, suicides and accidental deaths. After analysing the 1931-1938 data on deprivation of life, excluding abortions, homicides would vary from 200 to 300 per year. When comparing all the deprivations of life with homicides by the cause of death, it can be concluded that these figures included preparation for murder and attempted murder as well as persuasion and help to commit suicide. Thus, due to the change in the terminology and the inclusion of attempted murders, the police statistics can be considered unreliable. Homicides are the most precisely quantitatively defined by the rates of causes of death, which in 1932-1938 Lithuania were relatively stable with an amplitude of 93-139 murders per year. On a basis of the analysis of the police statistical data, it is possible to identify at least 4 main types of homicides in interwar Lithuania: reproductive – infanticide and abortions, aggressive – killings during brawls, economic - for profit as well as unintentional homicides that happened during accidents. K d hi t i l t ti ti h i id h i id t i l t d th i t Keywords: historical statistics, homicides, homicide types, violent deaths, interwar Lithuania. Nužudymų tendencijos ir tipai XX a. 3–4 dešimtmečio Lietuvoje: oficialios statistikos trūkumai Nužudymų tendencijos ir tipai XX a. 3–4 dešimtmečio Lietuvoje: oficialios statistikos trūkumai Santrauka. Šiame straipsnyje kritiškai analizuojama oficialių statistinių XX a. 3–4 de- šimtmečio nužudymų šaltinių patikimumas ir siekiama įvertinti jų trūkumus. Atsižvel- giant į gautus rezultatus, bus mėginama padaryti galimas prielaidas apie nužudymų tipus ir tendencijas tarpukario Lietuvoje. Straipsnyje susitelkiama į medicinos įstaigų duomenų su mirties priežastimis ir policijos skelbtų nužudymų statistikos analizę, atsi- ribojant nuo teismų pateikiamos nužudymų bylų kiekybinės informacijos. Nužudymų statistiniai duomenys nuo 1924 m. buvo skelbiami Statistikos biuleteniuose ir Lietuvos statistikos metraščiuose. Šie statistikos šaltiniai nužudymų tyrime kelia klausimų dėl savo patikimumo, dėl neaprašytos duomenų rinkimo metodologijos, skirtingos nužu- dymų terminijos vartojimo, dirbtinio miesto ir kaimo vietovių apibrėžimo, Lietuvos teritorijos ir gyventojų skaičiaus kitimo. XX a. 3 deš. nužudymų skaičiaus negalime 101 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 nustatyti dėl nužudymų terminijos kaitos ir jos turinio. 1924–1931 m. vartotas ter- minas prievartos mirtys, išskyrus savižudybes, o nužudymai nebuvo išskirti atskirai. 1932–1939 m. prievartos nusikaltimai skirstyti į keturias grupes: nusižudymus, nu- žudymus, nelaimingus atsitikimus ir kitokią prievartą. XX a. 4 deš. išsamesni duome- nys atskleidė, kad vyrai buvo nužudomi 3–4 kartus dažniau nei moterys ir priklau- sė 15–29 ir 29–44 metų amžiaus grupėms, o moterys – 15–29 metų amžiaus grupei. Labiausiai paplitęs nužudymo būdas – nušovimas. Policijos publikuotuose nužudymų statistiniuose duomenyse taip pat išryškėja sąvokų problema. Remiantis Lietuvos bau- džiamuoju statutu, kaip gyvybės atėmimas buvo apibrėžti nužudymai, pavainikių kūdi- kių nužudymai, abortai, rengimasis ir pasikėsinimas nužudyti, suteikimas priemonių ir kalbinimas nusižudyti. Tačiau policijos statistikoje ši terminija atsispindėjo tik iš dalies, 1927–1930 m. policija vartojo žmogžudysčių terminą ir jas skirstė į padarytas: pasi- pelnymo tikslu, per muštynes ir kitokias. Nesantuokinių vaikų žudymas buvo išskirtas atskirai. Jei neskaičiuotume kitokių nužudymų, infanticidas buvo dažniausias nužudy- mo tipas tarpukario Lietuvoje. Nuo 1931 m. policijos duomenyse gyvybės atėmimas buvo skirstytas į septynis tipus pagal įvykio motyvą: pasipelnymo tikslu, per muštynes, ginant savo ar kitų gyvybę, per neatsargumą, pavainikių kūdikių žudymas, gemalo sunai- kinimas ir kitais tikslais. Nusižudymai, nelaimingi atsitikimai ir rasti mirusiųjų kūnai į šias kategorijas neįėjo. Išanalizavus 1931–1938 m. gyvybės atėmimo duomenis, neska- ičiuojant abortų, nužudymai varijavo tarp 200–300 atvejų per metus, tačiau palyginus visus gyvybės atėmimus su nužudymais pagal mirties priežastį darytina išvada, kad šie skaičiai pateikti su rengimusi ir pasikėsinimu nužudyti bei suteikimu priemonių ir kalbinimu nusižudyti. Taigi, dėl sąvokų kaitos ir pasikėsinimų priskaičiavimo prie nu- žudymų policijos statistinius duomenis galima laikyti nepatikimais. Nužudymų tendencijos ir tipai XX a. 3–4 dešimtmečio Lietuvoje: oficialios statistikos trūkumai Tiksliausiai kieky- biškai nužudymus apibrėžia mirties priežasčių rodikliai, kurie 1932–1938 m. Lietuvo- je fiksavo gana stabilią 93–139 nužudymų per metus amplitudę. Remiantis policijos statistinių duomenų analize galima identifikuoti bent keturis pagrindinius nužudymų tipus tarpukario Lietuvoje: reprodukcinį – nesantuokinių kūdikių žudymą ir abortus, agresyvųjį – nužudymai muštynių metu, ekonominį – siekiant pasipelnyti, netyčinius nužudymus per nelaimingus atsitikimus. y p g Pagrindiniai žodžiai: istorinė statistika, nužudymai, nužudymų tipai, prievartos mir- tys, tarpukario Lietuva. 1 The Historical Homicide Monitor (HHM) has been developed in the framework of the project Nordic Homicide from Past to Present led by prof. Janne Kivivuori (University of Helsinki), which has brought together the Nordic criminologists and historians researching lethal violence. More about the project and the HHM: https://blogs.helsinki. fi/historicalhomicidemonitor/project-2/. Introduction Historical violence is one of the most favourite topics of nowadays crime historians, where homicide rates are generally considered as an indication of the level of serious violence (Spierenburg, 2001). However, when studying his- torical violence, researchers tend to rely on official statistics collected by state institutions; of course, it helps to provide a broader view of the phenomenon and helps with comparisons for different periods or levels of violence. Com- 102 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics parisons of crime levels and trends between different countries have been an object of statistical research since the 1820s. It is reported in the literature that homicides are the most accurately re- corded violent crimes, explaining the considerable body of literature on his- torical long–term trends of homicides that has researched murder since the Middle Ages to nowadays in the United States of America (Lane, 1979, Monk- konen, 2006) and western European countries (Aebi & Linde, 2016; Eisner, 2003; Spierenburg, 2008, 2012). Historical homicides are researched extensive- ly in the Nordic countries (Ylikangas, Karonen, Lehti, 2001; von Hofer, 1990, Vuorela, 2018). Here, a new quantitative research tool of historical homicide monitor1 has been developed and used to transform qualitative data to quan- titative, enabling comparisons cross-nationally and over time. The result of this tremendous work is a comparison of homicides in Denmark, Sweden and Finland from the early modern times to the present (Kivivuori, et al., 2022). However, only some of the research has used and critically evaluated of- ficial historical statistical data on violent crimes (Gurr, 1981; Monkkonen, 2001), others have used their own created database or focused on the inter- war period (Lehti, 2001b). The research on historical statistics of homicide in Eastern Europe remains limited; in particular, from the Baltic countries, only the interwar Estonian homicide trends have been analysed in comparison to Finland, concluding that the rise of criminal violence in 1930s Estonia was related to the modernisation of agriculture and pressures of uncertain future on youth, competitive values and normalised violence inherited from Czarist Russia (Lehti, 2001a). In the Lithuanian historiography, research of long-term historical homi- cide trends is still waiting for its turn, and only a few historical criminology studies have shown interest in the interwar period crime trends in Lithuania. 2 Original in Lithuanian: “Statistika – tai faktai, pakeisti skaičiais; tie skaičiai parodo mums kokiu keliu mes ėjom, kur mus tas kelias veda ir, jeigu reikalinga, kaip geriau tą kelią pakeisti kitu, nauju keliu”. Alfa. Kriminalinė statistika. Policija, 1931, No 11, p. 215. 3 Volumes of Statistical Yearbooks of Lithuania have been published on the site of the Official Statistics Portal: https://osp.stat.gov.lt/. 4 Statistical Bulletin. https://www.epaveldas.lt/preview?id=C1C1B0000078024. 5 Police. https://www.epaveldas.lt/preview?id=C1B0003778541. 6 Reference Book of Criminalistics. https://www.epaveldas.lt/preview?id=C10000221382. 7 The author is thankful for valuable comments and suggestions on the historical homicide statistics research for Prof. Aleksandras Dobryninas, Prof. Zenonas Norkus, Prof. Paul Knepper and Prof. Janne Kivivuori. Introduction The historical statistics of homicides registered by the police in 1931-1939 without critical assessment have been presented in an overview of the develop- ment of crime in Lithuania between 1918-1993 (Smaliukas & Urbelienė, 1994). 103 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 Babachinaitė and Paulikas (2002) have analysed the police-registered crime statistics without critically examining them. Even after noticing that only be- tween 1937-1938 attempted murders were included in the deprivations of life statistical data, the researchers have concluded that the rates of criminal depri- vation of life were not low in interwar Lithuania. In the history of the interwar criminalistics, the motives of murders have first been presented in the Lithu- anian historiography by Palskys (1995), the content of homicides punished by the death penalty has been investigated by Černevičiūtė and Kaubrys (2014), and the statistics of femicide have been briefly analysed by Černevičiūtė and Kareniauskaitė (2021). A key problem with much of the literature regarding statistical research of the interwar Lithuania’s homicides is that it makes only superficial overviews of the homicide trends without critically evaluating the methodology of data collection and misuses the statistics to make conclusions without credibility. Under the pseudonym Alfa, an author of an article on the crime statistics in 1930s Lithuania wrote: “Statistics are facts replaced by numbers; those num- bers show us what path we have been on, where that path is leading us and, if necessary, how to better replace that path with another – new – path.”2 How- ever, statistics should not be accepted without any doubts. Historical homicide research confronts many methodological challenges, which scholars do not always address. This article, for the first time in the Lithuanian historiography, calls into question the reliability of the official historical statistical sources of homicides in 1920s-1930s Lithuania and aims to evaluate the limitations of the mentioned sources. With this in mind, we will attempt to make some assump- tions about homicide types and trends in interwar Lithuania. When carrying out statistical research on the homicide phenomenon, there is no universal method for how to count murders. However, it could be studied from three different perspectives – a victim’s, a crime’s and from the perspective of a perpetrator’s punishment. The first one could be called counting the bodies – calculating murder vic- tims and checking body inspection reports. According to the crime historian 104 Sigita Černevičiūtė. Introduction Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics Pieter Spierenburg (2001), homicide rates should always be calculated accord- ing to the reports about bodies found. Medical statistics about the causes of death are available in most European countries from the end of the nineteenth century or the beginning of the twentieth, and it is difficult to hide a body. The second stage after the body is found is defining it as a crime and inves- tigating the circumstances to find the perpetrator. For our research it means studying the police-recorded homicides. And the third one, we can research homicides via court statistical data and calculate convicted or acquitted crim- inals. All of these three angles have their own advantages and disadvantages. They can provide a different overview of the trends and types of homicides in Lithuania in the 1920s and 1930s. In this article, we will analyse the medi- cal and police statistical records as one of the most reliable sources about the homicides’ phenomenon. The mentioned methodological problems have inspired the author of this article to start rather scrupulous work of critically analysing and comparing the data of homicides in the Official Statistical Yearbooks of Lithuania, official police periodicals, archival statistics and official governmental law editions related to the organisation of collection of statistics. It should be mentioned that at the beginning of the research on homicide statistics in interwar Lithu- ania, the primary sources were the Statistical Yearbooks of Lithuania (Lit. Lie tuvos Statistikos Metraščiai, Vol. 1-12)3, Bulletin of Statistics (Lit. Statistikos Biuletenis, 1923-1940)4, while the journals Police (Lit. Policija, 1924-1940)5 and Reference Book of Criminalistics (Lit. Kriminalistikos Žinynas, 1935-1940)6 had only been scanned and published on the internet in Portable Document Format or as images without a possibility to copy the data en masse. Because of that, all the sources had to be reviewed thoroughly and numbers had to be collected manually and recalculated by the author of this article7. 105 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 We have not used the data published by the authors of the first historical sta- tistics project in Lithuania8 in the present article, but it is important to acknowl- edge that at least 200 new datasets of the historical statistics about the Baltic States have been published, including the dataset Causes of Death of the Popula- tion in Lithuania, 1919-19399, where violent deaths have been mentioned. 8 The project Historical Sociology of Modern Restorations: A Cross-Time Comparative Study of Post-Communist Transformations in the Baltic States (2018-2022) led by the sociologist Zenonas Norkus. More about the project: https://lida.dataverse.lt/dataverse/ HistatData_Baltic. 9 Norkus, Z., Ambrulevičiūtė, A., Markevičiūtė, J., Morkevičius, V., Žvaliauskas, G. (2021) Causes of Death of the Population in Lithuania, 1919-1939. Lithuanian Data Archive for the HSS (LiDA), V1, https://hdl.handle.net/21.12137/K4ZXOA. Introduction The compilers of the table have grouped the data of 1925-1939 according to the In- ternational Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10 chapter, version 2019). However, the researchers should have kept the methodological limitations of the causes of death statistics in in- terwar Lithuania in mind and separate the violent deaths until 1932 from the homicides since then (more about it in this article p. 112). 10 Lietuvos statistika 1920-1930 metais. Kaunas: Centralinis statistikos biuras, 1931, p. 9. 8 The project Historical Sociology of Modern Restorations: A Cross-Time Comparative Study of Post-Communist Transformations in the Baltic States (2018-2022) led by the sociologist Zenonas Norkus. More about the project: https://lida.dataverse.lt/dataverse/ HistatData_Baltic. 9 Norkus, Z., Ambrulevičiūtė, A., Markevičiūtė, J., Morkevičius, V., Žvaliauskas, G. (2021) Causes of Death of the Population in Lithuania, 1919-1939. Lithuanian Data Archive for the HSS (LiDA), V1, https://hdl.handle.net/21.12137/K4ZXOA. 10 Lietuvos statistika 1920-1930 metais. Kaunas: Centralinis statistikos biuras, 1931, p. 9. Institutionalisation of Interwar Lithuanian Statistics Lithuania declared its Independence from the Russian Empire on February 16th, 1918. During the establishment of the First Republic of Lithuania, fol- lowing an example of Tsarist Russia, the collection of statistics was decentral- ised, and each ministry had its own, usually weak statistical units (Martišius. 2009). However, as early as 1919, the General Statistics Department was es- tablished on 6 September by the order No 370 of the Minister of Trade and Industry Jonas Šimkus. Soon it was reorganised into a new statistics depart- ment with 6 staff members at the Credit Office10. Thus, the collection of statis- tical data started with only a few individuals. In addition to this, all ministries maintained their own statistical divisions. Because of that, it was decided to centralise the process. The government’s resolutions establishing the Central Statistical Bureau and the Standing Statistics Committee entered into force on May 20th, 1921. 106 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics The committee became Lithuania’s chief statistical body, and the Central Sta- tistical Bureau was its executive body, functioning as a separate department under the Ministry of Finance. The objectives of the Central Statistical Bureau were: The committee became Lithuania’s chief statistical body, and the Central Sta- tistical Bureau was its executive body, functioning as a separate department under the Ministry of Finance. The objectives of the Central Statistical Bureau were: “a) to draw up plans for the work in the field of statistics and to submit them to the Standing Statistics Commission for review and final editing; b) to follow the work and research on statistics in foreign literature and to apply the most suitable methods of statistics to the State of Lithuania; c) to collect, verify and manage statistical material, publish individual press rele- ases and publish periodic statistical collections; d) to inform the State institutions about the statistical data; e) to perform other tasks at the request of the Minister”11. One of the first tasks of the Central Statistical Bureau was to organise the General Population Census of Lithuania, which was conducted in 1923. The first results were published in the monthly Bulletin of Statistics12 and later in a separate book about the data on the Census13. In Lithuania at that time (53,242 km²), excluding the Klaipėda Region, 2,028,971 inhabitants were registered, 47.7 % of which were men and 52.3 % were women. 12 Pirmieji surašymo daviniai. Statistikos biuletenis, 1923, No 1, p. 3-4. 13 Lietuvos gyventojai. Pirmojo 1923 m. rugsėjo 17 d. visuotino surašymo duomenys. Kaunas: Finansų ministerija. Centralinis statistikos biuras, 1926. 11 Centralinis statistikos biuras. Vyriausybės žinios, 1921 05 20, No 60-587. Lietuvos gyventojai. Pirmojo 1923 m. rugsėjo 17 d. visuotino surašymo duomenys. Kaunas Institutionalisation of Interwar Lithuanian Statistics When the Klaipėda Region was incorporated into Lithuania in 1923, it was also included in the activities of the Central Statistical Bureau since 1925. The work of the statistical bureau improved over the years, and its partici- pation in the international statistical organisations began. Lithuania joined the International Trade Statistics Bureau in Brussels in 1928. Although it should be borne in mind that Lithuania did not join many such international organi- sations simply because of finances – Lithuania was not a wealthy country and could not afford to pay a membership fee. Lithuania wanted to sign the Inter- national Convention on Statistics in 1929, but this decision was postponed for unknown reasons. It can be assumed that it was not signed because of a requirement to meet international standards that Lithuania was not eager to follow. 107 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 At the beginning of the 1920s, the Central Statistical Bureau had 16 people on staff and just 3 departments, but at the end of the decade it already had 6 departments: the office and the departments of demography, agriculture, in- dustry and labour, foreign trade, and economic market14. As a result, the staff increased twice – to 34 people, which allowed the bureau to improve the qual- ity of Lithuanian statistics. The end of the development of the statistical data collection system can be considered in 1930 when the Law on State Statistics was adopted. It aimed to solve the problems that had arisen so far: to clarify the centralisation of the col- lection of statistics and define the right to request statistics from institutions and individuals. Since 1930 the Central Bureau of Statistics was appointed as the highest executive body of statistics; the Statistical Committee replaced the Standing Statistical Committee under the Central Statistical Bureau. The law provided that the Central Statistical Bureau: “1) collects, verifies and processes statistical data required by law or the government; 2) conducts general population, agricultural and other censuses required by law 3) publishes state statistical data and issues periodical statistical bulletins and ye- arbooks and other statistical publications; 4) unifies the statistical data on the work of public law institutions of the entire state; 5) co-operates with international and foreign statistical institutions.”15 The law also stipulated that all institutions and persons had to provide statistical data to the statistical bureau upon request. 14 Lietuvos statistika 1920-1930 metais. Kaunas: Centralinis statistikos biuras, 1931, p. 15. 15 Valstybės statistikos įstatymas. Vyriausybės žinios, 1930 07 14, No 332-2272. Lietuvos statistika 1920 1930 metais. Kaunas: Centralinis statistikos biuras, 1931, p. 15. 15 Valstybės statistikos įstatymas. Vyriausybės žinios, 1930 07 14, No 332-2272. 14 Lietuvos statistika 1920-1930 metais. Kaunas: Centralinis statistikos biuras, 1931, p. 1 15 Valstybės statistikos įstatymas. Vyriausybės žinios, 1930 07 14, No 332-2272. 16 32 Nr. Mirimai mirties priežastimi. Didž. Lietuvos naturalinio gyventojų judėjimo 1924 m. daviniai. Statistikos biuletenis, 1925, No 5, p. 48-49. Institutionalisation of Interwar Lithuanian Statistics Persons who refused to provide such information or submitted false information without a legitimate reason had to be fined 250 litas. However, the new law on statistics was not perfect. The relations of the statistical bureau with other state institutions were not specified. It was not explained who could impose the fine for false infor- mation – administrative bodies or a court (Martišius, 2009). The bureau of statistics itself, until 1940, was poorly funded and, there- fore, poorly equipped. At that time, the calculations in Lithuania were per- formed using electric and manual arithmometers and perforating machines (Martišius, 2009). Although the statistical collection system had been im- 108 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics proved at the beginning of the 1930s and a breakthrough in the official statis- tics had taken place, the detailed and sufficiently precise statistics began to be published only in 1935. However, there were many problems in conducting a quantitative study of homicides, even with the 1930s data. Collection of Homicide Statistics of Lithuania in 1920s-1930s Collection of Homicide Statistics of Lithuania in 1920s-1930s According to the sociologist Zenonas Norkus, the beginning of statehood of the First Republic of Lithuania was “statistical darkness.” Most of the statisti- cal data about the period of 1918-1920 when Lithuania was battling against the Bolshevik forces, Bermontians and Poland have been collected retrospectively (Norkus, 2014). The Central Statistical Bureau started to continuously publish official statistics with the causes of death based on some kind of methodol- ogy only starting from 192416 up to 1939 in the Bulletin of Statistics and the Statistical Yearbooks of Lithuania. The police-recorded homicides were started to be published in the Statistical Yearbooks only since 1931 (with the data from 1929-1930). These two primary sources for the statistical research of homi- cides have a few different problems. First of all, the methodology of how the data had been collected was ex- plained only vaguely in the Bulletin of Statistics, where the violent deaths of 1924 were mentioned for the first time. The methodologies used in the Statisti- cal Yearbooks of Lithuania were not explained at all. Secondly, various terms to describe the homicide phenomenon without exact definitions were used in different parts of the yearbook and for different years: violent death, homicide, and deprivation of life. As a result of the chang- ing and vague methodologies, it is not clear what was included into the records and what was not. It should be mentioned that in the historical research of homicides, infanticide and abortions are usually not included because of their fundamental difference from other types of homicides (Lehti, 2001a). In this article, we attempt to understand the definition of homicide in the interwar Lithuanian statistics and include the mentioned types in the research. The ar- chival sources of the Central Statistical Bureau (which led the publishing of the 109 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 yearbooks) do not help as well – only a few documents with original statistical data of homicides by the cause of death from the years 1933-1934, 194017-1941 have survived until nowadays, but there is nothing to explain its methodolo- gies18. Consequently, only comparisons and a careful analysis of the periodi- cals that published only partial statistics could help.h Thirdly, there were no realistic concepts of urban and rural areas. Collection of Homicide Statistics of Lithuania in 1920s-1930s Accord- ing to the Bulletin of Statistics of 1924, cities and towns with more than 2,000 inhabitants were considered urban areas, and this decision was based on in- ternational practice19. However, in the Statistical Yearbooks of Lithuania, the statistics of homicides by the causes of death were only divided into urban and rural areas since 193220. It should be noted that the statistical data were also affected by Lithuanian territorial changes. Lithuania lost Vilnius in 1920 and regained it at the end of 1939, annexed the Klaipėda Region in 1923 and lost it in 1939 (Laurinavičius, 2009). Because of these territorial changes, we have the statistics in different years and different data sets; for example, homicide statistics by the cause of death were published with the Klaipėda Region for 1925, but without it be- tween 1926-1927, and then again with the Klaipėda Region between 1928- 1938, and without it in 1939. As a result, it is rather hard to have a complete set of homicide rates for the entire interwar period of Lithuania. Changes in population growth also impact the rates of crime statistics. As mentioned, the general census of the Lithuanian population was conducted in 1923, recording 2 million people in the territory without the Klaipėda Region. In the Statistical Yearbook of Lithuania, the changes in the population were cal- culated on the basis of natural increase only, and migration was not taken into account, although many people were going abroad every year (Gaučas, 1978). 17 In 1940 (full year from January through December), there were 31 homicides registered in the urban areas, 89 in the rural, in total – 120. Mirusieji miestuose ir kaimuose mirties priežastimi. Lietuvos centrinis valstybės archyvas [Lithuanian Central State Archives – later on LCVA], fund 418, inventory 1, file 4, page 6. 17 In 1940 (full year from January through December), there were 31 homicides registered in the urban areas, 89 in the rural, in total – 120. Mirusieji miestuose ir kaimuose mirties priežastimi. Lietuvos centrinis valstybės archyvas [Lithuanian Central State Archives – later on LCVA], fund 418, inventory 1, file 4, page 6. i 18 It should be mentioned that only 20 files of the Central Statistical Bureau have survived until nowadays in the fund of No 418, that is preserved in LCVA. None of these files explain methodologies of the statistical data collection. 20 Lietuvos statistikos metraštis 1932, T. 5, Kaunas: Spindulio b-vės sp., 1933, p. 32-33 Collection of Homicide Statistics of Lithuania in 1920s-1930s 18 It should be mentioned that only 20 files of the Central Statistical Bureau have survived until nowadays in the fund of No 418, that is preserved in LCVA. None of these files explain methodologies of the statistical data collection. 19 23 Nr. Miestų ir miestelių su gyventojų skaičiumi virš 2000 žm.) sąrašas. Statistikos biuletenis, 1924, No 6, p. 25. 19 23 Nr. Miestų ir miestelių su gyventojų skaičiumi virš 2000 žm.) sąrašas. Statistikos biuletenis, 1924, No 6, p. 25. 20 Lietuvos statistikos metraštis 1932, T. 5, Kaunas: Spindulio b-vės sp., 1933, p. 32-33. 110 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics A new census was planned to be completed in the 1930s. However, because of the global Great Depression of 1929-1933, the money was used for essentials with the thought that it would be possible to return to it later, but in 1940 the occupation of Lithuania by the Soviet Union changed these plans. Because of all these problems in this article, we have decided not to count homicide rates per 100,000 population and use only “bald” homicide numbers at the time. Counting the Bodies: Trends of Violent Deaths in Lithuania between 1924-1939 As mentioned before, the official statistics of Lithuania were compiled by the Central Statistical Bureau. Medical records on homicides were collected and processed in the Department of Demography, which collected the sta- tistics on the natural movement of the population – the information about the married, born and the deceased. Separate forms were to be sent to the Central Statistical Bureau by the registration points of the respective religions. For example, in 1930, there were 669 points in the main Lithuania (without the Klaipėda Region), 500 of whom were Catholic, 42 for Israelites, 53 for Old Believers, 27 for Orthodox, 26 for Evangelical Lutherans; 7 for Evangeli- cal Reformed, 5 for Methodists, 4 for Baptists; 3 for Mahomethones, one for Adventists and one for Karaits21. The statistical bureau received about 2,500 to 3,000 death certificates per month, divided into counties, towns and villages by gender, major age groups, causes of death and religions22. 21 Lietuvos statistika 1920-1930 metais. Kaunas: Centralinis statistikos biuras, 1931, p. 28-29. 22 Lietuvos statistika 1920-1930 metais. Kaunas: Centralinis statistikos biuras, 1931, p. 29. 23 Knibbs, G. H. The International Classification of Disease and Causes of Death and its Revision. The Medical Journal of Australia. 1929 01 05, Vol. 1, p. 8-9. Collection of Homicide Statistics of Lithuania in 1920s-1930s Although official Lithuanian statistics were published with French terms, international classifications were not followed, for example, in 1920, an in- ternational list of causes of death, entitled Chapter XIV, External causes, was divided into extremely specific categories, ranging from suicide, accidental deaths like drowning, accidental falling, right through to homicide by fire- arms, homicide with cutting or stabbing instruments, homicide by other means as well as infanticide. Also, violent deaths of unknown causation23 were also mentioned. 111 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 In Lithuania, this procedure was not followed. As mentioned before, there have been problems detected with the terminology of homicides in the official statistics. The first official statistical data with homicides as violent deaths in 1924 were published in the monthly Bulletin of Statistics in 1925, in a table of the deceased in 1924 sorted by months and causes of death. As the source states, only the minority of the deaths were stated by medical doctors; the ma- jority were based on the testimonies of relatives. It was also explained that all the death certificates that did not clearly state the cause of death or were un- readable were considered to be other illnesses or unknown causes, which could have meant that some of the homicide cases were included in these numbers as well24. The same decision was made in the third chapter of the Statistical Yearbook of Lithuania, Natural Movement of the Population in Lithuania, were the deceased by cause of death were also recorded. The term violent death was used in Lithuania between 1924-1931 in the Bulletin of Statistics and the Statistical Yearbook of Lithuania to describe homi- cides as violent deaths, except suicides with the French equivalent Morts vio- lentes (suicide excepté), amounting to 723 violent deaths in 1931(Figure 1a). Suicides, other illnesses and the unknown or unexplored causes were men- tioned separately. In these sources, homicides were not distinguished into a separate group. This complicates the study of the homicide statistics in inter- war Lithuania and the comparison of the data with foreign countries. Since 1932 violence was distinguished into 4 main groups of suicide, homi- cide, accidents and other violence25. Seeing homicides singled out from acci- dents in 1932, it can be assumed that until then violent deaths had included both homicides and accidental deaths. 24 32 Nr. Mirimai mirties priežastimi. Didž. Lietuvos naturalinio gyventojų judėjimo 1924 m. daviniai. Statistikos biuletenis, No 5, p. 48. 25 It is interesting to note that the execution of death sentences was mentioned among the other forms of violence, but no statistics were provided, although previous research on the death penalty proves that executions took place in Lithuania at least a few per year (Černevičiūtė, S., Kaubrys S. 2014). Ironically, the statistical yearbook explains, “Deaths from this violent death are included in other tables under the category of accidents.“ So, state violence was also recorded and executions were called accidental deaths. 24 32 Nr. Mirimai mirties priežastimi. Didž. Lietuvos naturalinio gyventojų judėjimo 1924 m. daviniai. Statistikos biuletenis, No 5, p. 48. Collection of Homicide Statistics of Lithuania in 1920s-1930s It should be mentioned that the term accidental death could be understood as involuntary manslaughter in the in- terwar Lithuanian context. As a result, in the first period of 1924-1931, before 112 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics Figure 1a. Violent deaths (without suicides) in Lithuania between 1924-1931 Source: Statistical Bulletin No 5; Statistical Yearbook of Lithuania, Vol. 1-4. 151 169 527 473 513 493 522 37 39 179 166 204 184 201 358 188 208 706 639 717 677 723 0 200 400 600 800 1000 1924 1925 1926 1927 1928 1929 1930 1931 Male Female Total Linear (Total) Figure 1a. Violent deaths (without suicides) in Lithuania between 1924-1931 Source: Statistical Bulletin No 5; Statistical Yearbook of Lithuania, Vol. 1-4. Figure 1b. Homicides in Lithuania between 1932-1939 (with the Klaipėda Region, except 1939) Source: Statistical Yearbook of Lithuania Vol 5 12 87 88 103 72 90 95 104 102 35 20 26 21 30 37 35 28 122 108 129 93 120 132 139 130 0 50 100 150 1932 1933 1934 1935 1936 1937 1938 1939 Male Female Total Linear (Total) Figure 1b. Homicides in Lithuania between 1932-1939 (with the Klaipėda Region, except 1939) Figure 1b. Homicides in Lithuania between 1932-1939 (with the Klaipėda Region, except 1939) Source: Statistical Yearbook of Lithuania, Vol. 5-12. Source: Statistical Yearbook of Lithuania, Vol. 5-12. Source: Statistical Yearbook of Lithuania, Vol. 5-12. homicide was separated from other violent deaths, we cannot determine the exact rate of homicide victims (Figure 1a and 1b). h homicide was separated from other violent deaths, we cannot determine the exact rate of homicide victims (Figure 1a and 1b). The period of 1932-1939 clearly shows that men were killed 3-4 times more often than women. When comparing the murder victims by gender and age, the most frequent victims of homicide were in the 15-29 and 29-44 age groups for men, while the 15-29 age group stands out for females (Table 1). The Finn- ish criminologist Veli Verkko has researched homicides in 1920s-1950s Fin- land and has been one of the first to discuss the need to analyse homicides by gender. In the so called Verkko’s laws, it was stated: “in countries with high 113 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 Table 1. Collection of Homicide Statistics of Lithuania in 1920s-1930s Homicides by the cause of death in Lithuania between 1932-1939 (with the Klaipėda Region, except 1939) by age group and gender Age group 1932 1933 1934 1935 1936 1937 1938 1939 M* F** M F M F M F M F M F M F M F 0 2 2 3 0 3 5 0 1 1 2 1 0 2 0 0 0 1-4 1 2 0 0 3 0 0 1 0 1 2 0 0 0 1 1 5-14 5 1 1 0 0 1 2 3 2 2 4 2 7 2 2 0 15-29 29 12 36 13 28 13 30 7 34 11 39 10 39 15 35 16 30-44 26 5 22 2 32 1 30 4 34 2 27 12 34 9 40 5 45-59 10 8 13 3 14 3 6 1 13 4 12 7 10 3 13 3 60-69 10 2 7 2 13 1 3 0 4 2 1 2 3 3 2 0 70-79 3 1 5 0 7 2 1 2 1 5 7 4 4 1 7 1 80 and more 0 2 0 0 2 0 0 0 1 1 2 0 0 0 0 0 Unknown 1 0 1 0 1 0 0 2 0 0 0 0 1 1 2 2 Total: 87 35 88 20 103 26 72 21 90 30 95 37 100 34 102 28 Source: Statistical Yearbooks of Lithuania, Vol. 5-12. M* – Male, F** – Female. homicide rates, the percentage of female offenders and victims is small, and vice versa, in countries with low overall homicide rates, the percentage of fe- male offenders and victims is high.” (Kivivuori, 2017, p. 148). The high in- terwar Lithuanian rates of homicides are an echo of the Verkko’s laws with a small percentage of female homicide victims and perpetrators. The murders of men can be explained by comparing the statistics with the police data, which shows a high number of deaths in brawls (Figure 3). The female murder vic- tims’ age group could be explained by the fact that at the peak of their repro- ductive age many women became victims of domestic violence (Černevičiūtė & Kareniauskaitė, 2021). Since 1932 homicides were divided on a basis of the method used to com- mit it: shooting, beating, murder by other means and unknown methods. 26 The arrest was a dangerous situation also for others involved or around: 10 policemen and 3 civilians were killed, 12 policemen and 11 civilians got injured. Lietuvos policija 1918- 1928. Ed. I. Tamašauskas, Kaunas: V. R. M. Piliečių apsaugos departamentas, 1930, p. 179 Collection of Homicide Statistics of Lithuania in 1920s-1930s Shooting was the most popular method of murder in 1930s Lithuania, and only the second one was beating (Figure 2). It can thus be reasonably assumed that the fact that shooting dominated reflected one of the technical factors of 114 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics Figure 2. Homicide victims by the method used to commit the homicide in Lithu- ania in 1932-1939 (with the Klaipėda Region, except 1938-1939) Source: Statistical Yearbooks of Lithuania, Vol. 5-12. 253 235 110 139 80 37 39 75 333 272 149 214 0 200 400 Shooting Beating Other methods Unknown methods Male Female Total Figure 2. Homicide victims by the method used to commit the homicide in Lithu- ania in 1932-1939 (with the Klaipėda Region, except 1938-1939) Source: Statistical Yearbooks of Lithuania, Vol. 5-12. Source: Statistical Yearbooks of Lithuania, Vol. 5-12. the homicides in interwar Lithuania since the First World War and the Lithu- anian Wars of Independence, as guns may have been widely accessible in the Lithuanian society. It is worthwhile noting that homicide here was probably understood only as an intentional murder except for infanticide and abortion. the homicides in interwar Lithuania since the First World War and the Lithu- anian Wars of Independence, as guns may have been widely accessible in the Lithuanian society. It is worthwhile noting that homicide here was probably understood only as an intentional murder except for infanticide and abortion. As mentioned earlier, infanticide in interwar Lithuania was one of the most common murder types, so strangulation and suffocation would probably be added to the picture. Police Records of Deprivation of Life: Types and Content of Homicides in 1930s Lithuania First, the crime-related statistics, including homicides, were published from the year of 1920. Based on the police records, 12,605 criminals were ar- rested that year, primarily murderers, robbers, thieves, deserters, illegal vodka producers and anti-state criminals. 258 of the cases were recorded as armed attacks, 77 % of these were carried out with the purpose of robbery and the rest – with a revenge motive. 183 men and 42 women were killed in these crimes among the total of 225 murder victims. There were 182 murderers ar- rested, and 5 of them were killed during the arrest26. 30 It should be mentioned that in Czarist Russia only the sections on political and religious crimes came into effect, as the rest of the code was considered too liberal for Russia. In Lithuania, the German military administration introduced the 1903 Russian Criminal Code in full in 1917 (Maksimaitis, 2001, p. 55-56). 29 For example, the police-provided data in the journal Police show that the total number of deprivation of life cases was 389 in 1931 and 400 in 1932, but the Statistical Yearbook of Lithuania recorded 386 in 1931 and 399 in 1932, so the discrepencies are by only a few numbers. Kriminalistikos žinynas, 1939, No. 27, p. 132; Lietuvos statistikos metraštis 1931, Vol. 4, Kaunas: Spindulio b-vės sp., 1932, p. 341; Lietuvos statistikos metraštis 1932, Vol. 5, Kaunas: Spindulio b-vės sp., 1933, p. 268-269. 28 Visiems apskričių viršininkams ir kriminalinės policijos viršininkui. 14417 nr. Krimina- linės Žinios Policijai. 1926, No 1, p. 8. 27 Ibid., p. 180. 28 Visiems apskričių viršininkams ir kriminalinės policijos viršininkui. 14417 nr. Krimina- linės Žinios Policijai. 1926, No 1, p. 8. 29 For example, the police-provided data in the journal Police show that the total number of deprivation of life cases was 389 in 1931 and 400 in 1932, but the Statistical Yearbook of Lithuania recorded 386 in 1931 and 399 in 1932, so the discrepencies are by only a few numbers. Kriminalistikos žinynas, 1939, No. 27, p. 132; Lietuvos statistikos metraštis 1931, Vol. 4, Kaunas: Spindulio b-vės sp., 1932, p. 341; Lietuvos statistikos metraštis 1932, Vol. 5, Kaunas: Spindulio b-vės sp., 1933, p. 268-269. 30 It should be mentioned that in Czarist Russia only the sections on political and religious crimes came into effect, as the rest of the code was considered too liberal for Russia. In Lithuania, the German military administration introduced the 1903 Russian Criminal Code in full in 1917 (Maksimaitis, 2001, p. 55-56). 27 Ibid., p. 180. 28 Collection of Homicide Statistics of Lithuania in 1920s-1930s The source states that 115 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 the data for 1920 were not complete and were sorted out only at the end of the year, and uncritically presumes that it could be higher by 50 %27. To sum up, the mentioned criminal statistics are very problematic and need a careful evaluation: there are no other sources to compare and verify the data. It is also sporadic, therefore, what is left is only a partial view of the homicides’ phe- nomenon of that period. The interwar Lithuanian police was subordinated to the Department of Civil Protection (the Police Department since 1934) and worked under the Ministry of Internal Affairs. The statistics used to be sent to the Department of Civil Protection28 every month. Even though there were some published crime statistics of the year 1920, complete and continuous official data sets were pub- lished of the years 1927-1930 in the journal Police, but the Statistical Yearbooks of Lithuania began to publish the statistical data collected by the police only from 1929. It should be mentioned that there were some discrepancies29 be- tween the data published in the journal Police and in the Statistical Yearbooks of Lithuania, probably because of the rates stemming from the Klaipėda Region. Taking this into account, we have decided to analyse both sources separately. Police, as a law enforcement agent, used crime classifications according to the Penal Statute of Lithuania. It should be explained that in 1918 the Lithu- anian government decided to adopt the 1903 Criminal Code of the Russian Empire in as much as it did not contradict the temporary Constitution enact- ed on the 2nd of November, 1918 (Maksimaitis, 2001). The imperial Russian Criminal Code of 1903 came into force in Lithuania in 191930. Homicides were included in the XXII chapter of Deprivation of Life (Rus. O лишеніи жизни) 116 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics and distinguished crimes into homicide; infanticide; abortion; preparation to murder; attempted murder; persuasion and help to commit suicide (defined in the articles 453-466). 31 “Kas nusikalto ką nužudęs, tas baudžiamas kalėti sunkiųjų darbų kalėjime ne trumpiau kaip aštuoneris metus.” Baudžiamasis statutas su papildomaisiais baudžiamaisiais įstaty- mais ir komentarais, sudarytais iš Rusijos Senato ir Lietuvos Vyriausiojo Tribunolo spren- dimų bei kitų aiškinimų. Red. Martynas Kavolis, Simanas Bieliackinas. Kaunas: D. Gut- mano knygynas, 1934, p. 371. 33 Statistikos žinios apie įvairius kriminalinius atsitikimus. Ibid. 32 Visiems apskričių viršininkams ir kriminalinės policijos viršininkui. 14417 nr. Krimina- linės Žinios Policijai. 1926, No 1, p. 8. kaip aštuoneris metus.” Baudžiamasis statutas su papildomaisiais baudžiamaisiais įstaty- mais ir komentarais, sudarytais iš Rusijos Senato ir Lietuvos Vyriausiojo Tribunolo spren- dimų bei kitų aiškinimų. Red. Martynas Kavolis, Simanas Bieliackinas. Kaunas: D. Gut- mano knygynas, 1934, p. 371. 32 Visiems apskričių viršininkams ir kriminalinės policijos viršininkui. 14417 nr. Krimina- linės Žinios Policijai. 1926, No 1, p. 8. 33 Statistikos žinios apie įvairius kriminalinius atsitikimus Ibid 34 Rimka, A. Policijos statistikos žinios. Policija, 1927, No 9, p. 8. 35 Povilaitis, A. Nusikaltimai, išeikvojimai ir jų baimė. Kova su piktu radijo bangomis, Kau- nas: Kriminalistikos žinynas, 1938, p. 247. 36 „Kai kurios policijos įstaigos, paduodančios žinias apie žmogžudystes, pritaikydavo prie žmogžudysčių ir savižudybes; vagystes su išlaužimu - prie plėšimų ir t.t. T. y. buvo išai- kinta, kad daugelis policijos valdininkų silpnai moka kvalifi kuoti nusikaltimus.” Budre- vičius, K. Policijos statistikos žinios. Policija, 1928, No 1, p. 27. Collection of Homicide Statistics of Lithuania in 1920s-1930s Homicide was defined in the article 453: “Whoever committed a crime by killing someone shall be punished by imprisonment with hard labour for no less than eight years.”31 It should be mentioned that although a definition of voluntary manslaughter was neither used in interwar Lithuania nor separated from other homicides in the statistics, the crime was defined in the article 458, describing it as a murder committed in a state of serious tension. The legal term of deprivation of life and the types of homicides did not properly unify the data of the police records. In different sources and years, there were different terms used as well. Research shows that until the begin- ning of 1927 the police used to gather statistical data about the crimes from the police districts every month. However, it did not show a complete picture of the criminogenic situation in the country. The police was already struggling with mistakes in the data when same crimes were counted as different types of offences. For example, in older statistical data suicides were counted as homi- cides as well as accidental deaths32. However, starting with 1927 new rules for statistical data of crimes were applied, the term homicide was used and distin- guished into three types of homicides committed: for-profit; during brawls; for other purposes. Infanticide was separated from homicides33. It should be noted that in the police sources, the exact term of infanticide was used as a murder of a bastard infant, already exposing a moral concept of the murder of the chil- dren born out of wedlock. The statistics of the crimes between 1927-1930 were already criticised by the well-known economist at the time Albinas Rimka. He noticed that homicides defined as others are committed more often than for-profit and during brawls. He suggested that homicides should be distinguished into 5 groups: (1) deliber- 117 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 ate murder for profi t; (2) deliberate murder out of personal revenge or jealousy without a motive for profi t; (3) unintentional murders during brawls; (4) ran- dom homicides when a victim and a perpetrator are the victims of an accident (for example: getting shot when playing with a gun or a driver kills someone in a street); (5) other homicides (low numbers for sadistic murders, etc.). In his opinion, the sum of all homicides was useful, along with infanticide rates34. Collection of Homicide Statistics of Lithuania in 1920s-1930s From this standpoint, while leaving out others, which probably included a variety of murders, including those committed for political reasons, infanticide was the most common murder type in interwar Lithuania. Th e majority of them were committed by single mothers, poor and unable to support a child fi nancial- ly and afraid of the consequences of having a bastard child in a conservative, re- ligious society of interwar Lithuania. Th e second most common homicide used to happen in brawls, where the perpetrator and the victim were usually both males. In this type of murder, the social problem of alcohol was evident. Alcohol abuse was one of the main technical factors of homicide, potentially provoking and accelerating the numbers of violence in drunken confl icts at home, parties, weddings, etc. (Figure 3). A low number of murders for-profi t may be explained by the fact that this type of homicide was punishable by death (Černevičiūtė & Kaubrys, 2014). Th e police rates provided evidence of the rise of homicides in 1931 (Figures 3, 5), when the world economic crisis of 1929 started to impact the Lithuanian economy35. Nevertheless, we should keep in mind that the police rates of homicides also included records of attempted murder. Th e problems of the statistics mentioned by Rimka were later addressed by the chief of criminal police Kazys Budrevičius. He explained that even though the procedure of collecting the statistics was not perfect, it was better than it had been until 1927 when it had been realised that the registration of diff erent crimes, including homicides, was really problematic: “Some police agencies, re- porting murders, classifi ed suicides as homicides and burglaries as robberies etc. Th at is to say, many police offi cers have a poor ability to classify crimes.”36 As a 118 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Offi cial Statistics FIGURE 3. Police-registered homicides by type between 1927-1930 Source: Police, 1937, No 2. 15 16 24 13 45 42 37 37 83 76 66 59 187 156 115 100 0 50 100 150 200 1927 1928 1929 1930 For profit During brawls Others Infanticide FIGURE 3. Police-registered homicides by type between 1927-1930 Source: Police, 1937, No 2. result, a table for the statistical data of crimes was created as simple as possible. 37 1930 m. lapkričio 21 d. visiems apskričių ir kriminalinės policijos rajonų viršininkams. Policija, 1930, No 23, p. 459; 1930 m. lapkričio 21 d. Piliečių apsaugos departamento ap- linkraštis No 37. 200a. Piliečių Apsaugos Departamento Aplinkraščių Rinkinys. 1918 m. vasario 16–1931 m. gegužės 31 d. Kaunas: Piliečių Apsaugos Departamento leidinys, 1931, p. 216. 38 Statistinės žinios apie nusikalstamuosius darbus. Policijos departamento aplinkraščių są- vadas. 1937 m. birželio mėn. 1 d. Kaunas: “Policijos” laikraščio leidinys, 1937, p. 54. Kai kurie stambesni, policijos užregistruoti, nusikalstamieji darbai 1931-1938 metais Kriminalistikos žinynas, 1939, No. 27, p. 132-133. 38 Statistinės žinios apie nusikalstamuosius darbus. Policijos departamento aplinkraščių są- vadas. 1937 m. birželio mėn. 1 d. Kaunas: “Policijos” laikraščio leidinys, 1937, p. 54. 39 Kai kurie stambesni, policijos užregistruoti, nusikalstamieji darbai 1931-1938 metais. 37 1930 m. lapkričio 21 d. visiems apskričių ir kriminalinės policijos rajonų viršininkams. Policija, 1930, No 23, p. 459; 1930 m. lapkričio 21 d. Piliečių apsaugos departamento ap- linkraštis No 37. 200a. Piliečių Apsaugos Departamento Aplinkraščių Rinkinys. 1918 m. vasario 16–1931 m. gegužės 31 d. Kaunas: Piliečių Apsaugos Departamento leidinys, 1931, p. 216. 38 Statistinės žinios apie nusikalstamuosius darbus. Policijos departamento aplinkraščių są- vadas. 1937 m. birželio mėn. 1 d. Kaunas: “Policijos” laikraščio leidinys, 1937, p. 54. 39 Kai kurie stambesni, policijos užregistruoti, nusikalstamieji darbai 1931-1938 metais. Kriminalistikos žinynas, 1939, No. 27, p. 132-133. 40 Policijos užregistruotų nusikalstamų darbų statistika 1939 metų. Lentelių paaiškinimas. Kriminalistikos žinynas, 1940, No 33, p. 138. 41 Ibid. Collection of Homicide Statistics of Lithuania in 1920s-1930s However, Budrevičius expected that the collection of the crime statistics would be taken over by the statistics professionals in the Central Statistical Bureau. Public criticism may explain why new instructions for the statistical data collection were created just aft er a few years and came into force on January 1st, 1931. Th e statistics of the police already used the term of deprivation of life and distinguished it into 7 types: for-profi t; during brawls; defending one’s own life or the lives of others; involuntary; infanticide; abortion and for other purposes37. It was clarifi ed that these classifi cations had been made according to the causes of deprivation of life, and it did not include dead bodies found, suicides and accidental deaths38. Note that abortions at the time were consid- ered deprivation of life. It may be assumed that as a result of the new rules of the police statistics’ collection the most informative records of the police on homicides were cre- ated between 1931-1938, where the mentioned 7 types of deprivation of life (Figure 4)39 give us a general view of the homicides in Lithuania. If we ex- 119 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 FIGURE 4. Deprivation of life according to the causes recorded by the police in Lithu- ania between 1931-1938 Source: Police, 1939, No 27. 9 14 20 22 8 15 17 13 45 27 31 30 43 44 41 38 107 114 161 142 147 174 109 88 108 94 88 72 67 80 50 66 6 4 5 5 8 2 2 6 44 38 66 57 43 81 60 58 70 109 127 144 126 153 158 149 0 50 100 150 200 1931 1932 1933 1934 1935 1936 1937 1938 For profit During brawls For other purposes Infanticide Defending their own and other lives Involuntary Abortion FIGURE 4. Deprivation of life according to the causes recorded by the police in Lithu- ania between 1931-1938 Source: Police, 1939, No 27. 41 Ibid. Collection of Homicide Statistics of Lithuania in 1920s-1930s 9 14 20 22 8 15 17 13 45 27 31 30 43 44 41 38 107 114 161 142 147 174 109 88 108 94 88 72 67 80 50 66 6 4 5 5 8 2 2 6 44 38 66 57 43 81 60 58 70 109 127 144 126 153 158 149 0 50 100 150 200 1931 1932 1933 1934 1935 1936 1937 1938 For profit During brawls For other purposes Infanticide Defending their own and other lives Involuntary Abortion FIGURE 4. Deprivation of life according to the causes recorded by the police in Lithu- ania between 1931-1938 Source: Police, 1939, No 27. cluded abortion (which is usually not considered as a homicide, but we should take into account the concept of interwar Lithuania), it would vary from 200 to 300 murders per year. However, in the data provided by the source, there were some discrepancies in the sums of the diff erent kinds of homicides, making the source only partially reliable. Also, when comparing the police records of deprivation of life with the homicides by the causes of death (from medical records), it must be pointed out that there are very signifi cant diff erences between the actual victims and the police-reported homicides (Figure 5). 3-4 times bigger numbers of homi- cides reported by the police, apparently, show that the police records included not only homicide, infanticide and abortion but also preparation for murder and attempted murder as well as persuasion and help to commit suicide40. For example, in 1939, the police reported 136 homicides, 66 infanticide cases, 154 abortions, 87 cases of preparation to murder and attempted murder and 3 cases of persuasion and help to commit suicide41. It means that at least 20 % of the police-recorded cases of deprivation of life were attempted murders. Th e 120 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics Figure 5. Homicide victims versus deprivation of life in Lithuania between 1932-1938 Source: Statistical Yearbooks of Lithuania, Vol. 5-11; Police, 1939, No 27. 122 108 129 93 120 132 139 400 498 472 442 549 437 418 0 500 1000 1932 1933 1934 1935 1936 1937 1938 Homicides (by number of deaths) Deprivation of life (police records) Figure 5. Homicide victims versus deprivation of life in Lithuania between 1932-1938 Source: Statistical Yearbooks of Lithuania, Vol. 5-11; Police, 1939, No 27. Collection of Homicide Statistics of Lithuania in 1920s-1930s 122 108 129 93 120 132 139 400 498 472 442 549 437 418 0 500 1000 1932 1933 1934 1935 1936 1937 1938 Homicides (by number of deaths) Deprivation of life (police records) gure 5. Homicide victims versus deprivation of life in Lithuania between 1932-1938 Source: Statistical Yearbooks of Lithuania, Vol. 5-11; Police, 1939, No 27. Source: Statistical Yearbooks of Lithuania, Vol. 5-11; Police, 1939, No 27. Figure 6. Police-registered cases of deprivation of life by type between 1931-1934 (without the Klaipėda Region) Source: Statistical Yearbooks of Lithuania, Vol. 4-7. 9 14 20 22 42 27 31 30 108 95 85 73 70 108 128 143 157 155 218 205 0 200 400 1931 1932 1933 1934 For profit During brawls Infanticide Abortion Others Figure 6. Police-registered cases of deprivation of life by type between 1931-1934 (without the Klaipėda Region) Source: Statistical Yearbooks of Lithuania, Vol. 4-7. Figure 6. Police-registered cases of deprivation of life by type between 1931-1934 (without the Klaipėda Region) Source: Statistical Yearbooks of Lithuania, Vol. 4-7. aforementioned attempted murders and homicides related to suicides were not collected until 1937, making medical records the only reliable source for statistical homicide research. It is important to highlight the fact that medi- cal records do not reflect infanticide rates, simply meaning “pure” numbers of homicides in the 1930s Lithuania. Our data show that “pure” homicide rates were relatively stable at the time, varying from 122 in 1932, going down to 93 homicides in 1935 and rising to 139 in 1938 (Figure 5). The most popular source in the Lithuanian historiography for crime statis- tics is the Statistical Yearbooks of Lithuania. Yet, the usage of the terms to de- fine homicide, counting attempted murders and taking the territorial change (with the Klaipėda Region or without) into account, shows its disadvantages in comparison to the more accurate police data. In the police crime data of 1931-1934, the term deprivation of life was used and distinguished into 5 types: 121 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 Figure 7. Police-registered deprivations of life by type between 1935-1939 (with the Klaipėda Region between 1933-1935, without – between 1936-1939) Source: Statistical Yearbooks of Lithuania, Vol. 6-12. 42 „Tiesa, Lietuvoje kerštauti yra labai įprasta. Kai kada dėl menkiausių priežasčių, sakysime, dėl paliudijimo teisme jo nenaudai, dėl įžeidimo arba šiaip dėl kokių nesvarbių dalykų atsiteisiama gyvybe.” Valaitis, J. Žmogžudysčių priežastys Lietuvoje. Kriminalistikos žinynas. 1937, No 14, p. 84. Collection of Homicide Statistics of Lithuania in 1920s-1930s 401 364 344 413 377 268 226 244 253 255 260 256 215 220 645 617 599 673 633 483 446 0 500 1000 1933 1934 1935 1936 1937 1938 1939 Deprivation of life Abortion and infanticide Total Figure 7. Police-registered deprivations of life by type between 1935-1939 (with the Klaipėda Region between 1933-1935, without – between 1936-1939) Source: Statistical Yearbooks of Lithuania, Vol. 6-12. Source: Statistical Yearbooks of Lithuania, Vol. 6-12. for profit, during brawls, infanticide, abortion and others (Figure 6). Between 1935-1939 the homicide-related police statistical data were distinguished only into two groups: deprivation of life and abortion and infanticide, making it even more complicated to use the data for actual research of homicides at the time (Figure 7). Without critical evaluation, this data was used by Smaliukas and Urbelienė (1994). They have not explained their work methodology, only stating that they have analysed the Statistical Yearbooks of Lithuania, but the statistics that they have published only partially match the yearbooks. The scholars have used the term deprivation of life without any explanation, which is quite clear for the years 1935-1939 but is not in the period of 1931-1934, making the analysis of Smaliukas and Urbelienė untrustworthy. However, even the unreliable police records reflect some tendencies of homicides as a social phenomenon. For example, statistics for the 1935-1936 murder motives in Lithuania show that revenge was the most common mo- tive for murder. According to the source “It is true, it is very common to take revenge in Lithuania, sometimes for the smallest reasons, let’s say, for testifying against him in court, for an insult or when for some otherwise unimportant things one pays off his life.”42 It should be mentioned that the violence in the 122 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics Table 2. 44 Povilaitis, A. Nusikaltimai, išeikvojimai ir jų baimė. Kova su piktu radijo bangomis, Kau- nas: Kriminalistikos žinynas, 1938, p. 247-248. 43 Budrevičius K. Ar nusikaltimų skaičius Lietuvoje didėja ar mažėja. Policija, 1931, No 2, p. 36. Collection of Homicide Statistics of Lithuania in 1920s-1930s The police-registered homicides and attempted homicides by motives in Lithuania between 1935-1936 Motive 1935 1936 Revenge 38 24 Family conflict 16 24 Unrequited love 10 20 Quarrels 14 17 In brawl fights 16 9 In order to get an inheritance 12 7 During a robbery 5 8 Due to negligence 4 2 For-profit 5 2 Murders committed by the mentally ill 2 4 Out of hate - 5 To avoid paying a debt to a debtor - 1 To avoid paying alimony and out of shame 8 1 In an attempt to rape 1 2 Self-defence - 1 Obsession to kill 1 - Source: Reference Book of Criminalistics, 1937, No 14. Source: Reference Book of Criminalistics, 1937, No 14. society was explained as a legacy of the First World War, believing that it de- moralised the society, people lost the sense of responsibility for their actions and started to live in the present day without thinking about the future43. Fam- ily conflicts, unrequited love, quarrels and murders out of hate were typical homicides at that time, which signals domestic violence problems in Interwar Lithuania (Černevičiūtė & Kareniauskaitė, 2021). Most of the homicides were committed in the rural areas (except for infanticide) (Table 2), for example, in 1931, 1932, 1933, 1934 only 2, 3, 1 and 4 homicides were committed in the capital Kaunas, respectively. In other cities, there were one or two homicides registered per year44. These tendencies could be explained by the agricultural 123 ISSN 2351-6097 eISSN 2538-8754 KRIMINOLOGIJOS STUDIJOS 2021/9 society of Interwar Lithuania, where most of the population was living in the villages but not in the cities. The critical analysis of the official statistical data of medical and police re- cords provides only a partial view of the homicides’ phenomenon in inter- war Lithuania. Further analysis is welcome, the police-provided data of the 1930s could be recalculated taking only homicides committed for profit, dur- ing brawls and others in consideration and comparing them to the data of homicides by a cause of death. Additionally, the homicide rates per population with estimated errors would provide an opportunity for cross-national com- parisons. Also, a study of the court statistical data records on homicides would reveal the characteristics of perpetrators and provide observations about the interwar Lithuania’s penal policy. Conclusion The Central Statistical Bureau had been publishing statistical data relevant to the investigation of homicides in Statistical Bulletins and the Statistical Year- books of Lithuania since 1924. In the homicide studies, these statistical sources are problematic due to an unclear data collection methodology, the use of dif- ferent homicide terms, unrealistic definitions of urban and rural areas and the changes in Lithuania’s territory and population. We cannot determine the exact rates of the 1930s homicides due to the change in the homicide terminology and its content. Between 1924-1931 the term vio- lent deaths, except suicides, was used, and homicides were not singled out. The analysis also shows that until 1931 violent deaths also included homicides and accidental deaths. From 1932 to 1939 violent deaths were divided into 4 groups: suicide, homicide, accidents and other violence. The more detailed data of the 1930s have revealed that most frequent victims of homicide were in the 15-29 and 29-44 age groups for men, while the 15-29 age group stands out for fe- males. The most common method of killing was shooting. The police-published homicide statistics also reveal a problem of termi- nology. According to the Penal Statute of Lithuania, deprivation of life dis- tinguished crimes into homicide; infanticide; abortion; preparation to mur- der; attempted murder; persuasion and help to commit suicide. However, this terminology was only partially reflected in the police statistics, as between 124 Sigita Černevičiūtė. Homicide Trends and Types in 1920s-1930s Lithuania: Limitations of Official Statistics 1927-1930 the police used the term homicides and classified them into the ones committed: for-profit; during brawls and for other purposes. Infanticide was separated from homicides. If not counting others, infanticide was the most common murder type in interwar Lithuania. Since 1931, the statistics of the police used the term of deprivation of life and distinguished it into 7 types according to the motive: for-profit; during brawls; defending one’s own life or the lives of others; involuntary; infanticide; abortion and for other purposes. It did not include dead bodies found, suicides and accidental deaths. After analysing the 1931-1938 data on deprivation of life, excluding abor- tions, homicides would vary from 200 to 300 per year. When comparing all deprivations of life with homicides by the cause of death, it can be concluded that these figures included preparation for murder and attempted murder as well as persuasion and help to commit suicide. Aebi M. F., Linde, A. (2016) Long-Term Trends in Crime: Continuity and Change, P. Knepper, A. Johansen (Eds.), The Oxford handbook of the history of crime and criminal justice, Oxford: Oxford University Press, pp. 57-87; Conclusion Thus, due to the change in the terminology and the inclusion of attempted murders, the police statistics can be considered unreliable. Homicides are the most precisely quantitatively defined by the rates of causes of death, which in 1932-1938 Lithuania were relatively stable with an amplitude of 93-139 murders per year. On a basis of the analysis of the police statistical data, it is possible to identify at least 4 main types of homicides in interwar Lithuania: reproductive – infanticide and abortions, aggressive – kill- ings during brawls, economic – for profit as well as unintentional homicides that happened during accidents. Funding. This article is a part of the postdoctoral fellowship project Homicides and Punishments in Lithuania in 1918-1940. 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https://openalex.org/W2070261555	https://europepmc.org/articles/pmc4398387?pdf=render	English	null	The Role of Lipoprotein-Associated Phospholipase A₂ in a Murine Model of Experimental Autoimmune Uveoretinitis	PloS one	2,015	cc-by	9,973	RESEARCH ARTICLE G. L. Crawford1, J. Boldison2, D. A. Copland1, P. Adamson3, D. Gale4, M. Brandt5, L. B. Nicholson1,2, A. D. Dick1,2* 1 Academic unit of Ophthalmology, School of Clinical Sciences, University of Bristol, Bristol, United Kingdom, 2 School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom, 3 Ophthiris Discovery Performance Unit, GlaxoSmithKline, Stevenage, United Kingdom, 4 Ophthiris Discovery Performance Unit, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America 5 Platform Technology Sciences, King of Prussia, Pennsylvania, United States of America * a.dick@bristol.ac.uk OPEN ACCESS Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipopro- teins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macro- phage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp- PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-defi- cient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to deter- mine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1–20 or 161–180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tis- sue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp- PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in dis- ease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity. Citation: Crawford GL, Boldison J, Copland DA, Adamson P, Gale D, Brandt M, et al. (2015) The Role of Lipoprotein-Associated Phospholipase A₂ in a Murine Model of Experimental Autoimmune Uveoretinitis. PLoS ONE 10(4): e0122093. doi:10.1371/journal.pone.0122093 Academic Editor: Andrew W Taylor, Boston University School of Medicine, UNITED STATES Received: January 1, 2015 Accepted: February 21, 2015 Published: April 15, 2015 Copyright: © 2015 Crawford 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. Academic Editor: Andrew W Taylor, Boston University School of Medicine, UNITED STATES Received: January 1, 2015 Accepted: February 21, 2015 Published: April 15, 2015 Academic Editor: Andrew W Taylor, Boston University School of Medicine, UNITED STATES Received: January 1, 2015 Accepted: February 21, 2015 Published: April 15, 2015 Copyright: © 2015 Crawford et al. * a.dick@bristol.ac.uk The Role of Lipoprotein-Associated Phospholipase A₂ in a Murine Model of Experimental Autoimmune Uveoretinitis G. L. Crawford1, J. Boldison2, D. A. Copland1, P. Adamson3, D. Gale4, M. Brandt5, L. B. Nicholson1,2, A. D. Dick1,2* G. L. Crawford1, J. Boldison2, D. A. Copland1, P. Adamson3, D. Gale4, M. Brandt5, L. B. Nicholson1,2, A. D. Dick1,2* Introduction The specific roles of these authors are articulated in the 'author contributions' section. Non-anterior uveitis (posterior, pan and intermediate uveitis) is a collective term used to de- scribe a range of intraocular inflammatory disorders affecting the uvea and retina, and whilst more rare than anterior uveitis, is significantly more sight threatening [1]. Uveitis accounts for 10% of the 285 million visually impaired patients worldwide [2] with considerable financial and social implications, as it predominantly effects the working age population [3]. Uveitis is presumed autoimmune or immune mediated when infectious aetiology has been excluded [1, 4–6]. In mice, experimental autoimmune uveoretinitis (EAU) is an antigen-specific Th1/Th17 CD4+ T cell-directed murine model, employed to mimic human non-infectious, non-anterior uveitis, which has been utilised extensively to develop a detailed understanding of the immuno-pathogenesis of vascular inflammation, retinal leukocyte infiltration and mechanisms of tissue damage [6–8]. In this model, tissue destruction is dependent upon activation and infil- tration of mononuclear cell infiltration [6, 9–12]. Competing Interests: PA, DG and MB are employees of GlaxoSmithKline, whose company partly funded this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Tissue inflammation is held in check and homeostasis maintained, in part through micro- glia (resident retinal myeloid cell population) but following breakdown of blood ocular barrier [13] circulating macrophages infiltrate the retina early in the course of EAU [7, 14]. The migra- tion of CD4+ T cells to the retina causes activation and further accumulation of CD11b+ mac- rophages. Macrophages display broad heterogeneity and their phenotype and behaviour is regulated by a plethora of stimuli found in the local microenvironment. For example, macro- phages can inhibit T-cell proliferation in the eye but also produce tissue-damaging superoxides [7, 15, 16]. Notwithstanding, regulating macrophage activation will reduce inflammation [17, 18] and as such, an ability to manipulate macrophage phenotype and/or migration has the po- tential to abrogate EAU onset. One means of repressing macrophage activation is through prevention of hydrolysis of oxi- dised low density lipoproteins (oxLDL), catalysed by the enzyme Lipoprotein Associated Phos- pholipase A2 (Lp-PLA2). This reaction produces lysophosphatidylcholine (LPC) and oxidised non-esterified fatty acids (ox-NEFA) two potent pro-inflammatory mediators, which up-regu- late expression of chemokines and adhesion molecules, induce macrophage migration and pro- mote pro-inflammatory cytokine release [19, 20]. The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis OPEN ACCESS 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: © 2015 Crawford 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: http://www.bbsrc.ac.uk/home/home.aspx, Biotechnology and biological sciences research council: BB/J500197/1, received by GLC (nee Gemma Beers) and http://www.gsk.com/ GlaxoSmithKline: STU100020289, received by GLC (nee Gemma Beers). The funder GlaxoSmithKline provided support in the form of salaries for authors PA, DG & MB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 Competing Interests: PA, DG and MB are employees of GlaxoSmithKline, whose company partly funded this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 Introduction The phospholipase A2 (PLA2) family are a group of enzymes involved in catalysing the hydrolysis of fatty acyl moieties from the sn-2 po- sition of oxidized phospholipid molecules [19]. Lp-PLA2 is a 45kDa secreted enzyme, express- ed by monocytes, macrophages, T cells and mast cells. This enzyme contains a catalytic serine residue [21] and is active in basal physiological conditions. Unlike other PLA2 family members, Lp-PLA2 functions independently of calcium and is highly specific for substrates with a short chain fatty acyl residue at the sn-2 position, meaning it does not hydrolyse membrane phos- pholipids [22]. Lp-PLA2 suppression has been used to modulate disease in both atherosclerosis and diabetic macular oedema models [23], which in common with uveitis, present with macrophage medi- ated tissue damage and underlying vascular pathology. It was first noted that Lp-PLA2 expres- sion was high in atherosclerotic plaques, particularly those which were necrotic and prone to rupture and a meta-analysis of nearly 80,000 people demonstrated a continuous association be- tween the mass and activity of Lp-PLA2 and risk of coronary heart disease [24]. A diabetic and hypercholesterolemic swine model was used to test the efficacy of Lp-PLA2 depletion in athero- sclerosis. In short, treatment of atherosclerotic pigs with Darapladib, a selective, reversible in- hibitor of Lp-PLA2 [25] induced a significant reduction in lesion development and necrotic core area [26]. Similar results were also noted in the human Integrated Biomarker and Imaging Study 2 (IBIS-2) [27] whereby ultrasound analysis confirmed that Darapladib treated patients had smaller coronary artery lesions than those receiving a placebo control. Furthermore, PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 2 / 20 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Darapladib has been shown to attenuate expression of monocyte chemoattractant protein 1 (MCP-1), VCAM-1 and TNF-α gene expression, [28] and demonstrate reduced cerebral vascu- lar permeability in the diabetic/hypercholesterolemic swine model [23]. These data further im- plicate Lp-PLA2 inhibition as a promising therapeutic target for prevention or resolution of inflammation and vascular permeability. Darapladib has been shown to attenuate expression of monocyte chemoattractant protein 1 (MCP-1), VCAM-1 and TNF-α gene expression, [28] and demonstrate reduced cerebral vascu- lar permeability in the diabetic/hypercholesterolemic swine model [23]. These data further im- plicate Lp-PLA2 inhibition as a promising therapeutic target for prevention or resolution of inflammation and vascular permeability. Methods Mice C57BL/6 Lp-PLA2 knockout mice (-/-, KO), and heterozygous (-/+, HET) and WT littermate controls were obtained from GlaxoSmithKline (Philadelphia, USA and Stevenage, UK). The Lp-PLA2 KO animals were generated by GlaxoSmithKline using 129/BL6 ES cells. Single nucle- otide polymorphism (SNP) analysis was used throughout breeding to generate animals that were >98% C57BL/6. The colony was then established in the UK and SNP analysis was re-run to confirm the genetic background of the mice (>99% C57BL/6) [29]. The animals have no physical or immunological phenotype. B10 RIII mice are inbred colonies at University of Bris- tol Animal Services Unit. All mice were housed in specific pathogen free conditions with con- tinual access to food and water. Female mice aged 6–8 weeks were immunised for EAU, and housed according to Home Office regulations in the University of Bristol animal house facility. Treatment of animals was carried out in accordance with the European Directive 86/609/EEC, the GlaxoSmithKline Policy on the Care, Welfare and Treatment of Animals and conformed to the Association for Research in Vision and Ophthalmology animal policy (ARVO statement for the use of Animals in Ophthalmic and Vision Research). Mice were sacrificed by recognised schedule one methods and all animal work was approved by the University of Bristol ethical approval group. Introduction In light of success in other models of macrophage mediated vascular inflammatory patholo- gies, albeit not in autoimmune disease, and given that tissue damage in uveitis is dependent upon macrophage infiltration and activation, we utilised Lp-PLA2 knockout (KO) mice and a specific Lp-PLA2 pharmacological inhibitor SB-435495 (referred to herein as Lp-PLA2 deletion or inhibition respectively) to determine the role of Lp-PLA2 in autoimmune retinal inflamma- tion. The prediction being that enzyme depletion would suppress macrophage activation in the face of a T cell driven disease. However, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, in the murine EAU model, suppression of Lp-PLA2 activity was unable to attenuate disease or show delayed dis- ease onset. Human RBP-3161–180 (SGIPYIISYLHPGNTILHVD) and RBP-31–20 peptide (GPTHLFQPSLVLDMAKVLLD) were synthesised by Sigma-Aldrich (Poole, U.K) and Severn Biotech (Worcester, U.K) respectively. High performance liquid chromatography was used to confirm peptide purity of >95%, and aliquots of peptide preparations were stored at -80°C. SB-435495 and vehicle solution were provided by GlaxoSmithKline (Stevenage, UK). Complete media comprising Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat inactivated foetal calf serum (FCS), 100U/ml penicillin-streptomycin, 2mmol/L L-Glutamine and Sodium pyruvate (Invitrogen, Paisley, U.K). EAU clinical assessment Digital images of the retina were obtained by topical endoscopic fundus imaging (TEFI) using a protocol described previously by Paques et al [30] and subsequently validated for EAU [8]. In short, a Nikon D80 digital camera, with a 10-million pixel charge-coupled device image sensor and Nikkor AF 85 /F1.8 D objective and additional +4.00 dioptre magnifying lens, (Nikon, Tokyo, Japan), 5cm long, 3mm diameter teleotoscope (1218AA: Karl Storz, Tuttlington, Ger- many), and xenon lamp were used to capture colour images of the retinal fundus. Topical ap- plication of phenylephrine 2.5% and tropicamide 1% (Minims; Chauvin Pharmaceuticals, Romford,UK), was used to dilate the pupil, while corneal anaesthesia was achieved by topical application of oxybuprocaine 0.4% (Minims). Viscous eye gel (Novartis Pharmaceuticals, Cam- berley, UK) allowed digital images to be obtained by direct corneal contact with the endoscope. All images were cropped using Photoshop CS software (Adobe, Mountain view, California, USA). Using an adapted clinical grading system, fundal images were scored according to in- flammatory changes to the optic disc and retinal vessels in addition to retinal lesions and struc- tural damage [31]. Reagents Human RBP-3161–180 (SGIPYIISYLHPGNTILHVD) and RBP-31–20 peptide (GPTHLFQPSLVLDMAKVLLD) were synthesised by Sigma-Aldrich (Poole, U.K) and Severn Biotech (Worcester, U.K) respectively. High performance liquid chromatography was used to confirm peptide purity of >95%, and aliquots of peptide preparations were stored at -80°C. SB-435495 and vehicle solution were provided by GlaxoSmithKline (Stevenage, UK). Complete media comprising Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat inactivated foetal calf serum (FCS), 100U/ml penicillin-streptomycin, 2mmol/L L-Glutamine and Sodium pyruvate (Invitrogen, Paisley, U.K). 3 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Sample collection and cell preparation Blood samples were obtained via cardiac puncture, collected in neonatal KE/1.3 1.3ml EDTA tubes and centrifuged for 8 minutes at 1800rpm and 4°C. The plasma fraction was removed and stored at -80°C for plasma Lp-PLA2 enzyme activity and plasma SB-435495 analysis (Glax- oSmithKline). The enucleated right eye was placed in sterile DMEM for retinal dissection and flow cytometric analysis, the left eye was orientated in optimal cutting temperature (OCT) compound and snap frozen in liquid nitrogen for histology. Spleens were also removed for flow cytometry and histology. Spleen and thymus were mashed and filtered through a 40μm cell strainer to remove cellular debris. Splenocytes were re-suspended in 1ml ACK salt solution (Ammonium-chloride-potas- sium lysing buffer, 8,024mg/L NH4CL, 1,001mg/L KHCO3, 3.722mg/L EDTA.Na2.2H20) to eliminate erythrocytes, then all samples were washed, re-suspended in 10 ml sterile DMEM and filtered again to remove aggregated cells. Eyes were enucleated, washed and all extraneous vascular and connective tissue was re- moved. The retina was then dissected microscopically in HBSS and retinas were physically dis- aggregated through a 40μM cell strainer and re-suspended in 200μl FACS buffer. EAU induction and treatment C57BL/6 and B10RIII mice were immunised subcutaneously (s.c) in one flank with 50μg RBP- 3 1–20 or 50μg RBP-3 161–180 respectively. Peptides were prepared in water (2% DMSO), emulsi- fied in CFA (1mg/ml 1:1 v/v) and supplemented with 1.5mg.ml Mycobacterium tuberculosis complete H37 Ra (BD Biosciences, Oxford, U.K.). A simultaneous I.P injection of 1.5μg Bor- della pertussis toxin (Tocris, Bristol, UK) was also administered. Doses of SB-435495, phos- phate buffered saline (PBS) or vehicle solution were administered by I.P injection once or twice daily (9am or 9am/pm respectively) as described (S4 Fig). Flow cytometric analysis Cells were incubated with 24G2 cell supernatant for 20 minutes at 4°C before incubation with fluorochrome-conjugated monoclonal antibodies (BD Pharmingen, Oxford, UK) against cell surface markers including, CD4 (553051, 1/100), CD8 (553029, 1/100), CD11b (557657, 1/ 400), Ly6G (551461, 1/100), Ly6C (553104, 1/100) and CD45 (552848, 1/1000) at 4°C for 20 4 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis minutes. A streptavidin secondary antibody was added to all samples, (48-4317-82, eBioscience, San Diego, USA, 1/1000) then cell suspensions were acquired using a 3-laser BD LSR-II flow cytometer (BD Cytometry Systems, Oxford, UK). Analysis was performed using FlowJo software (Treestar, San Carlos, California, USA). Cell numbers were calculated by refer- ence to a known cell–standard, as previously reported [14]. In brief, splenocytes at a series of known cell concentrations were acquired using a fixed and stable flow rate for 45 seconds. Based on total cell number acquired during this time, a standard curve was generated and used to interpolate cell concentrations of ocular infiltrating cells acquired at the same flow rate and time. Immunofluorescent staining Staining for oxLDL was performed using a polyclonal rabbit anti-mouse antibody (ABIN1385598, Antibodies-online, Georgia USA, 1/100). 12-μm sections (as described above) were fixed using 4% PFA, then permeabilised and blocked using PBS supplemented with 0.25% Triton X-100, 1% BSA and 5% normal goat serum (Vector Laboratories, Burlingame, Califor- nia, USA). Slides were washed in staining buffer (0.1% Triton X-100, 1% BSA in PBS) and incu- bated overnight at 4°C with 50μl staining buffer containing the oxLDL antibody plus a rat anti- mouse CD31 antibody directly conjugated with FITC (553372, BD Pharmingen, Oxford, UK, 1/100). Negative controls were treated with the latter only. The following day, slides were washed and incubated for one hour with 50μl staining buffer containing goat anti-rabbit Alexa Fluor 568 (A-11011, Invitrogen, Paisley, UK, 1/200). Sections then received a final wash and were mounted using VECTASHIELD HardSet mounting media with DAPI (Vector Laboratories, Burlingame, California, USA). Immunofluorescence staining was examined using a confocal scanning laser imaging system fitted with krypton-argon lasers (TCS-SP2-AOBS; Leica microsystems, Wetzlar, Germany). Immunohistochemical analysis 12μm sections were cut from eyes snap frozen in OCT medium at -20°C, and mounted onto poly-L-lysine (Sigma, Poole, UK) coated glass slides. Sections were fixed in acetone for 10 min- utes and endogenous peroxidases were blocked with 0.3% hydrogen peroxide and methanol for 30 minutes then washed in PBS. Blocking was achieved using normal rabbit serum (Vector Laboratories, Burlingame, California, USA) diluted in 2% bovine serum albumin (BSA) diluted in PBS (PBSA) for 30 minutes, followed by the addition of a rat anti-mouse primary antibody (MCA1388, AbDSerotec, Oxford, UK, 1/250), in 2% PBSA and left to incubate overnight at 4°C. A biotinylated rabbit anti-rat IgG secondary antibody (BA-4001, Vector, UK, 1/200), di- luted in 0.1% PBSA was added to all sections for 30 minutes at room temperature then slides were coated with Avidin/Biotinylated enzyme complex (ABC kit, Vector Laboratories, Burlin- game, California, USA) for 30 minutes. Sections were washed, coated with DAB reagent (DAKO, Denmark) for 2 minutes, and then immersed in haematoxylin stain. Slides were im- mediately washed and dehydrated by passage through 75% and 100% ethanol and Histoclear (National Diagnostics, Georgia, USA). Histomount (National Diagnostics, Georgia, USA) mounting media was used to affix coverslips and sections were examined under a light micro- scope. Clinical scoring was performed blind by an independent assessor based on cellular infil- trate and structural damage to the retina. Nitrite Assay 50μl Griess reagent (Sigma-Aldrich, St Louis, USA) was added to BMDM supernatants (tripli- cate) and standards (9 point curve with 10,000nm top standard). Plates were read after five minutes, using a Spectramax-190 plate reader (Molecular Devices, California, USA) and Soft- max-pro software, with absorbance wavelengths of 630nm and 540nm and a linear standard curve. Lp-PLA2 assay Quantification of Lp-PLA2 enzyme activity in murine plasma was performed at GlaxoSmithK- line via isolation of enzyme catalyzed release of [3H-] acetate from the substrate 1-O-Hexade- cyl-2-O-[3H-] acetyl-sn-glycer-3-phophorylcholine ([3H-]PAF obtained from Perkin Elmer, Waltham, MA. Product number: NET910250UC). Twenty microliters (20μL) of plasma was used for a single activity measurement using a 1 minute reaction time at 37°C. The Lp-PLA2 enzyme reaction was quenched through chloroform:methanol (2:1 ratio) extraction of each in- dividual reaction. Isolated activity (disintegrations per unit time (DPMs)) were extracted into the aqueous layer and converted to nanomoles of product through normalization to a [3H-] PAF standard. Radioactivity (DPMs) was counted using a liquid scintillation counter (Beck- man, Brea, CA). Inhibition of activity was gauged according to the activity level in the presence of inhibitor (“inhibited”), relative to blank or control plasma. SB-435495 plasma analysis All plasma analysis was performed at GlaxoSmithKline. Briefly, SB-435495 was extracted from plasma (25 μL aliquot) using a protein precipitation method. Following vortexing and 5 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis centrifugation, an aliquot of the supernatant (100μL) was removed and diluted with 50μL of acetonitrile/10mM Ammonium Formate, pH3 (50/50, v/v) and mixed. Samples were quantitat- ed for SB-435495 using a liquid chromatography mass spectrometry (UPLC-MS/MS) method. The LC-MS/MS system consisted of a Waters Acquity UPLC (Milford, MA) and an AB Sciex API 4000 triple quadrupole mass spectrometer (Framingham, MA) operating under Analyst 1.4.2 software (AB Sciex, Framingham, MA). The lower limit of quantification of SB-435495 was 1ng/ml and the upper limit of quantification was 1000ng/ml in the assay. Culture of bone marrow derived macrophages (BMDM) Mice were sacrificed by cervical dislocation; the hind legs excised using aseptic technique, were placed in sterile DMEM and kept on ice. Following removal of the bone heads with a scalpel, bone marrow was flushed with un-supplemented DMEM. The marrow was then filtered through a 70μm cell strainer to remove debris. Cells were cultured in Teflon culture bags (pro- vided by Dr. M. Munder, University of Heidelberg, Heidelberg, Germany) for eight days at 5–- 10x106 cells per bag in 50ml of complete culture media supplemented with 50ng/ml macrophage colony stimulating factor and 5% Horse serum (Invitrogen). Stimulation of bone marrow derived macrophages Macrophages were seeded at a concentration of 1 x106 cells/500μl per well in a 24 well plate. Cells were treated with either media alone, IL-4 (20ng/ml Peprotech, New Jersey, USA), prosta- glandin-E2 (PGE2) (0.5 μg/ml Sigma-Aldrich, St Louis, USA), LPS (1–100ng/ml Sigma-Aldrich, St Louis, USA) or IFN-γ (0.5–50ng/ml Peprotech, New Jersey, USA) for 2, 4 or 24 hours as specified. Supernatants were removed and stored at -20°C for further assays. Results To determine basal enzyme activity status of the Lp-PLA2 KO, HET and WT mice, cardiac blood samples were processed for plasma analysis, which confirmed that Lp-PLA2 activity was highest in WT animals, reduced in HETs and absent in Lp-PLA2 KO mice (Fig 1A). Bone mar- row derived macrophages (BMDM), stimulated with LPS (1ng/ml) or media alone for 2 or 4 hours, were used for RT-qPCR quantification of Pla2g7 gene expression. The results confirmed Pla2g7 gene expression was absent in Lp-PLA2 KO mice, with the highest expression observed in Lp-PLA2 WT mice (Fig 1B). To further determine expression and functionality of the secret- ed enzyme, an in vitro extracellular Lp-PLA2 activity assay was performed (Cayman chemicals, Michigan, USA). Supernatants from media alone and LPS (1ng/ml) stimulated BMDM secret- ed detectable quantities of functional Lp-PLA2, as indicated by the enzyme induced hydrolysis of a 2-Thio-PAF substrate and subsequent colorimetric change to the supernatant. Further- more, when BMDM were pre-treated with the SB-435495 inhibitor at a range of doses before stimulation, the enzymatic reaction was absent or reduced (S1 Fig) indicating efficacy of the SB-435495 in vitro. Prior to assessing the in vivo therapeutic efficacy of either Lp-PLA2 deletion or inhibition, we wished to ascertain whether Lp-PLA2 enzyme inhibition altered classical macrophage acti- vation. ELISA estimation of IL-6 and MCP-1 was determined following LPS-stimulation of BMDM treated with a dose range of SB-435495. A trend towards a reduction in IL-6 protein expression was identified in macrophages treated with 10μM SB-435495, when compared to the untreated controls; however the inhibitor failed to significantly alter IL-6 and MCP-1 pro- tein expression, (S2 Fig). To mimic physiological conditions the assay was further developed by spiking with endogenous oxLDL, as Lp-PLA2 is transported in vivo on oxPL molecules with- in oxLDL. However the simultaneous addition of SB-435495 and exogenous oxLDL did not significantly alter IL-6 protein expression when compared to controls (Fig 2A). Given our ob- served sustained IL-6 response, we wished to confirm that IL-6 production was independent of Lp-PLA2 and showed that LPS-stimulated BMDM from Lp-PLA2 WT, HET and KO mice were equivalent and maintained an IL-6 dose response (Fig 2B). qPCR analysis was utilised to examine expression of a number of characteristic phenotypic markers of macrophage activation status. Statistical analysis Data are displayed using mean ± SEM. Analysis of continuous data with two variables was cal- culated using a T test. Those with 3 or more variables were calculated using a one or two way ANOVA as appropriate with a secondary Tukey test to determine the differences between treatment groups or Lp-PLA2 genotype. Differences between groups were defined as significant at P<0.05. Extracellular Lp-PLA2 assay Supernatants from BMDM samples, stimulated for 24 hours as described previously, were mixed with a 2-Thiol PAF substrate for one minute. In the presence of Lp-PLA2 the substrate 6 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis was hydrolysed, producing a colorimetric change. Enzyme activity was calculated from the change in absorbance values read over a 15 minute period, using a Spectramax-190 plate reader and Softmax-pro software. PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 Results Data showed no significant difference in IL-1β and IL-10 gene expression, as a signature of M1 and M2 macrophage activation respectively, in LPS stimulated BMDM from Lp-PLA2 WT, HET and KO mice after both 2 and 4 hours in culture (Fig 3A and 3B). Quantification of the expression of Arginase-1 (Arg-1) and Nitric oxide synthase (iNOS or NOS2) was used as a further indicator of macrophage phenotype [32, 33]. Our data showed no significant difference in Arg-1 expression from Lp-PLA2 WT, HET or KO derived BMDM (Fig 3C). Further, a nitrite assay was used to determine whether Lp-PLA2 dele- tion altered NOS2-dependent nitrite production as an indicator of M1 phenotype. Data 7 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis showed a dose-dependent, LPS mediated nitrite induction, which was independent of Lp-PLA2 expression (Fig 3D). Nitrite production was also unchanged in LPS and IFNγ stimulated Fig 1. Quantification of Lp-PLA2 enzyme activity and gene expression in murine plasmas and BMDM. A) Enzyme analysis of Lp-PLA2 activity from murine cardiac blood samples (n = 15–17) **** p<0.001. B) RT-qPCR quantification of BMDM Pla2g7 expression (n = 3). Lp-PLA2 WT, HET and KO BMMO were stimulated with LPS or media only for 2 or 4 hours, and gene expression analysed. Pla2g7 mRNA expression normalized by GAPDH. * p<0.05 p<0.01 p<0.005 doi:10.1371/journal.pone.0122093.g001 Fig 1. Quantification of Lp-PLA2 enzyme activity and gene expression in murine plasmas and BMDM. A) Enzyme analysis of Lp-PLA2 activity from murine cardiac blood samples (n = 15–17) *** p<0.001. B) RT-qPCR quantification of BMDM Pla2g7 expression (n = 3). Lp-PLA2 WT, HET and KO BMMO were stimulated with LPS or media only for 2 or 4 hours, and gene expression analysed. Pla2g7 mRNA expression normalized by GAPDH. * p<0.05 p<0.01 p<0.005 Fig 1. Quantification of Lp-PLA2 enzyme activity and gene expression in murine plasmas and BMDM. A) Enzyme analysis of Lp-PLA2 activity from murine cardiac blood samples (n = 15–17) *** p<0.001. B) RT-qPCR quantification of BMDM Pla2g7 expression (n = 3). Lp-PLA2 WT, HET and KO BMMO were stimulated with LPS or media only for 2 or 4 hours, and gene expression analysed. Pla2g7 mRNA expression normalized by GAPDH. * p<0.05 p<0.01 p<0.005 showed a dose-dependent, LPS mediated nitrite induction, which was independent of Lp-PLA2 expression (Fig 3D). PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Fig 3. Effect of Lp-PLA2 enzyme ablation on macrophage activation markers as an indicator of macrophage activation status. Expression of IL-1β, IL-10 and Arginase-1 in Lp-PLA2 WT, HET and KO mice by RT-qPCR. Data from BMDM, following 2 or 4 hour stimulation with LPS (1ng/ml) or media control A) IL-1β (n = 3–4) B) IL-10(n = 3) C) Arginase-1 (n = 3–4). D) A nitrite assay performed as described, using supernatants of BMDM from Lp-PLA2 WT, HET and KO mice cultured in presence of LPS/IFNγ for 24 hours, showed no significant difference between genotypes (n = 9 from 5 mice). E) Data collected from nitrite assays using supernatants of BMDM treated with either LPS or IFNγ plus SB-435495 (1μM or 0.1μM) showed no significant difference between genotypes (n = 3) *p<0.001 Fig 3. Effect of Lp-PLA2 enzyme ablation on macrophage activation markers as an indicator of macrophage activation status. Expression of IL-1β, IL-10 and Arginase-1 in Lp-PLA2 WT, HET and KO mice by RT-qPCR. Data from BMDM, following 2 or 4 hour stimulation with LPS (1ng/ml) or media control A) IL-1β (n = 3–4) B) IL-10(n = 3) C) Arginase-1 (n = 3–4). D) A nitrite assay performed as described, using supernatants of BMDM from Lp-PLA2 WT, HET and KO mice cultured in presence of LPS/IFNγ for 24 hours, showed no significant difference between genotypes (n = 9 from 5 mice). E) Data collected from nitrite assays using supernatants of BMDM treated with either LPS or IFNγ plus SB-435495 (1μM or 0.1μM) showed no significant difference between genotypes (n = 3) *p<0.001 doi:10.1371/journal.pone.0122093.g003 doi:10.1371/journal.pone.0122093.g003 expression was identified in LPS treated macrophages derived from Lp-PLA2 HET and KO mice, when compared to WT control cells (Fig 4). Flow cytometric analysis of B and T cell marker expression in the thymus and spleen was also undertaken (CD45, CD3ε, B220, CD4, CD8, CD44, CD62L, CD69, CD25) which showed no significant difference between genotypes in naïve animals (S3 Fig). Having ascertained the efficacy and functionality of SB-435495 in vitro we then wished to determine its effects in vivo using the well-established B10 RIII murine model of EAU. Follow- ing induction of EAU, mice were randomly allocated into treatment groups and received IP in- jections of SB-435495 or vehicle control at the doses and time points depicted in S4 Fig. Results Nitrite production was also unchanged in LPS and IFNγ stimulated BMDM from WT mice, cultured with doses of SB-435495 known to give optimal Lp-PLA2 in- hibition (Fig 3E). 8 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Fig 2. Effect of Lp-PLA2 depletion on macrophage activation and phenotype on BMDM IL-6 protein expression. Supernatants from BMDM cultured for 24 hours with SB-435495 treated oxLDL plus 1ng/ml LPS or media control were used to quantify IL-6 protein expression (n = 3). A) SB-435495 was not able to significantly alter IL-6 expression in LPS treated BMDM. IL-6 was undetected in media only controls. BMDM derived from Lp-PLA2 WT, HET and KO mice were cultured with a titration of LPS. Supernatants were collected at 24 hours and used to quantify IL-6 protein expression by ELISA. B) There was no significant difference in IL-6 expression between Lp-PLA2 WT, HET or KO BMDM. doi:10.1371/journal.pone.0122093.g002 Fig 2. Effect of Lp-PLA2 depletion on macrophage activation and phenotype on BMDM IL-6 protein expression. Supernatants from BMDM cultured for 24 hours with SB-435495 treated oxLDL plus 1ng/ml LPS or media control were used to quantify IL-6 protein expression (n = 3). A) SB-435495 was not able to significantly alter IL-6 expression in LPS treated BMDM. IL-6 was undetected in media only controls. BMDM derived from Lp-PLA2 WT, HET and KO mice were cultured with a titration of LPS. Supernatants were collected at 24 hours and used to quantify IL-6 protein expression by ELISA. B) There was no significant difference in IL-6 expression between Lp-PLA2 WT, HET or KO BMDM. doi:10.1371/journal.pone.0122093.g002 doi:10.1371/journal.pone.0122093.g002 To further characterise the effect of Lp-PLA2 deletion on macrophage phenotype, flow cy- tometry was used to quantify the expression of macrophage activation markers (CD68+ MHCII+ and CD40+ expression) in BMDM cultured from Lp-PLA2 KO, HET and WT mice and stimulated with a panel of cytokines for 24 hours. The expression of MHCII and CD68 was unchanged when Lp-PLA2 was deleted, however a significant reduction in CD40 9 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 Treatment continued until either day 14 (peak disease) or day 21 (disease resolution) when ani- mals were sacrificed for analysis of retinal cellular infiltrate by flow cytometry and histology. An average Lp-PLA2 inhibition of 50.2% and 54.3% (S5A Fig) was measured in plasma samples PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 10 / 20 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Fig 4. Flow cytometric analysis of BMDM phenotype from cells cultured for 24 hours in the presence of a panel of cytokines. Geometric mean fluorescence intensity values for A) intracellular CD68 expression (n = 4), B) cell surface MHCII expression (n = 3) and C) cell surface CD40 expression (n = 5) p<0.05 p<0.01 p<0.005 doi:10 1371/journal pone 0122093 g004 Fig 4. Flow cytometric analysis of BMDM phenotype from cells cultured for 24 hours in the presen of a panel of cytokines. Geometric mean fluorescence intensity values for A) intracellular CD68 expres (n = 4), B) cell surface MHCII expression (n = 3) and C) cell surface CD40 expression (n = 5) p<0.05 p<0.01 p<0.005 doi:10 1371/journal pone 0122093 g004 Fig 4. Flow cytometric analysis of BMDM phenotype from cells cultured for 24 hours in the pres of a panel of cytokines. Geometric mean fluorescence intensity values for A) intracellular CD68 expre ( 4) B) ll f MHCII i ( 3) d C) ll f CD40 i ( 5) 0 05 Fig 4. Flow cytometric analysis of BMDM phenotype from cells cultured for 24 hours in the presence of a panel of cytokines. Geometric mean fluorescence intensity values for A) intracellular CD68 expression (n = 4), B) cell surface MHCII expression (n = 3) and C) cell surface CD40 expression (n = 5) * p<0.05 p<0.01 *p<0.005 doi:10.1371/journal.pone.0122093.g004 11 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis taken at trough dosing (24h post previous dose) from B10 RIII mice treated once daily with ei- ther 20mg/kg or 35mg/kg SB-435495 respectively. Flow cytometry and histology data revealed that treatment of mice with this dose of SB-435495 did not reduce the number of infiltrating cells in the retinas of animals at day 14 or 21 post immunisation when compared to control an- imals (S5B Fig). To achieve a more robust inhibition of Lp-PLA2 activity, mice were treated twice daily from day 5 post immunisation, with SB-435495 in the hope of maintaining more consistent enzyme inhibition. Mice were given IP injections of 30mg/kg of SB-435495, every 12 hours (S4 Fig). Analysis of plasma samples taken from mice at day 14 post immunisation showed 89.9% inhi- bition of Lp-PLA2 at trough dosing (12 hours post previous dose) (S6A Fig). In spite of this re- duction in enzyme activity, there were equivalent numbers of CD45+ infiltrating leukocytes in the retina of these mice by flow cytometry (Fig 5A) and histology (Fig 5B). Furthermore, clini- cal scoring of TEFI images showed no change to longitudinal disease progression and no signif- icant difference in EAU between treatment groups (Fig 5C). As the data suggested partial Lp-PLA2 inhibition was insufficient to abrogate retinal leuko- cyte infiltration, we then wished to assess the effect of permanent and complete deletion of Lp- PLA2 in EAU. As such, C57BL/6 Lp-PLA2 KO mice were utilised for subsequent experiments, with HET and WT littermates as controls. Mice were sacrificed at day 26 (peak disease) for analysis by flow cytometry and histology. However, in spite of the systemic deletion of Lp- PLA2, a comparable number of cells (CD45+, CD11b+, CD4+, CD8+) were found to infiltrate the retina during EAU in Lp-PLA2 KO mice and HET/WT littermate controls (Fig 6). Oxidation of LDL is an important step in the onset and progression of atherosclerosis. Oxi- dation of the lipoprotein molecule prevents binding to the LDL receptors and infers an in- creased affinity for macrophage scavenger receptors such as CD36 and LOX-1[34]. This is turn leads to reduced macrophage migration and the formation of lipid-laden foam cells, which are key features of an atherosclerotic plaque. However, less is known about the role of oxLDL in EAU and as such immunohistochemical staining of the Lp-PLA2 substrate was used to charac- terise its availability and distribution in both a healthy and uveitic eye. Data revealed that oxLDL was not detected in the naïve retina (Fig 7A), but in the uveitic retina appeared to co-lo- calise to the vasculature and outer plexiform layer (Fig 7B). PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 Discussion We have successfully shown that SB-435495 is an effective, reversible inhibitor of Lp-PLA2 both in BMDM in vitro and in an in vivo murine model of EAU. A small amount of platelet ac- tivating factor acetyl hydrolase (PAF-AH) activity was detected in the serum of Lp-PLA2 KO mice (Fig 1A), however this is not thought to be Lp-PLA2 as it is not inhibited with SB-435495 at concentrations >20uM which are 200% effective on the isolated enzyme [29]. Significant Lp-PLA2 inhibition with SB-435495 was not able to significantly alter the cytokine profile of BMDM in vitro, but cells treated with the inhibitor did show a trend towards a dose dependent reduction in the expression of IL-6. Furthermore, in vivo, Lp-PLA2 deletion or inhibition had no effect on EAU induction or onset. Lp-PLA2 is so called because in humans 80% of the circulating enzyme is carried on apoli- poprotein-B100 moieties on low density lipoprotein (LDL) molecules [35]. The remaining 20% is carried on high density lipoprotein (HDL) and very low density lipoprotein (VLDL) mole- cules, while in mice the ratios are reversed, due to minimal expression of LDL in this species. Increased Lp-PLA2 mass and activity has been identified as a biomarker for coronary heart dis- ease, and is found in patients with high cardiovascular risk factors [24]. It is also known to be associated with major adverse events in patients with coronary artery disease in a CRP 12 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Fig 5. Quantification of retinal cellular infiltrate and onset of clinical disease in a murine model of EAU; effective of SB-435454. A) Flow cytometry data showing CD45+, CD11b+, CD4+ and CD8+ cellular infiltrate in retinal digests from animals at day 14 post immunisation, treated twice daily with either SB-435495 or controls (n = 9–10 eyes). B) Histological staining scores from 12μm ocular sections taken from eyes enucleated at day 14 post immunisation, stained for CD45+ (DAB/haematoxylin) (n = 9–10 eyes). C) TEFI clinical score, day 13 post immunisation, (average scores provided by two independent assessors from two identical repeat experiments. n = 18–20). d i 10 1371/j l 0122093 005 p p p p Fig 5. Quantification of retinal cellular infiltrate and onset of clinical disease in a murine model of EAU; effective of SB-435454. A) Flow cytometry data showing CD45+, CD11b+, CD4+ and CD8+ cellular infiltrate in retinal digests from animals at day 14 post immunisation, treated twice daily with either SB-435495 or controls (n = 9–10 eyes). B) Histological staining scores from 12μm ocular sections taken from eyes enucleated at day 14 post immunisation, stained for CD45+ (DAB/haematoxylin) (n = 9–10 eyes). C) TEFI clinical score, day 13 post immunisation, (average scores provided by two independent assessors from two identical repeat experiments. n = 18–20). doi:10.1371/journal.pone.0122093.g005 Fig 5. Quantification of retinal cellular infiltrate and onset of clinical disease in a murine model of EAU; effective of SB-435454. A) Flow cytometry data showing CD45+, CD11b+, CD4+ and CD8+ cellular infiltrate in retinal digests from animals at day 14 post immunisation, treated twice daily with either SB-435495 or controls (n = 9–10 eyes). B) Histological staining scores from 12μm ocular sections taken from eyes enucleated at day 14 post immunisation, stained for CD45+ (DAB/haematoxylin) (n = 9–10 eyes). C) TEFI clinical score, day 13 post immunisation, (average scores provided by two independent assessors from two identical repeat experiments. n = 18–20). doi:10.1371/journal.pone.0122093.g005 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 13 / 20 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Fig 6. Quantification of retinal cellular infiltrate at day 26 post immunisation in a C57BL/6 murine model of EAU carrying Lp-PLA2 WT, heterozygous and homozygous gene-deletion genotypes. Flow cytometry data showing CD45+, CD11b+, CD4+ and CD8+ cellular infiltrate in retinal digests from Lp-PLA2 KO, HET, WT animals with EAU at day 26 post immunisation. (n = 14–15 eyes). doi:10.1371/journal.pone.0122093.g006 Fig 6. Quantification of retinal cellular infiltrate at day 26 post immunisation in a C57BL/6 murine model of EAU carrying Lp-PLA2 WT, heterozygous and homozygous gene-deletion genotypes. Flow cytometry data showing CD45+, CD11b+, CD4+ and CD8+ cellular infiltrate in retinal digests from Lp-PLA2 KO, HET, WT animals with EAU at day 26 post immunisation. (n = 14–15 eyes). Fig 6. Quantification of retinal cellular infiltrate at day 26 post immunisation in a C57BL/6 murine model of EAU carrying Lp-PLA2 WT, heterozygous and homozygous gene-deletion genotypes. Flow cytometry data showing CD45+, CD11b+, CD4+ and CD8+ cellular infiltrate in retinal digests from Lp-PLA2 KO, HET, WT animals with EAU at day 26 post immunisation. (n = 14–15 eyes). doi:10.1371/journal.pone.0122093.g006 independent manner [35]. Lp-PLA2 is considered a key enzyme in vascular inflammation, due to its ability to catalyse the hydrolysis of oxPL on oxLDL molecules in the intima of blood ves- sels resulting in the production of ox-NEFA and LPC, which are cytotoxic and have a potent ef- fect on many different cell types. As such, the notion of Lp-PLA2 suppression as a therapeutic target has received much inter- est in recent years. A porcine model, using streptozotocin treatment and a high fat, high choles- terol diet was used to induce diabetes and accelerate subsequent onset of atherosclerosis. Treatment with the Lp-PLA2 inhibitor, Darapladib, resulted in a reduction of Lp-PLA2 activity and atherosclerotic plaque size, IL-6 expression and systemic hCRP. Whether such response would be evident in a T cell driven autoimmune model, where both macrophage activation and inflammatory vasculopathy are evident, was the rationale for the current work. As such, the notion of Lp-PLA2 suppression as a therapeutic target has received much inter- est in recent years. A porcine model, using streptozotocin treatment and a high fat, high choles- terol diet was used to induce diabetes and accelerate subsequent onset of atherosclerosis. Treatment with the Lp-PLA2 inhibitor, Darapladib, resulted in a reduction of Lp-PLA2 activity and atherosclerotic plaque size, IL-6 expression and systemic hCRP. PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 Whether such response would be evident in a T cell driven autoimmune model, where both macrophage activation and inflammatory vasculopathy are evident, was the rationale for the current work. oxLDL molecules have been shown not only to carry the enzyme, but also to up-regulate Lp-PLA2 expression in the human THP-1 monocyte cell line [36] and data show that when 14 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 15 / 20 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Fig 7. Ocular oxLDL in naive and uveitic mice. Immunofluorescence staining of DAPI (blue) oxLDL (red) and CD31 (green) using 12μm ocular sections from A) naïve mice or B-C) those immunised for EAU (sequential slides). Ganglion cell layer (GCL), inner nuclear layer (INL), inner plexiform layer (IPL), outer nuclear layer (ONL). Fig 7. Ocular oxLDL in naive and uveitic mice. Immunofluorescence staining of DAPI (blue) oxLDL (red) and CD31 (green) using 12μm ocular sections from A) naïve mice or B-C) those immunised for EAU (sequential slides). Ganglion cell layer (GCL), inner nuclear layer (INL), inner plexiform layer (IPL), outer nuclear layer (ONL). doi:10.1371/journal.pone.0122093.g007 oxLDL is treated with SB-435495, Lp-PLA2 activity is reduced [36]. Furthermore, macrophage expression of Lp-PLA2 is increased by TLR4 ligation with LPS, due to a P38 MAPK mediated transcriptional up-regulation [37], suggesting that the enzyme is likely to be highly expressed during inflammation. As such, we hypothesised that by suppressing Lp-PLA2 enzyme activity it may be possible to alter macrophage phenotype and subvert the subsequent macrophage-me- diated tissue damage that occurs in EAU. To this end, both LPS and oxLDL were used to stimu- late BMDM, and we did note a trend towards a dose response of SB-435495 in IL-6 expression (Fig 2A). MCP-1 expression plays an important role in myeloid cell migration in uveitis and the prod- ucts of oxLDL breakdown, LPC and NEFA, up-regulate macrophage MCP-1 production, a re- sponse we hoped to attenuate by depleting Lp-PLA2. However, SB-435495 failed to reduce MCP-1 expression from LPS stimulated BMDM (S2B Fig). oxLDL itself is also capable of in- ducing MCP-1 expression in macrophages [38] and we therefore postulate the possibility that an equilibrium exists between the influence of oxLDL and the associated hydrolytic bi-products on myeloid cells in vitro. PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 16 / 20 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Whilst EAU is comparable in many respects to both atherosclerosis and diabetic macular oedema, demonstrating macrophage and TH17+ cell mediated vascular pathology, EAU does not share the same underlying dyslipidaemic traits and therefore it is possible this is why we did not observe an effect in EAU despite the involvement of inflammation, macrophage infil- tration and vessel inflammation in this model. In the normal retina there appears to be limited expression of oxLDL (Fig 7A), which in- creases when disease is fulminant (Fig 7B). We had predicted that activation, phenotype and migration of circulating myeloid cells might be altered by systemic Lp-PLA2 depletion and hence the ocular availability of the substrate would be unimportant. However quantification of retinal leukocyte infiltration during peak disease, by both flow cytometry and histology re- vealed no difference between Lp-PLA2 depleted samples and controls, indicating that the con- trol of migration of cells into the tissue, driven by antigen specific T cell activation, was independent of Lp-PLA2 activity. Interestingly, and in accordance with our work, recent publications have stated that in spite of promising preliminary findings, Darapladib treatment and associated Lp-PLA2 reduction failed to significantly reduce the incidence of myocardial infarction, stroke and cardiovascular death in a cohort of patients with coronary heart disease, when compared to a placebo treated control group following an extensive randomised, multicentre phase III clinical trial, [43]. Sim- ilarly, whilst we were able to show a clear reduction in systemic Lp-PLA2, which resulted in some alteration to macrophage cytokine profiles, our data show that manipulation of Lp-PLA2 does not alter the onset or progression of EAU. It seems therefore that the acute inflammatory pathways involved in EAU onset proceed independently of Lp-PLA2 mediated mechanisms. Lp-PLA2 depletion may cause oxLDL to accumulate, with the poten- tial to induce MCP-1 production, hence negating any beneficial effects of SB-43545. oxLDL also induces myeloid IL-1β production [39] and hence could explain why Lp-PLA2 HET BMDM, with intermediate enzyme activity, show the lowest gene expression of IL-1β see by qPCR (Fig 3A). In addition to the effect on MCP-1 and IL-1β expression, recent publications have identified a link between oxLDL, and differentiation of cells of the CD4+ TH17 lineage. Using an in vitro co-culture of T cells and dendritic cells treated with TGF-β and oxLDL, an up-regulation of TH17 cell differentiation has been identified, which was absent in samples treated with native LDL or an oxLDL depletion antibody [40]. As such, it is possible that both the substrate and bi-products of the Lp-PLA2 reaction have the potential to exacerbate autoimmune disease. Our data show no significant change to in vitro Arg-1 expression in response to altered Lp- PLA2 activity (Fig 3C) and similarly, BMDM expression of IL-10, an anti-inflammatory cyto- kine, was unchanged in KO mice when compared to WT control cells (Fig 3B). Furthermore, in BMDM derived from Lp-PLA2 WT, HET and KO mice, there was a dose-dependent LPS mediated nitrite production, but this remained independent of Lp-PLA2 activity (Fig 3D). As such, these data infer that depletion of the Lp-PLA2 enzyme does not significantly alter macrophage phenotype. The activity of Lp-PLA2 is regulated to an extent by the degree of HDL and LDL binding, which delivers either anti or pro inflammatory/atherosclerotic behaviour respectively as ob- served in man. Unlike LDL, HDL acts to remove cholesterol from lipid laden foam cells in the vessel intima and acts as an anti-oxidant, reducing the ability of LDL to become oxidised by re- moval of lipid hydroperoxidases [41]. The behaviour and phenotype of HDL and LDL carrier molecules can be altered in chronic conditions such as type II diabetes, where increased small dense LDL (sdLDL) molecules afford greater affinity for Lp-PLA2 [42]. Similarly, diabetic pa- tients have been shown to have HDL molecules with reduced anti-oxidant behaviour owing to an imbalance of lipid/protein and apoA-I/apoA-II ratios [42]. These lipid modifications con- tribute to the onset of atherosclerosis, but are unlikely to be observed in the context of sub- acute T cell driven tissue inflammation such as that seen uveitis. Supporting Information S1 Fig. Expression and functionality of secreted Lp-PLA2. Supernatants from A) media alone and B) LPS (1ng/ml) stimulated BMDM were found to secrete detectable quantities of functional Lp-PLA2, as indicated by a colorimetric change to the supernatant as a result of hy- drolysis of a labelled 2-Thio PAF substrate. This response was abrogated in the presence of SB- 435495 treatment. (TIF) S2 Fig. Effect of Lp-PLA2 depletion on macrophage activation and phenotype. Supernatants from BMDM pre-treated with a dose titration of SB-435495 plus 1ng/ml LPS were used to quantify expression of IL-6 and MCP-1 by ELISA. n = 3 p>0.05. SB-435495 did not significant- ly reduce either A) IL-6 or B) MCP-1 protein expression from LPS treated BMDM (n = 3) (TIF) S3 Fig. Immune phenotype in naïve Lp-PLA2 WT, HET and KO mice. Quantification of B and T cell populations from A) the thymus and B) the spleen using geometric mean fluores- cence intensity (GMFI). GMFI quantification of CD4+ T cell activation markers from C) thy- mocytes and D) splenocytes and CD8+ T cell activation markers from E) thymocytes and F) splenocytes. (TIF) S4 Fig. Dosing regimen for in vivo EAU experiments using SB-435495. Mice were immu- nised and randomly allocated to a treatment group. Each mouse was given a standardised daily or twice daily volume of SB-435495 by i.p injection and the dose calculated based on the weight of the animal. (TIF) 17 / 20 PLOS ONE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 NE \| DOI:10.1371/journal.pone.0122093 April 15, 2015 The Role of Lipoprotein-Associated Phospholipase A₂ in Uveoretinitis Author Contributions Conceived and designed the experiments: GLC JB DAC PA DG MB LBN ADD. Performed the experiments: GLC JB DAC DG MB. Analyzed the data: GLC JB DAC PA DG MB LBN ADD. Contributed reagents/materials/analysis tools: PA DG MB. Wrote the paper: GLC JB DAC PA DG MB LBN ADD. DG MB LBN ADD. 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https://openalex.org/W3197900533	https://pusdikra-publishing.com/index.php/jrss/article/download/240/203	Indonesian	null	Pemahaman Konsep Bentuk Aljabar Dan Kaitannya Dengan Hasil Belajar Matematika Pada Siswa Kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020	Cybernetics	2,021	cc-by	3,311	ABSTRACT Tujuan penelitian ini adalah untuk mengetahui : (1) Bagaimana pemahaman konsep operasi bentuk aljabar di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020, (2) Bagaimana hasil belajar matematika siswa di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020, (3) Bagaimana hubungannya antara pemahaman konsep operasi bentuk aljabar dengan hasil belajar matematika siswa di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020. Penelitian ini merupakan penelitian menggunakan metode dekripsi Kuantitatif. Populasi dalam penelitian ini adalah siswa/I kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020. Populasi dalam penelitian ini berjumlah 360 orang siswa/I kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020 sedangkan sampel dalam penelitian ini mengambil sebesar 20% dari jumlah populasi yaitu 78 orang siswa/i. Intrumen pengumpulan data dalam penelitian ini adalah tes. Berdasarkan hasil penelitian, diperoleh kedua variabel penelitian berdistribusi normal. Untuk variabel pemahaman konsep bentuk aljabar (X) diperoleh x2hitung =-252,11 dan variabel hasil belajar matematika siswa (Y) diperoleh x2hitung = -13625,02, sedangkan x2tabel pada taraf signifikan α = 5% adalah sebesar 11,070. Sehingga untuk kedua variabel penelitian diperoleh x2 hitung > x2 tabel dan ini berarti bahwa kedua variabel penelitian adalah berdistribusi normal. Dalam penelitian ini juga diperoleh koefisien korelasi rxy = 0,974, sedangkan t hitung = 37,48. Nilai t tabel (dk 76 : α = 0,05) adalah sebesar 1,995 sehingga diperoleh t hitung = 37,48 > t tabel = 1,995. Sedangkan koefisien determinan I = 0,94 atau 94% yang berarti besarnya sumbangan variabel bebas X terhadap variabel terikat Y adalah 94 % sedangkan 6% lagi ditentukan oleh faktor lain. Jadi yang menjadi kesimpulan dalam penelitian ini adalah : ada hubungan antara pemahaman konsep bentuk aljabar dengan hasil belajar matematika siswa kelas VII MTsN 2 Medan. Pemahaman Konsep Hasil Belajar Siswa Kata Kunci Cybernetics: Journal Educational Research and Social Studies Volume 2, Nomor 3, Juli 2021 Journal Homepage: http://pusdikra-publishing.com/index.php/jrss Cybernetics: Journal Educational Research and Social Studies Volume 2, Nomor 3, Juli 2021 Journal Homepage: http://pusdikra-publishing.com/index.php/jrss Pemahaman Konsep Bentuk Aljabar Dan Kaitannya Dengan Hasil Belajar Matematika Pada Siswa Kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020 Lailatun Nur Kamalia Siregar Universitas Islam Negeri Sumatera Utara Corresponding Author : PENDAHULUAN Pendidikan adalah proses pengubahan sikap dan tata laku seseorang atau kelompok orang dalam usaha mendewasakan manusia melalui upaya pelajaran dan pelatihan. Pendidikan mempunyai peranan yang sangat menentukan bagi perkembangan dan perwujudan diri individu, terutama bagi perkembangan bangsa dan negara. Kemajuan suatu kebudayaan bergantung kepada cara kebudayaan tersebut mengenali, menghargai, dan memanfaatkan sumber daya manusia. Hal ini berkaitan erat dengan kualitas pendidikan yang diberikan kepada anggota masyarakat dan kepada peserta didiknya. 84 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 Dari kedua tujuan pendidikan di atas, dapat disimpulkan bahwa proses pendidikan yang ideal adalah proses pendidikan yang dikemas dengan memperhatikan adanya sebagai aspek, yaitu aspek kognitif, afektif, dan psikomotorik. Apabila proses pendidikan dapat dilaksanakan dengan memperhatikan adanya keseimbangan ketiga aspek tersebut, maka pendidikan akan mampu mengantisipasi perubahan dan kemajuan masyarakat. Matematika merupakan salah satu ilmu pengetahuan yang paling penting dalam pengembangan proses pendidikan. Ilmu-ilmu pengetahuan lainnya tidak akan berjalan dengan sempurna, karena matematika dapat mengembangkan dirinya mencapai keberhasilan. Namun pada kenyataannya, masalah yang sering muncul adalah kemampuan siswa dalam pemahaman konsep matematika masih rendah. Siswa beranggapan bahwa matematika hanyalah kumpulan angka-angka yang sulit dipahami. Selain rendahnya hasil belajar matematika dan perhatian siswa pada pelajaran matematika juga dapat menghambat siswa untuk memahami materi yang mereka pelajari. Dalam belajar bentuk aljabar khususnya operasi bentuk aljabar, kesulitan yang sering dialami siswa dan cenderung membuat siswa merasa bosan, bisa jadi disebabkan oleh beberapa hal diantaranya : metode mengajar yang kurang tepat, suasana belajar yang menantang untuk membangun imajinasi anak, dan guru kurang memotivasi kemampuan abstraksi anak. Karena hal tersebut, nilai matematika siswa dari tahun ke tahun selalu rendah jika dibandingkan dengan pelajaran yang lain seperti pelajaran Bahasa Indonesia, Bahasa Inggris dan Agama. Belajar juga mendapatkan suatu proses internal dan kondisi lingkungan luar yang essensial bagi berbagai macam hasil belajar dan keterampilan, terutama belajar matematika. Faktor-faktor yang mempengaruhi belajar matematika adalah faktor yang mempengaruhi belajar kognitif disamping pengetahuan umum, kemampuan penalaran deduktif dan kemampuan keterampilan. Berdasarkan penyataan tersebut dapat disimpulkan bahwa struktur mental itu bukan hanya menerima informasi baru. Asimilasi dan Akomodasi merupakan dua aspek yang sama dari proses yang sama. PENDAHULUAN Kedua aspek itu adalah dua aspek dari aktifitas intelektual yang dasarnya adalah suatu proses yang melibatkan interaksi antara pikiran dan kenyataan serta kemampuan- kemampuan siswa dari hasil pelajaran, ada 3 tujuan pendidikan yang mendasar, yaitu 1) Tujuan kognitif berkenaan dengan tingkah laku dari perubahan berbagai proses mental, 2) Tujuan efektif berkenaan dengan perubahan tingkah laku dalam sikap, dan 3) Tujuan psikomotorik berkenaan dengan kemampuan memanipulasi secara fisik. 85 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 Berdasarkan uraian pada latar belakang masalah, penelitian mengidentifikasi berbagai masalah sebagai berikut : Hasil belajar matematika siswa masih renda, Kemampuan siswa dalam penguasaan konsep matematika masih rendah, Metode yang digunakan guru dalam mengajar tidak bervariasi, Siswa menganggap matematika merupakan pelajaran yang sulit, Kurangnya motivasi guru kepada siswa. Sehubungan dengan batasan masalah di atas, maka yang menjadi rumusan masalah dalam penelitian ini adalah sebagai berikut : Bagaimana pemahaman konsep bentuk Persegi Panjang di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020, Bagaimana hasil belajar matematika siswa di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020, Bagaimana hubungannya antara pemahaman konsep bentuk persegi panjang dengan hasil belajar matematika siswa di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020. Adapun tujuan penelitian ini adalah sebagai berikut : Untuk mengetahui pemahaman konsep bentuk persegi panjang di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020. Untuk mengetahui hasil belajar matematika siswa di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020. Untuk mengetahui hubungannya antara pemahaman konsep bentuk persegi panjang dengan hasil belajar matematika siswa di kelas VII MTsN 2 Medan Tahun Pelajaran 2019/2020. Adapun manfaat dari penelitian ini adalah : Manfaat Teoritis : Manfaat teoritis penelitian ini adalah memberikan masukan pengetahuan tentang pemahaman konsep bentuk aljabar dan kaitannya dengan hasil belajar matematika pada siswa. Manfaat Paktis : Sebagai bahan masukan bagi kepala sekolah terutama VII MTsN 2 Medan untuk bisa mengingatkan hasil belajar siswa, Sebagai bahan masukan bagi guru dan orang tua dalam memberikan motivasi yang maksimal tentang belajar matematika, Sebagai bahan masukan bagi siswa yang mengalami kesulitan belajar khususnya belajar matematika, Sebagai bahan masukan bagi penelitian dan pembaca dalam mengembangkan wawasan dan ilmu pengetahuan dalam bidang pendidikan. METODE PENELITIAN Jenis penelitian yang digunakan adalah studi literatur. Metode studi literatur adalah serangkaian kegiatan yang berkenaan dengan metode pengumpulan data pustaka, membaca dan mencatat, serta mengelolah bahan penelitian (Zed, 2008:3). Studi kepustakaan merupakan kegiatan yang diwajibkan dalam penelitian, khususnya penelitian akademik yang tujuan utamanya adalah mengembangkan aspek teoritis maupun aspek manfaat 86 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 praktis. Studi kepustakaan dilakukan oleh setiap peneliti dengan tujuan utama yaitu mencari dasar pijakan / fondasi utnuk memperoleh dan membangun landasan teori, kerangka berpikir, dan menentukandugaan sementara atau disebut juga dengan hipotesis penelitian. Sehingga para penelitidapat menggelompokkan, mengalokasikan mengorganisasikan, dan menggunakan variasi pustaka dalam bidangnya. Dengan melakukan studi kepustakaan, para peneliti mempunyai pendalaman yang lebih luas dan mendalam terhadap masalah yang hendak diteliti. Melakukan studi literatur ini dilakukan oleh peneliti antara setelah mereka menentukan topik penelitian dan ditetapkannya rumusan permasalahan, sebelum mereka terjun ke lapangan untuk mengumpulkan data yang diperlukan (Darmadi, 2011) HASIL DAN PEMBAHASAN a. Pengertian Pemahaman Konsep a. Pengertian Pemahaman Konsep Pemahaman menurut Kamus Besar Indonesia berarti “Proses, Perbuatan, Cara memahami atau memahamkan”. Sudjono (2003 : 50) menyatakan bahwa “Pemahaman (Comprehension) adalah kemampuan seseorang untuk mengerti atau memahami sesuatu setelah sesuatu itu diketahui dan di ingat. Dengan kata lain memahami adalah mengetahui tentang sesuatu dan dapat melihatnya dari berbagai segi”. “Pemahaman atau komprehensi adalah tingkat kemampuan yang mengharapkan siswa mampu memahami arti dalam konsep, situasi serta fakta yang diketahuinya. Dalam hal ini tes tidak hanya menghafal secara verbalitas, tapi memahami konsep dari masalah atau fakta yang dinyatakan”. Pemahaman adalah mencakup kemampuan untuk menangkap makna dan arti dari bahan yang dipelajari. Adanya kemampuan ini dinyatakan dalam menguraian isi pokok dari suatu bacaan, mengubah data yang disajikan dalam bentuk tertentu ke bentuk lain seperti rumus matematika ke dalam bentuk kata- kata, membuat perkiraan tentang kecenderungan yang nampak dalam data tertentu seperti dalam grafik. Pemahaman yaitu taraf ini mencakup bentuk pengertian yang paling rendah, taraf ini berlangsung dengan sejenis pemahaman yang menunjukkan bahwa siswa mengetahui apa yang sedang dikomunikasikan dan dapat digunakan bahan pengetahuan atau ide tertentu tanpa perlu menghubungkannya dengan bahan lain tanpa perlu melihat seluruh implikasinya. Pemahaman dapat dibedakan ke dalam tiga kategoti yaitu : Tingkat terendah adalah pemahaman terjemahan, mulai dari terjemahan dalam arti sebenarnya, misalnya dari bahasa Inggris ke dalam bahasa Indonesia, mengartikan Bhineka Tunggal Ika, mengartikan Merah putih, menerapkan prinsip-prinsip listrik dalam memasang sakelar. 87 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 Tingkat kedua adalah pemahaman penafsiran, yakni menghubungkan bagian-bagian terdahulu yang diketahui berikutnya, atau menghubungkan beberapa bagian dari grafik dengan kejadian, membedakan yang pokok yang bukan pokok. Menghubungkan pengetahuan tentang konjungsi kata kerja, subjek dan possesive pronoun sehingga menyusun kalimat “My friend is studying”, bukan My friend studying”, merupakan contoh pemahaman penafsiran. Pemahaman tingkat ketiga atau tingkat tertinggi adalah pemahaman ekstrapolasi. Dengan ekstrapolasi diharapkan seseorang mampu melihat di balik yang tertulis, dapat membuat ramalan tentang konsekuensi atau dapat memperluas presepsi dalam arti waktu, dimensi, kasus, ataupun masalahnya. Pemahaman adalah suatu dasar bagi segala tindakan seseorang. Ia memberikan kontribusi yang besar bagi sukses tidaknya seseorang lebih jauh, pemahaman menjadikan seseorang saling mengerti, dan lebih lanjut lagi saling menghargai. Pemahaman sekaligus mencegah timbulnya saling curiga dan lebih jauh lagi mencegah timbulnya saling bentrokan. Agar pemahaman akan konsep-konsep matematika dapat dipahami oleh anak-anak lebih mendasar harus diadakan pendekatan belajar dalam mengajar, antara lain : 1. a. Pengertian Pemahaman Konsep Anak/peserta didik yang belajar matematika harus menggunakan benda-benda kongkrit dan membuat abstraksinya dari konsep- konsepnya. 2. Materi atau pelajaran yang akan diajarkan harus ada hubungannya atau pengaitan dengan yang sudah dipelajari. 3. Supaya anak/peserta didik memperoleh sesuatu dari belajar matematika harus mengubah suasana abstrak dengan menggunakan simbol. 4. Matematika adalah ilmu seni kreatif karena itu baru dipelajari dan diajarkan sebagai ilmu seni. Dari kutipan di atas dapat disimpulkan bahwa pemahaman adalah proses sesuatu setelah sesuatu itu diketahui dan diingatnya, abstraksi dalam situasi yang khusus atau kongkret berbentuk prosedur, gagasan umum atau metodologi yang digeneralisasikan, ide-ide, prinsip teknis atau teori-teori yang harus diingat dan diterapkan. Juga bisa dikatakan pemahaman adalah tingkat kemampuan siswa memahami arti, konsep, situasi serta fakta yang diketahui, siswa tidak hanya hafal secara verbalitas, tapi memahami konsep dari masalah atau fakta yang dinyatakan, menyerap arti bahan materi yang dipelajari sehingga dapat mengingat kembali dan menginterprestasikan. 88 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 b. Pengertian Bentuk Aljabar Aljabar berasal dari bahasa arab “al-jabar” yang berarti “Pertemuan”, “Hubungan” adalah cabang matematika yang dapat dicirikan sebagai generalisasi dari bidang Aritmatika. Aljabar juga merupakan nama sebuah struktur aljabar abstrak, yaitu aljabar dalam sebuah bidang. Huruf-huruf dalam aljabar digunakan sebagai pengganti angka. Bentuk aljabar sering melibatkan angka (disebut Konstanta), huruf (disebut Peubah atau Variabel), dan operasi hitung. Hal ini penting untuk kita ketahui dan mengerti agar penulisan singkat dalam aljabar dapat kita gunakan untuk menyelesaikan masalah sehingga lebih mudah dipahami. c. Pengertian Hasil Belajar Matematika Belajar adalah suatu proses yang kompleks yang terjadi pada semua orang dan berlangsung seumur hidup, sejak dia masih bayi hingga ke liang lahat nanti. Belajar juga dapat diartikan sebagai suatu proses usaha yang dilakukan seseorang untuk memperoleh perubahan tingkah laku yang baru secara keseluruhan, sebagai hasil pengalaman sendiri dan interaksi lingkungannya. Perubahan tingkah laku tersebut menyembut perubahan yang bersifat pengetahuan (kognitif), keterampilan (psikomotorik) dan perubahan yang menyangkut nilai dan sikap (afektif). Belajar juga berarti perbaikan dalam tingkah laku dan kecakapan-kecakapan (manusia) atau memperoleh kecakapan- kecakapan tingkah laku yang baru. Hasil belajar adalah kemampuan yang diperoleh anak setelah melalui kegiatan belajar. Belajar itu sendiri merupakan suatu proses dari seseorang yang berusaha untuk memperoleh suatu bentuk perubahan perilaku yang relatif menetap. Anak yang berhasil dalam belajar adalah yang berhasil mencapai tujuan-tujuan pembelajaran atau tujuan-tujuan intruksional. d. Hasil Belajar Matematika Pada Bentuk Aljabar d. Hasil Belajar Matematika Pada Bentuk Aljabar Hasil belajar matematika adalah dapat membuat seorang anak berhasil dalam belajar yang berhasil mencapai tujuan-tujuan pembelajaran atau tujuan- tujuan interaksional. Hasil belajar yang ditunjukkan oleh siswa dapat menunjukkan kesulitan-kesulitan yang dialami oleh siswa. Kesulitan belajar yang dialami oleh siswa biasanya nampak dari kekeliruan-kekeliruan yang dilakukan oleh siswa ketika proses belajar. Contohnya pada mengerjakan soal- soal latihan yang ada pada buku matematika kelas VII yaitu mengenai operasi bentuk aljabar. Kajian standar isi pada pelajaran semester ganjil dan semester genap di MT i Kajian standar isi pada pelajaran semester ganjil dan semester genap di MTs yaitu : MTs yaitu : a. Bilangan 89 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 ▪ Memahami sifat-sifat operasi hitung bilangan dan penggunaannya dalam pemecahan masalah. b. Aljabar ▪ Memahami bentuk aljabar, persamaaan dan pertidaksamaan linier satu variabel. ▪ Menggunakan bentuk aljabar, persamaan dan pertidaksamaan linier satu variabel, dan perbandingan dalam pemecahan masalah. ▪ Menggunakan konsep himpunan dan diagram Venn dalam pemecahan masalah. masalah. . Geometri ▪ Memahami hubungan garis dengan garis, garis dengan sudut, sudut dengan sudut, serta menentukan ukurannya. ▪ Memahami konsep segi empat dan segitiga serta menentukan ukurannya. c. Pengertian Koefisien dan Konstanta Perhatikan bentuk aljabar 8 7 5 6 3 2 3 4 + + + + a a a a . Bilangan-bilangan 3,6,5,7 dan 8 disebut koefisien dari bentuk aljabar. Dalam hal dapat diterangkan sebagai berikut : 4 3a mempunyai koefisien 3 a 7 mempunyai koefisien 7 3 6a mempunyai koefisien 6 8 merupakan konstanta 2 5a mempunyai koefisien 5 2 5a mempunyai koefisien 5 Jadi, faktor 3c). (b dan , 2, adalah 3c) (b 2 + + a a Jadi, faktor 3c). (b dan , 2, adalah 3c) (b 2 + + a a b. Pengertian Suku dan Suku Sejenis Perhatikan bentuk-bentuk aljabar .8 6 6 3 2 − + p dan a a Dalam hal ini a dan a 6 3 2 disebut suku-suku dari 8 6 6 3 2 − + dan p dan a a disebut suku-suku dari .8 6 − p Suku-suku dalam bentuk bentuk aljabar 6x – 7y – 3z misalnya, dapat diubah ke bentuk 6x + (-7y) + 3z. Dengan demikian, suku-suku dari 6x + (-7y) + 3z adalah 6x, -7y, dan 3z. Sekarang perhatikan bentuk-bentuk aljabar a a 4 , 3 + 7b, dan 3p – 2q – r. bentuk-bentuk tersebut terturut-turut disebut suku tunggal, suku dua, dan suku tiga. Pemberian nama ini bersesuaian dengan banyak suku bentuk-bentuk aljabar tersebut. Bentuk aljabar x a x 6 3 4 + + mempunyai suku-suku 4x, 3 a , dan 6x. Suku-suku 4x dan 6x memuat variabel yang sama, yaitu x. Suku-suku tersebut diberikan nama suku-suku sejenis, sedangkan 4x dan 3a disebut suku- suku tidak sejenis. a. Faktor-Faktor Internal Faktor internal adalah semua faktor yang timbul dari luar diri anak itu sendiri, baik yang berkenan dengan segi jasmani anak maupun dengan segi rohani anak. a. Pengertian Faktor Perkalian Pada pembahasan tentang faktor bilangan cacah dinyatakan bahwa 14 = 2 x 7, maka 2 dan 7 disebut faktor dari 14. Pengertian ini akan kita pakai sebagai dasar untuk membahas faktor perkalian dari suatu bentuk aljabar. Bentuk aljabar , 3 3 a x a = maka a 3 memiliki faktor-faktor, yaitu 3 dan a . Faktor 3 disebut faktor angka atau faktor numerik. Faktor ini sering disebut juga koefisien dari a . Faktor a disebut faktor huruf atau faktor alfabetik. Agar lebih mengerti, perhatikan contoh-contoh berikut . 3 faktor numerik q x p x p x q p 3 3 2 = p2 faktor huruf q faktor huruf Jadi, faktor dari 3p2q adalah 3, p2, dan q. Pada p2, bilangan 2 disebut pangkat atau eksponen. 2 faktor numerik ( ) 3c) (b x x 2 3c 2 + = + a b a a faktor huruf (b + 3c) faktor aljabar 2 faktor numerik ( ) 3c) (b x x 2 3c 2 + = + a b a a faktor huruf (b + 3c) faktor aljabar huruf 90 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 Jadi, faktor 3c). (b dan , 2, adalah 3c) (b 2 + + a a Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 KESIMPULAN 1. Setelah dikonsultasikan dengan predikat pemahaman konsep bentuk aljabar siswa diatas maka pemahaman konsep bentuk aljabar siswa di kelas VII MTsN 2 Medan Tahun Ajaran 2019/2020 dapat digolongkan bahwa 3,06% termasuk dalam katagori sangat kurang baik. Dari sudut ini sebenarnya secara umum siswa sekolah ini telah mempunyai pemahaman konsep bentuk aljabar siswa, sehingga mempunyai potensi untuk aktif, ulet, sedia, sabar dan disiplin mengikuti aktifitas belajar matematika di sekolah. Namun perlu mendapatkan perhatian, bahwa selain data ditemukan pula jumlah 31,53% tergolong kurang pemahaman atau belum mempunyai pemahaman untuk belajar. 2. Hasil belajar siswa yang dicapai siswa disekolah ini dapat digolongkan bahwa siswa yang prestasinya tinggi adalah 26 orang atau 33,33%, cukup 43 orang atau 55,13% kurang 8 orang atau 10,26% dan rendah 1 orang atau 1,28%. Untuk kategori tinggi yaitu siswa memiliki skor 10-14, sedangkan untuk kategori cukup yaitu siswa memiliki skor 4 ke bawah. 3. Ditemukan adanya hubungan yang positif antara pemahaman konsep belajar dan hasil belajar matematika pada siswa kelas VII MTsN 2 Medan. Hal ini dapat dilihat dari perhitungan yang diperoleh, yaitu nilai thitung = 37,48, sedangkan nilai ttabel pada taraf signifikan α = 0,05 dengan derajat kebebasan = n-2 = 78-2 76, ttabel = 1,990. Sehingga diperoleh thitung = 37,48 > ttabel = 1,990. Demikian Ho ditolak dan Ha diterima yang menyatakan bahwa ada hubungan yang signifikan antara pemahaman konsep bentuk aljabar dengan hasil belajar matematika siswa. 3. Ditemukan adanya hubungan yang positif antara pemahaman konsep belajar dan hasil belajar matematika pada siswa kelas VII MTsN 2 Medan. Hal ini dapat dilihat dari perhitungan yang diperoleh, yaitu nilai thitung = 37,48, sedangkan nilai ttabel pada taraf signifikan α = 0,05 dengan derajat kebebasan = n-2 = 78-2 76, ttabel = 1,990. Sehingga diperoleh thitung = 37,48 > ttabel = 1,990. Demikian Ho ditolak dan Ha diterima yang menyatakan bahwa ada hubungan yang signifikan antara pemahaman konsep bentuk aljabar dengan hasil belajar matematika siswa. b. Faktor-Faktor Eksternal Faktor eksternal adalah faktor yang bersumber dari luar diri anak didik, faktor ini disebut juga dengan faktor lingkungan. 91 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 DAFTAR PUSTAKA Ali Imron, Belajar & Pembelajaran, Jakarta : Pustaka Jaya, 1996 Ali Imron, Belajar & Pembelajaran, Jakarta : Pustaka Jaya, 1996. Irma Fitria Amalia, Model Pembelajaran Kooperatif Teknik Think-Pair-Share Untuk Meningkatkan Pemahaman Konsep Fisika Siswa, Bandung : Skripsi Pada FPMIPA UPI, 2008. Mulyono Abdurrahman, Pendidikan Bagi Anak Berkesulitan Belajar, Jakarta : Rineka Cipta, 1999. Nana Sudjana, Penilaian Hasil Proses Belajar Mengajar, Bandung : Remaja Rosdakarya, 2005. Pusat Bahasa Departemen Pendidikan Nasional, Kamus Besar Bahasa Indonesia, Jakarta : Balai Pustaka, 2005. Pusat Bahasa Departemen Pendidikan Nasional, Kamus Besar Bahasa Indonesia, Jakarta : Balai Pustaka, 2001, Edisi Ketiga. Sardiman, Media Pendidikan, Jakarta : Rajawali Pers, 1999. Sardiman, Media Pendidikan, Jakarta : Rajawali Pers, 1999. 92 Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 Slameto, Belajar dan Faktor-Faktor yang Mempengaruhinya, Jakarta : Rineka Cipta, 2002. W. James Popham dan Eva L. Baker, Teknik Mengajar Secara Sistematis, Jakarta : Rineka Cipta, 2003. W.S. Winkel, Psikologi Pengajaran, Yogyakarta : Media Abadi, 2004. Cybernetics: Journal of Research and Educational Studies Volume 2 Nomor 3, Juli 2021 Halaman 84-93 W.S. Winkel, Psikologi Pengajaran, Yogyakarta : Media Abadi, 2004. 93
W2901319272.txt	null	de		Donald R. Dickey Field Notes, 1916	null	1,916	cc-by-sa	0
https://openalex.org/W3183642544	https://www.scielo.br/j/sa/a/XmkyzgNbBybYXdsdRSWtPbt/?lang=en&format=pdf	English	null	Phenotypic diversity of agromorphological characteristics of quinoa (Chenopodium quinoa Willd.) germplasm in Colombia	Scientia Agrícola	2,022	cc-by	5,218	Genetics and Plant Breeding DOI: http://doi.org/10.1590/1678-992X-2021-0017 Research Article Ana Cruz Morillo Coronado1* , Elsa Helena Manjarres Hernández1 , Yacenia Morillo Coronado2 na Cruz Morillo Coronado1* , Elsa Helena Manjarres Hernández1 , Yacenia Morillo Coronado2 1Universidad Pedagógica y Tecnológica de Colombia/ Facultad de Ciencias Agropecuarias, Avenida Central Norte # 39-115 – Tunja – Colombia. 2Corporación Colombiana de Investigación Agropecuaria/ AGROSAVIA, Cra 36a–23, Palmira – Valle del Cauca – Colombia. Corresponding author <ana.morillo@uptc.edu.co> Edited by: Alencar Xavier Received January 22, 2021 Accepted April 22, 2021 ABSTRACT: Chenopodium quinoa Willd. is an Andean crop with great nutritional value, economic potential, and a broad genetic and phenotypic diversity with adaptation to different conditions. In Colombia, C. quinoa is cultivated mainly in Cundinamarca, Nariño and Boyacá, where studies have been conducted to characterize the germplasm and lack of seed materials, some challenges for the quinoa crop. This work assessed agromorphological characteristics of 50 quinoa genotypes from the germplasm collection of Boyacá the using a completely randomized design on the farm in Tunja. The multivariate analysis followed by a clustering approach were conducted on agromorphological descriptors, in which 16 were qualitative descriptors (e.g, panicle shape, episperm color, leaf shape) and five quantitative descriptors (e.g, plant height, panicle number). The results showed that higher coefficients of variation were found in characteristics associated to yield. The principal component analysis (PCA) of the quantitative variables showed that the first two components explained 88 % of the total variation with the characteristics of plant height, length, diameter, and panicle number showing the highest variability. The quantitative characteristic clusters comprised length and diameter panicle, weight 1,000 seeds, and plant height, while the qualitative characteristic clusters comprised stem shape, branching habit, panicle shape, and color of the axils. The factorial analysis of mixed data discriminated the materials with outstanding morphoagronomic characteristics. Agromorphological characterization revealed a broad variability, which should be conserved and used in genetic improvement programs of C. quinoa. Received January 22, 2021 Accepted April 22, 2021 Received January 22, 2021 Accepted April 22, 2021 Keywords: Andean cultivation, phenotypic variability, genetic diversity, yield Materials and Methods Materials and Methods Phenotypic diversity of agromorphological characteristics of quinoa (Chenopodium quinoa Willd.) germplasm in Colombia Ana Cruz Morillo Coronado1 , Elsa Helena Manjarres Hernández1 , Yacenia Morillo Coronado2 Introduction quinoa varieties from of Boyacá, considering different variables in certain stages of plant development and establishing a taxonomic key for the identification of varieties. Morillo et al. (2020) studied the intrapopulation phenotypic variation of Piartal material and found that phenotypic variation is concentrated within populations because of high variations at the inter–individual level. Studies have reported a mixture of materials in the fields (Salazar et al., 2019), leading to high phenotypic variability with no certified cultivated material thus requiring more efficient seed selection processes to obtain quinoa genotypes or varieties (Morillo et al., 2020). Quinoa (Chenopodium quinoa Willd.) is a native species to the Andes with great nutritional value because of its high protein content, essential amino acids, vitamins, minerals, isoflavones, carbohydrates and unsaturated fats (Jaikishun et al., 2019; Sampaio et al., 2020). It has efficient water use, tolerance to low soil moisture, wide adaptation to different climates, including deserts (Bazile et al., 2016). Therefore, quinoa can contribute to food security in various regions of the world (Pinto et al., 2020). Quinoa has been cultivated in the Andes from Colombia to Chile. In Colombia, it is cultivated mainly in Cundinamarca, Boyacá, Cauca and Nariño (Jager, 2015). However, Colombia does not have certified seeds or commercial varieties, which has led to a mixture of varieties in the fields (Morillo et al., 2020). Therefore, characterization, conservation, and use of this phytogenetic resource is of great strategic importance for Colombia. This crop has great genetic potential thus providing a solution to the lack of seed materials, an issue in the productive chain in Colombia. In Colombia, few studies have investigated the morphological characterization of quinoa genotypes. The first studies were carried out in Bogotá and Nariño. This research studied the morphological and agronomic characteristics of 50 quinoa genotypes from the germplasm collection of Boyacá to improve varietal identification of cultivated germplasm quinoa and contribute to its breeding and conservation in Colombia. Statistical analysis The statistical analyses were carried out in three steps: (1) descriptive, (2) multivariate, and (3) clustering. First, data from morphoagronomic characterization was used in the descriptive analysis for the quantitative and qualitative variables in the statistical software InfoStat 2020 (Di Rienzo et al., 2020). We computed the Pearson’s correlation for the quantitative variables and its significance was assessed using the t–test (null hyphothesis is H0: r = 0, with 5 % of significance). Second, the principal component analysis (PCA) was performed on the quantitative variables using the correlation matrix between the characteristics, which were graphed on a two–dimensional plane to group the accessions with the R Core Team Software (2020). For the qualitative variables, we carried out the multiple correspondence analysis (MCA) and the correlation analysis. Third, the distance matrix was performed using the Euclidean distance for the cluster analysis. We used the minimum Ward’s distance as the grouping method where each cluster was given by the smallest increase in the total sum value of the squares of existing differences within each cluster of each observation concerning the cluster centroid. The analyses were carried out with the algorithms included in the extra- factor package of the R program version 1.07 for the cluster (Kassambara and Mundt, 2020). For the joint analysis of the quantitative and qualitative variables, a factorial analysis of mixed data was carried out with the factoextra package in the R program. Additionally, a For the evaluation of the quantitative characteristics, the number of panicles was the most variable descriptor, with a coefficient of variation of 39.43 % and a range from 2 to 32 panicles per plant. The least variable characteristic was plant height, with a coefficient of 27.1 % and a range from 43.3 to 131.5 cm (Table 3). Plant height is highly influenced by the environment variables; thus, G × E should be also observed for this characteristic. Lopez et al. (2012) reported that ecotypes cultivated in valleys are higher than on higher ground. Studies have shown that quinoa heights in Colombia range from 70 to 180 cm, consistent with our results (Morillo et al., 2020). The length of panicles in quinoa plants depends on the growth and differentiation processes that occur in the apical meristem (Garcia et al., 2019). These processes are determined by the genotype, inflorescence type, and fertility conditions in the soil, among others (Temel and Keskin, 2020). Morphoagronomic characterization We evaluated 16 qualitative and five quantitative descriptors (Table 2). Measurements were made on four individuals from each material. The FAO for quinoa and its wild relatives (Morillo et al., 2020) defined the descriptors evaluated. Plant Material We evaluated 50 quinoa (C. quinoa) cultivated materials from Boyacá under a completely randomized design (CRD) with three repetitions, where the experiment unit comprised 30 m by 30 m with 28 rows. The materials were sown during the first and second semesters of 2018 on a farm in Tunja, at 2,820 m above sea level, In Boyacá, few studies have estimated the agromorphological parameters of the cultivated materials. Infante et al. (2018) evaluated the physiological parameters in the vegetative and reproductive phase in Sci. Agric. v.79, n.4, e20210017, 2022 Agromorphological evaluation of quinoa Morillo et al. dendrogram was generated using the Euclidean distance and hierarchical grouping method of Ward’s minimum variance with the FactoMineR package (Le et al., 2008). with an average annual temperature 13 °C, relative humidity 78 %, photoperiod 12:12, and rainfall 1,216 mm. The soils in the experiment site were loamy– clay with good drainage. The agronomic management was conventional, with organic fertilization, biological management of pests and diseases, and manual control of weeds. The harvest was carried out manually when the plants reached physiological maturity (Table 1). Results and Discussion In Colombia, quinoa is produced with unidentified genetic materials because of varietal mixtures with morphological, phenological, and physiological changes that depend on edaphoclimatic conditions (Morillo et al., 2020). According to the qualitative and quantitative variables evaluated in this study, a broad phenotypic variability was observed in the quinoa materials from Boyacá, which has a direct effect on yield (Hancco, 2003). Understanding the evolutionary processes that determined the genetic diversity of this species allow management, conservation, and efficient use within a sustainable and economically profitable production scheme (Morillo et al., 2020). Statistical analysis Panicle lengths between 22 and 40 cm have been reported (Lopez et al., 2012) and, in this study, we found panicles between 12 and 45.6 cm in length. Studies have shown a strong correlation between the panicle diameter and yield (Hussain et al., 2020). Panicle diameters in this study ranged from 7.5 to 38.2 cm, which corroborates Morillo et al. (2020) in the study on the intrapopulation phenotypic variation in the material cultivated of Boyacá, Piartal, where a high diversity was found at the interindividual level, mainly in length, diameter, and color of the panicle. Table 1 – Origin Sites of the evaluated quinoa (C. quinoa) materials. Origen Quantity Altitude North latitude West Longitude Nariño (Gen1, Gen3, Gen4, Gen5, Gen6, Gen7, Gen8, Gen9, Gen11, Gen13, Gen14, Gen16, Gen22, Gen25, Gen26, Gen27, Gen32, Gen34, Gen35, Gen39, Gen40) 21 2,817 3°17´20” 77°21´28” Perú (Gen45) 1 3,259 12°04´15” 75°12´24” Boyacá (Gen38, Gen41, Gen43, Gen44, Gen46, Gen47, Gen48, Gen50, Gen51, Gen52, Gen53, Gen54, Gen55, Gen56, Gen57, Gen63, Gen65, Gen66, Gen69, Gen71, Gen72, Gen73, Gen77, Gen80, Gen86, Gen87, Gen106, Gen107) 28 2,942 5°30´02” 73°19´59” Table 1 – Origin Sites of the evaluated quinoa (C. quinoa) materials. Perú (Gen45) Perú (Gen45) 2 Sci. Agric. v.79, n.4, e20210017, 2022 Agromorphological evaluation of quinoa Morillo et al. The cluster analysis (Figure 1) formed three clusters related to the quantitative variables. The first (a) twenty materials were grouped (46, 56, 48, 65, 27, 32, 53, 50, 51, 6, 9, 4, 5, 38, 43, 8, 25, 47, 57 and 77), characterized by panicle length between 28.9 and 45.6 cm, panicle diameter from 22.5 to 38.2 cm, number of panicles from 11 to 32, plant height between 73.3 and 131.5 cm and weight of 1,000 seeds from 1.9 to 2.4 g. The second group (b) comprised eleven materials (52, 71, 72, 44, 55, 106, 69, 107, 80, 86, 87), which were characterized by a panicle length between 22.7 and 37.7 cm, panicle diameter from 17 to 28.6 cm, number of panicles between 7 to 20, plant height between 59 and 108.3 cm and weight of 1,000 seeds from 1.7 to 2, 1 g. The third group (c) comprised materials (11, 26, 13, 1, 22, 14, 16, 3, 7, 63, 66, 41, 39, 40, 45, 34, 35, 54, 73) with a panicle length between 12 and 26.3 cm, panicle Table 4 presents the Pearson correlation matrix between each pair of variables, which shows three highly significant coefficients (p < 0.05). The associated characteristics included plant height and panicle length (r = 0.94), panicle diameter and length (r = 0.97) and plant height and panicle diameter (r = 0.9). These results agree with Spehar and Barros (2005) who evaluated quinoa varieties from Peru and Bolivia and reported a strong correlation between plant height, length, and diameter. Similarly, Morillo et al. (2020) evaluated quinoa materials from Boyacá and found a positive correlation between plant height and panicle length (r = 0.91) and plant height and panicle diameter (r = 0.78). Montes et al. (2018) also observed a positive correlation between plant height and panicle length, which means that taller plants also have a greater panicle length. The PCA of the quantitative variables showed that 88 % of the total variation was explained by the first two components. In PC1, variables with greater contribution included plant height, length, diameter, and number of the panicles. In PC2, weight of 1,000 seeds was the most representative variable (Table 5). In general, quantitative variables that contribute more to the percentage of variance are related to yield (Morillo et al., 2020). Perú (Gen45) Table 4 – Simple correlation matrix between quantitative variables. Variable PL PDI NP WS PH Panicule lenght 1 Panicule diameter 0.97 1 N° panicule plants 0.61 0.63 1 Weight of 100 seeds –0.09 –0.08 –0.17 1 Plant height 0.94 0.90 0.64 –0.05 1 PL PDI NP WS PH Highly significant values (p < 0.05). Variables evaluated: PL = Panicle length; PDI = Panicle diameter; NP = Number of panicles; WS = Weight of the 1,000 seeds; PH = Plant height. Table 4 – Simple correlation matrix between quantitative variables. Variable PL PDI NP WS PH Panicule lenght 1 Panicule diameter 0.97 1 N° panicule plants 0.61 0.63 1 Weight of 100 seeds –0.09 –0.08 –0.17 1 Plant height 0.94 0.90 0.64 –0.05 1 PL PDI NP WS PH Highly significant values (p < 0.05). Variables evaluated: PL = Panicle length; PDI = Panicle diameter; NP = Number of panicles; WS = Weight of the 1,000 seeds; PH = Plant height. Table 2 – Morphoagronomic descriptors evaluated for the characterization of quinoa material from Boyacá. Table 3 – Descriptive statistics for the quantitative variables. Variable Average D.E. CV Mínimum Maximum PL 28.93 9.15 31.64 12.00 45.60 PDI 22.57 8.50 37.66 7.50 38.20 NP 13.65 5.38 39.43 2.30 31.70 WS 2.10 0.17 8.30 1.72 2.63 PH 79.64 21.62 27.15 43.30 131.50 D.E.=Standard deviation; *CV=coefficients of variation; PL= Panicle length; PDI P i l di t NP N b i l WS W i ht f 100 d Table 2 – Morphoagronomic descriptors evaluated for the characterization of quinoa material from Boyacá. Qualitative Initials Quantitative Initials Units of measurement Growth habit (H) Panicle length (PL) cm Presence of striae (PS) Panicle diameter (PDI) cm Strie color (SC) N ° plant panicles (NP) # Stem shape (SS) Weight of 1,000 seeds (WS) g Stem color (SCO) Plant height (PH) cm Pigmented axil (PA) Axil color (AC) Branch (B) Leaf shape (LS) Leaf margin (LM) Panicle shape (PSA) Panicle density (PD) Panicle color at flowering (PCF) Panicle color at physiological maturity (PCM) Grain shape (GS) Episperm color (EC) Table 3 – Descriptive statistics for the quantitative variables. Variable Average D.E. Perú (Gen45) *CV Mínimum Maximum PL 28.93 9.15 31.64 12.00 45.60 PDI 22.57 8.50 37.66 7.50 38.20 NP 13.65 5.38 39.43 2.30 31.70 WS 2.10 0.17 8.30 1.72 2.63 PH 79.64 21.62 27.15 43.30 131.50 D.E.=Standard deviation; CV=coefficients of variation; PL= Panicle length; PDI = Panicle diameter; NP = Number panicle; WS = Weight of 100 seeds and PH = Plant height. Table 2 – Morphoagronomic descriptors evaluated for the characterization of quinoa material from Boyacá. Qualitative Initials Quantitative Initials Units of measurement Growth habit (H) Panicle length (PL) cm Presence of striae (PS) Panicle diameter (PDI) cm Strie color (SC) N ° plant panicles (NP) # Stem shape (SS) Weight of 1,000 seeds (WS) g Stem color (SCO) Plant height (PH) cm Pigmented axil (PA) Axil color (AC) Branch (B) Leaf shape (LS) Leaf margin (LM) Panicle shape (PSA) Panicle density (PD) Panicle color at flowering (PCF) Panicle color at physiological maturity (PCM) Grain shape (GS) Episperm color (EC) Table 2 – Morphoagronomic descriptors evaluated for the characterization of quinoa material from Boyacá. Qualitative Initials Quantitative Initials Units of measurement Growth habit (H) Panicle length (PL) cm Presence of striae (PS) Panicle diameter (PDI) cm Strie color (SC) N ° plant panicles (NP) # Stem shape (SS) Weight of 1,000 seeds (WS) g Stem color (SCO) Plant height (PH) cm Pigmented axil (PA) Axil color (AC) Branch (B) Leaf shape (LS) Leaf margin (LM) Panicle shape (PSA) Panicle density (PD) Panicle color at flowering (PCF) Panicle color at physiological maturity (PCM) Grain shape (GS) Episperm color (EC) Highly significant values (p < 0.05). Variables evaluated: PL = Panicle length; PDI = Panicle diameter; NP = Number of panicles; WS = Weight of the 1,000 seeds; PH = Plant height. *Highly significant values (p < 0.05). Variables evaluated: PL = Panicle length; PDI = Panicle diameter; NP = Number of panicles; WS = Weight of the 1,000 seeds; PH = Plant height. Table 5 – Principal components and percentages of total variance explained. Table 5 – Principal components and percentages of total variance explained. Table 5 – Principal components and percentages of total variance explained. Table 5 – Principal components and percentages of total variance explained. Component Eigenvalues Total % de variance % acumulate 1 3.38 68.00 68.00 2 1.00 20.00 88.00 3 0.48 10.00 97.00 4 0.12 2.00 1.00 5 0.02 0.00 1.00 Figure 1 – The cluster analysis of the quinoa materials evaluated for quantitative traits. The letters correspond to each group formed according to the characteristics evaluated. Component Eigenvalues Total % de variance % acumulate 1 3.38 68.00 68.00 2 1.00 20.00 88.00 3 0.48 10.00 97.00 4 0.12 2.00 1.00 5 0.02 0.00 1.00 Figure 1 – The cluster analysis of the quinoa materials evaluated for quantitative traits. The letters correspond to each group formed according to the characteristics evaluated. Table 3 – Descriptive statistics for the quantitative variables. Variable Average D.E. *CV Mínimum Maximum PL 28.93 9.15 31.64 12.00 45.60 PDI 22.57 8.50 37.66 7.50 38.20 NP 13.65 5.38 39.43 2.30 31.70 WS 2.10 0.17 8.30 1.72 2.63 PH 79.64 21.62 27.15 43.30 131.50 D.E.=Standard deviation; **CV=coefficients of variation; PL= Panicle length; PDI = Panicle diameter; NP = Number panicle; WS = Weight of 100 seeds and PH = Plant height. Figure 1 – The cluster analysis of the quinoa materials evaluated for quantitative traits. The letters correspond to each group formed according to the characteristics evaluated. Sci. Agric. v.79, n.4, e20210017, 2022 3 Agromorphological evaluation of quinoa Morillo et al. branches. A simple branching habit has been observed in materials from highlands and salt flats (Kir and Temel, 2016). In this study, 10 % of the materials had a simple growth habit, while 84 % was branched in the lower third, which is common in valley quinoa. On the other hand, the branching habit is influenced by planting density, nutrients, and other environmental factors (Kir and Temel, 2016). diameter from 7.5 to 20.3 cm, number of panicles from 2 to 16, plant height between 43.3 and 74.7 cm and weight of 1,000 seeds from 1.7 to 2.6 g. In general, the groupings were conditioned by the characteristics associated to the panicle, seeds, and plant height. In this study, the morphological characteristics showed a wide range of variation. The evaluated germplasm was very diverse, possibly because of its coevolution process with the environment. Table 5 – Principal components and percentages of total variance explained. The frequency analysis showed that the panicle color had most variability, evaluated twice during the phenological cycle. The results showed that 46 % of the materials had purple panicles, 28 % had green ones, and 14 % had a mixture of green and yellow panicles (Figure 2). Panicle colors changed during physiological maturity. Hence, other studies on quinoa reported significant decreases in plant height, stem thickness, number of branches, and panicle ratios, as the sowing time progressed (Hirich et al., 2014; Temel and Keskin, 2020). Montes et al. (2018) reported that the wide range of colors in the materials evaluated is because the panicle is covered by granular vesicular pubescence rich in calcium oxalate with white, pink, and purple tones that contribute to panicle coloration of each variety. Similarly, leaf shape characteristic varies depending on the material. In the materials studied, 50 % of the plants had a rhomboidal leaf shape, 32 % was oval and 18 % was triangular. For the leaf margin, 68 % of the plants had an entire margin, while 32 % had a dentate margin. The panicle density is associated to the grain size and the smallest is formed in compact panicles (Gómez and Aguilar, 2016). In this study, 42 % of the quinoa evaluated had intermediate heads, between loose and compact, 40 % of the plants had compact heads, and 18 % had lax heads. The panicle shape in this study was mainly glomerular (80 %), 10 % was amarantiform, and the other 10 % was intermediate. Studies have shown an association between panicle density and its shape and genotype yield, since panicles with an intermediate shape tend to be compact with good yield (Pinto et al., 2020). This association with yield is because the flowers are sessile or pedicellate and are grouped in glomeruli. The glomerulus position in inflorescence and the position of flowers within glomerulus determine the size and number of grains or fruits (Chura et al., 2019). In quinoa, characteristics associated to the stem, such as color, striae and/or axils, can be used to identify varieties (Kir and Temel, 2016), since combinations of these characteristics differentiate the materials. In this study, stem color was variable: 54 % yellow, 26 % purple, and 10 % red. Purple was the predominant color in 84 % of the plants had axils. Table 5 – Principal components and percentages of total variance explained. Striae in 92 % of the plants varied in color according to the material: 56 % was yellow, 12 % was purple, and 12 % was dark green (Figure 2). An important aspect in quinoa commercialization is the grain color. In Colombia, white quinoa is preferred, but in countries such as Bolivia and Peru, black and brown varieties are desired in gourmet cuisine. In this study, grain color was variable: 48 % of the materials had beige grains, and 18 % of the plants had yellow grains, which is why they are acceptable materials for commercialization in Colombia. On the other hand, the branching habit of quinoa must be evaluated because mechanical harvesting tasks are difficult when the material presents certain types of Figure 2 – Phenotypic diversity in panicle color (A). Quinoa material with compact panicles and purple color; (B) Quinoa material with intermediate panicles and green color; (C) Quinoa material with loose panicles and purple color; (D) Purple axils on the stem, yellow striae. Figure 2 – Phenotypic diversity in panicle color (A). Quinoa material with compact panicles and purple color; (B) Quinoa material with intermediate panicles and green color; (C) Quinoa material with loose panicles and purple color; (D) Purple axils on the stem, yellow striae. Sci. Agric. v.79, n.4, e20210017, 2022 4 Agromorphological evaluation of quinoa Morillo et al. Figure 3 – Correlations for qualitative variables measured in materials of quinoa evaluated. H= Growth habit; PCM = Panicle color at physiological maturity; AC = Axil color; SC = Strie color; SCO = Stem color; PSA = Panicle shape; PD Panicle = density; SS = Stem shape; PCF = Panicle color at flowering. Figure 3 – Correlations for qualitative variables measured in materials of quinoa evaluated. H= Growth habit; PCM = Panicle color at physiological maturity; AC = Axil color; SC = Strie color; SCO = Stem color; PSA = Panicle shape; PD Panicle = density; SS = Stem shape; PCF = Panicle color at flowering. Figure 4 – The cluster analysis of quinoa materials evaluated for qualitative characteristics. The letters correspond to each group formed according to the characteristics evaluated. Figure 4 – The cluster analysis of quinoa materials evaluated for qualitative characteristics. The letters correspond to each group formed according to the characteristics evaluated. Figure 5 – The cluster analysis of quinoa materials evaluated for qualitative and quantitative characteristics. Table 5 – Principal components and percentages of total variance explained. The letters correspond to each group formed according to the characteristics evaluated. The correlation analysis of qualitative variables was high and positive for characteristics, such as panicle shape, stem color, and striae color, while the axil color had little contribution to characterizing the materials, since 78 % of the evaluated plants had purple color (Figure 3). Correlation studies are important for quinoa improvement because of the selection response and allow significant genetic gains in less time (Al–Naggar et al., 2018). ) In the dendrogram for qualitative characteristics, three clearly differentiated groups were shown (Figure 4). Group (a) had the materials (43, 86, 46 and 107) where most outstanding morphological characteristics were the shape of an angular stem, the color of the green axils, and the amaranthiform panicle shape. Group (b) had the materials (44, 87, 71, 69, 77, 22, 45, 66, 72, 73, 11, 5, 6, 26, 13, 63, 7, 27, 14, 16, 39, 34, 40, 8, 9, 1 and 4) with branching habits in the lower third, a glomerular panicle shape, and purple axils. Group (c) comprised materials (80, 57, 50, 51, 52, 41, 53, 3, 32, 35, 106, 47, 48, 56, 25, 38, 65, 54, and 55) that were characterized by a cylindrical stem, branching in the first third, glomerular panicle shape, and green axils. In general, the clusters were formed by characteristics, such as stem shape, branching habit, panicle shape, and axil color. Figure 4 – The cluster analysis of quinoa materials evaluated for qualitative characteristics. The letters correspond to each group formed according to the characteristics evaluated. The factorial analysis of mixed data considered all quantitative and qualitative variables and discriminated the materials with outstanding morphoagronomic characteristics (Figure 5). The cluster analysis formed four groups. First, material was grouped (3), characterized by the panicle yellow color and the number of panicles of 23 per plant. The second group comprised 21 materials, which were characterized by an average of 14 panicles per plant, plant height 79.63 cm, panicle length 28.93 cm, and density of the compact panicle. Group 3 was characterized by glomerulate panicle shape, cylindrical stem shape, plant height between 74.3 and 132.0 cm, and weight of 1,000 seeds from 1.7 to 2.4 g. Group 3 was formed by materials with glomerulate panicle shape, cylindrical stem shape, plant height ranging from Figure 5 – The cluster analysis of quinoa materials evaluated for qualitative and quantitative characteristics. Table 5 – Principal components and percentages of total variance explained. The letters correspond to each group formed according to the characteristics evaluated. 74.3 to 132.0 cm and weight of 1,000 seeds from 1.7 to 2.4 g. The last group it is characterized by growth habit of branching to the lower third, panicle color at physiological maturity green and purple, panicle length from 12 to 35 cm, and plant height from 43.3 to 86 cm. Studies on the morphoagronomic characterization of quinoa in Colombia have shown diverse quinoa 74.3 to 132.0 cm and weight of 1,000 seeds from 1.7 to 2.4 g. The last group it is characterized by growth habit of branching to the lower third, panicle color at physiological maturity green and purple, panicle length from 12 to 35 cm, and plant height from 43.3 to 86 cm. Studies on the morphoagronomic characterization of quinoa in Colombia have shown diverse quinoa 5 Sci. Agric. v.79, n.4, e20210017, 2022 Agromorphological evaluation of quinoa Morillo et al. germplasm with potential use. The studies report that producers require more stable materials in terms of phenotypic traits that allow optimal development of harvesting and postharvest processes and yields that are economically viable. Hussain, M.; Muscolo, A.; Ahmed, M.; Ahsan, M.; Al-Dakheel, A. 2020. Agro-morphological, yield and quality traits and interrelation with yield stability in quinoa (Chenopodium quinoa Willd.) genotypes under saline marginal environment. Plants 9: 1763. Jaikishun, S.; Li, W.; Yang, Z.; Song, S. 2019. Quinoa: in perspective of global challenges. Agronomy 9: 1–15. The phenotypic characterization carried out on the 50 quinoa materials suggest a broad genetic diversity, confirming that these materials result from mixtures that farmers keep crop after crop. However, it is necessary to consider the experimental conditions of the study, which was conducted in a single location and two semesters in a year. It is thus convenient to determine the genotype– by–environment interaction of the materials selected in the study for more accurate conclusions. Therefore, genetic improvement processes are needed to facilitate sustainable quinoa production in Colombia through the identification of elite materials that respond to the needs of farmers, producers and consumers. Kassambara, A.; Mundt, F. 2020. Factoextra: Extract and Visualize the Results of Multivariate Data Analyses. (R package version 1.07). Kir, A.E.; Temel, S. 2016. Determination of seed yield and some agronomical characteristics of different quinoa (Chenopodium quinoa Willd.) variety and populations under dry conditions of Iğdır Plain. Acknowledgments Pinto, K.; Cobo de la Peña, T.; Ostria, E.; Ibánez, C.; Briones, V.; Vergara, A.; Alvarez, R.; Castro, C.; Sanhueza, C.; Castro, P. 2020. Seed characterization and early nitrogen metabolism performance of seedlings from Altiplano and coastal ecotypes of quinoa. BMC Plant Biology 20: 343. The authors gratefully acknowledge the: “Patrimonio Autónomo Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación Francisco José de Caldas” –MinCiencias. ID. 63924, for the research financial support. Salazar, J., Torres, M.; Gutierrez, B.; Torres, A.F. 2019. Molecular characterization of Ecuadorian quinoa (Chenopodium quinoa Willd.) diversity: implications for conservation and breeding. Euphytica 215: 215:60. Table 5 – Principal components and percentages of total variance explained. Iğdır University Journal of the Institute of Science and Technology 4: 145-154. Le, S.; Josse, J.; Husson, F. 2008. FactoMineR: An R package for multivariate analysis. Journal of Statistical Software 25: 1-18. Lopez, J.P.; Timaran, M.F.; Betancourth, C. 2012. Evaluation of 16 selections of sweet quinoa (Chenopodium quinoa Willd.) in the municipality of Guaitarilla, Nariño. Revista de Ciencias Agrícolas 25: 130–149 (in Spanish, with abstract in English). Authors’ Contributions Conceptualization: Morillo Coronado, A.C.; Manjarres Hernández, E.H.; Morillo Coronado, Y. Data acquisition: Morillo Coronado, A.C.; Manjarres Hernández, E.H. Design of methodology: Morillo Coronado, A.C.; Manjarres Hernández, E.H. Writing and editing: Morillo Coronado, A.C; Manjarres Hernández, E.H.; Morillo Coronado, Y. Montes, C.; Burbano, G.A.; Muñoz, E.F.; Calderón, Y. 2018. Description of the phenological cycle of four ecotypes of (Chenopodium quinoa Willd.), In Puracé - Cauca, Colombia. Biotecnología en el Sector Agropecuario y Agroindustrial 16: 26–37 (in Spanish, with abstract in English). Morillo, A.; Manjarres, E.; Reyes, W.; Morillo, Y. 2020. Intrapopulation phenotypic variation in Piartal (Chenopodium quinoa Willd.) from the Departament of Boyacá, Colombia. African Journal of Agricultural Research 16: 1195-1203. References Bazile, D.; Jacobsen, S.E.; Verniau, A. 2016. The global expansion of quinoa: trends and limits. Frontiers in Plant Science 7: 1–6. Sampaio, S.; Fernandes, Á.; Pereira, C.; Calhelha, R.; Sokovic, M.; Santos, C.; Barros, L.; Ferreira, I. 2020. Nutritional value, physicochemical characterization and bioactive properties of the Brazilian quinoa BRS Piabiru. Food & Function 4: 2969- 2977. Chura, E.; Mujica, Á.; Haussmann, B.; Smith, K.; Flores, S.; Flores, A.L. 2019. Agronomic characterization of quinoa (Chenopodium quinoa Willd.) progeny from close and distant self-fertilized s5 simple crosses. Ciencia e Investigacion Agraria 46: 154–165. Spehar, C.R.; Barros, R.L. 2005. Agronomic performance of quinoa selected in the Brazilian Savannah. Pesquisa Agropecuária Brasileira 40: 609–612. Di Rienzo, J,; Casanoves, F., Balzarini, M.; González, L.; Tablada, M.; Robledo, C. InfoStat. Version 2012, Windows. Grupo InfoStat: Universidad Nacional de Córdoba, Argentina. Temel, S.; Keskin, B. 2020. Effect of morfological componentes on the herbage yield and quality of quinoa (Chenopodium quinoa Willd.) grown at different dates. Turkish Journal of Agriculture and Forestry 44: 533-542. Garcia, M.; García, J.; Deaquiz, Y. 2019. Physiological performance of quinoa (Chenopodium quinoa Willd.) under agricultural climatic conditions in Boyacá, Colombia. Agronomía Colombiana 37: 144-152. Hirich, A.; Choukr, A.; Jacobsen, S.E. 2014. Quinoa in Morocco- effect of sowing dates on development and yield. Journal of Agronomy and Crop Science 200: 371-377. 6 Sci. Agric. v.79, n.4, e20210017, 2022
https://openalex.org/W4289262779	https://zenodo.org/record/6985203/files/Opening%20the%20Future%20-%20COPIM%20WP3%20Final%20v2%20Toolkit%20Aug%202022.pdf	English	null	Opening the Future: How to Implement an Equitable Revenue Model for Open Access Monographs	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	12,541	1/34 DOI 10.5281/zenodo.6907707 Version 2 - updated Aug 2022 to incorporate minor clarifications 2/34 Table of Contents Table of Contents Introduction and Background Context ............................................................................................ 3 About the COPIM Project .......................................................................................................... 4 What is Opening the Future? ...................................................................................................... 4 What Opening the Future is Not ................................................................................................ 5 Assumptions ............................................................................................................................... 5 Why are Presses Interested in Opening the Future? ................................................................... 6 Financial Modelling and Scenario Planning ................................................................................... 8 The Membership Modeller ......................................................................................................... 8 Realistic Modelling .................................................................................................................. 10 Tax Implications ....................................................................................................................... 11 Organizational Partnerships .......................................................................................................... 12 Billing and Sales Partnerships .................................................................................................. 12 Jisc Collections .................................................................................................................... 12 Knowledge Unlatched ......................................................................................................... 12 LYRASIS ............................................................................................................................. 12 Open Book Collective ......................................................................................................... 13 Delivery Partnerships ............................................................................................................... 13 OAPEN ................................................................................................................................ 13 Project MUSE ...................................................................................................................... 13 Thoth .................................................................................................................................... 14 Marketing and Outreach................................................................................................................ 15 Outreach to Backlist Authors ................................................................................................... 15 Sample Email to Author ...................................................................................................... 15 Sample Template Email to Library from Author ................................................................. 16 Outreach to Frontlist Authors ................................................................................................... 16 Outreach to Open Access or Scholarly Communications Librarians ....................................... 17 Outreach to Acquisitions/Collection Librarians and Using Worldcat Data .............................. 17 Outreach to Library Consortia .................................................................................................. 18 Press Releases about New Signups .......................................................................................... 18 Conferences and Events ........................................................................................................... 19 Signup Workflow .......................................................................................................................... 21 Process Diagram ....................................................................................................................... 21 Actors and Actions ................................................................................................................... 22 Delivery of Subscription Content ................................................................................................. 22 Fixed Packages Should Not Change ........................................................................................ 23 Licensing: Use SERU Where Possible ..................................................................................... 23 Digital Preservation and Perpetual Access ............................................................................... 24 Delivery of OA Content ................................................................................................................ 24 Selection Procedures and Timescale ........................................................................................ 24 Technical Infrastructures ............................................................................................................... 25 ‘Signup’ Infrastructure ............................................................................................................. 26 Open Book Collective .............................................................................................................. 26 Appendix A: Jisc Collections Licensing Template ....................................................................... 27 Appendix B: Financial Modelling Spreadsheet ............................................................................ 28 Appendix C: Frequently Asked Questions .................................................................................... 29 Bibliography.................................................................................................................................. 34 3/34 1 Izabella Penier, Martin Paul Eve, and Tom Grady, ‘Revenue Models for Open Access Monographs 2020’, Community-Led Open Publication Infrastructures for Monographs, 2020 <https://doi.org/10.5281/zenodo.4011836>. About the COPIM Project COPIM (Community-led Open Publication Infrastructures for Monographs) is an international partnership of researchers, universities, librarians, open access book publishers and infrastructure providers. It is building community-owned, open systems and infrastructures to enable OA book publishing to flourish. COPIM is developing a significantly enriched not-for-profit and open source ecosystem for OA book publishing that supports and sustains a diversity of publishing initiatives and models, in the UK and internationally. To achieve its aims, the project is divided into seven work packages ranging from the technical (building open-source, community-based infrastructures that support the publication and dissemination of OA books), through to advocacy and knowledge sharing activities (establishing and consolidating partnerships between HE institutions and OA book publishers). Work Package 3 (WP3) of COPIM is dedicated to knowledge exchange and transfer, as part of the project’s central commitment to dissemination. It builds pathways to impact with relevant stakeholders from across the sector, including publishers ((New) University Presses, library-presses, scholar-led presses), academics, learned societies, and open technology developers. As part of its activities, WP3 is working to transition the business models of two university presses: the Central European University Press and Liverpool University Press. Introduction and Background Context Covid-19 has thrown many aspects of university research culture into acute relief. As the reality of the virus dawned and campuses worldwide went into lockdown, publishers scrambled to unpaywall their research. Publishers made topical works and more general material openly available, through their own sites and platforms such as Project Muse and JSTOR. Physical collections became inaccessible and demand for openly accessible research skyrocketed. It is unclear that it is desirable to return to the previous systems of scholarly communication in the book publishing world, in which physical copies may remain affordable, even while e-licensing agreements for libraries are not. This has been recognised in several recent global policy announcements including the cOAlition S/Plan S guidelines, and the recent UKRI consultation on OA. The latter’s proposed measures include the possibility of zero-embargo green OA, more liberal open licensing, and the long- vaunted requirement for funded monographs to be in scope. This last element built on a longstanding policy history in the UK foreshadowing a mandate for OA monographs. That said, the path to OA monographs is not free of obstacles. Among the many issues, the most frequently raised is the business model of Book Processing Charges (BPCs) and their apparent unaffordability, mostly due to distributional allocation of library resources. Happily, several recent reports have detailed non-BPC OA revenue and business models that presses could use to transition to OA - one of the most recent being COPIM’s Revenue models for Open Access monographs 2020.1 That report describes a variation on the journal ‘Subscribe to Open’ model whereby members ‘subscribe to a backlist, with the revenue then used to make the frontlist openly accessible’. This constitutes a new business model for OA monographs that had not previously been implemented. We implemented this model, dubbed ‘Opening the Future,’ in a partnership between the COPIM project, the Central European University Press (CEUP), and Liverpool University Press (LUP). This model presents a potential route for the mass and sustainable transition to OA of many small-to-mid sized university presses. This document sets out how we implemented this model, including the documentation of challenges, resources, timetables, and activities. It is intended as a roadmap for other presses that wish to implement an ‘Opening the Future’-esque model. Of course, this document is unlikely to cover everything, but the authors are happy to respond to individual queries where this will prove helpful. 4/34 What is Opening the Future? Opening the Future is a revenue model for open access monographs in which a press solicits a relatively small financial contribution from a moderate number of academic libraries, in order to cover the costs of new OA publications. In return, the member libraries receive special access to elements of the press’s backlist, as well as frontlist titles that are made OA. Libraries will retain access to the backlist for as long as they are members and with the option for the press to offer perpetual access to libraries after a certain period of membership. Membership will last a minimum of three years, with the option to renew after that. The revenue from the membership – that grants access to the backlist – will be used to fund the frontlist to be OA. The aim of this approach is to continue to yield a sustainable source of revenue for a press while achieving the desired commitment to making more titles OA. Given the current global library environment and already existing budget pressures that have now been exacerbated by Covid-19, a consortial model of funding promises a cost-effective solution for OA that means no single institution bears a disproportionate burden. The model also yields an elastic approach to open access that can grow with the success of the model. 5/34 What Opening the Future is Not Opening the Future is not like a ‘transformative agreement’ in the journal world. There is no linkage between an institution supporting the model and their own authors being allowed to publish OA. Opening the Future, as a result, is trying to break the link between institutions paying and their own authors being allowed to publish openly, in favour of the press securing a 100% open frontlist and so achieving the former by default. Opening the Future is better thought of as an attempt to build a one-time, open, global ‘collection’ that is shared by libraries in common, around the world. Assumptions There are several assumptions behind this model that make it possible and that show the aspirations for scaling: • The target output volume is 25 new OA titles per year for a press-level conversion. For a series-level conversion, we are aiming for 4-5 titles. • The model can work at the level of converting the entire press or on a single series. • It costs between approximately €6,500 to €8,900 to produce the ‘first copy’ of each new title. • The press can define and curate an attractive collection of backlist titles to offer exclusively to members. • Library members will be banded according to their size - meaning their payments will be tiered high, medium, and low. • Titles will be made OA only as and when enough members join and scaling is elastic – they would not be published upfront first and retrospectively funded. • Membership will last a minimum of three years, with the option to renew for another three – access to the backlist is granted in perpetuity, after three years of membership or an upfront payment of three years’ worth. • Membership will initially build over a three-year period, achieving the target revenue at end of year three. The number of OA titles will grow commensurately and gradually as more membership revenue allows more titles to be funded and published. • Membership will initially build over a three-year period, achieving the target revenue at end of year three. The number of OA titles will grow commensurately and gradually as more membership revenue allows more titles to be funded and published. • There will be some flexibility around which books a library subscribes to each year. This model has a number of characteristics in its favour, including pleasing library economics. For example, if an annual membership of €812 gives access to 50 backlist titles, and funds a further 25 OA per year - with only 200 member libraries joining this equates to around €15 per library, per title. The backlist/frontlist combination also provides a unique dual-route to rationalisation for acquisition librarians. Many institutions do not have dedicated OA funds, particularly for monographs: the fact that the backlist remains a subscription means that these libraries are able to 6/34 justify the expenditure as a purchase of books to which they would otherwise not have access. Assumptions At the same time, every year of membership will grow their (open) collection by (in this instance) 25 titles. And those libraries with OA budgets are able to justify this expenditure because the new frontlist titles are openly accessible. This will also allow institutions that have access to block grants for OA to use this pot to fund the purchase. It is further possible for funders directly to support such consortial membership schemes. justify the expenditure as a purchase of books to which they would otherwise not have access. At Two of the major advantages of this model are, first, that the number of OA titles can grow (or contract) elastically with the revenue stream, and, second, that once the target volume is reached, membership/subscription prices can decrease in future years. Pricing will, of course, be specific to the press that implements the model, but for CEUP we proposed three membership tiers with the highest band paying €1,200, the middle band at €800, and the lowest at €350. This pricing should be within the affordability of many academic libraries worldwide. We have modelled financial scenarios towards a target membership of 270 libraries, including slow and steady projections, enthusiastic ‘early adopter’ subscriptions, and even cancellations. While this last is not ideal for takeup of OA, it demonstrates a model that will allow flexibility: if the revenue is not in place, or if institutions cancel, then the volume of OA simply decreases. Why are Presses Interested in Opening the Future? This has an inherent level of complexity, which we hope we will reduce over time, for publishers as well as librarians.” – Emily Poznanski, Central European University Press – Emily Poznanski, Central European University Press – Emily Poznanski, Central European University Press “ University presses exist to disseminate scholarship. A majority of university presses have long-standing programmes rooted in the humanities, disciplines for which the monograph remains a significant vehicle both to conceptualise research and to share it. Repeated narratives of the decline of the monograph ignore the strength of supply, with an unending pipeline of authors driven by a compelling mixture of intellectual hunger and career necessity. They ignore a growing potential audience, as the drive for impact has created ever more urgent and engaging long-form research outputs. The problem, of course, is the model. Open access has for some years served to unite authors with wider potential readership for journals but it has not been systematically adopted for books because of the substantial costs arising from length, complexity and curation. Faced with the possibility of a well-funded, openly available STEM and a closed-access, narrowly available HSS, scholarly book publishers should be willing to explore new models that maximise the audience for the monograph. “ Opening the Future, with its realistic acknowledgement of publisher costs and of the realities of library budgets and policies, offers a potentially sustainable model to reform the dissemination of scholarly books. Liverpool University Press is ideally placed to test that assumption, as a mission-based publisher operating entirely without subsidy or endowment. If we can make the model work without a financial safety net then it should be possible for any publisher to make it work. To test the hypothesis behind OtF we have opted to work at series/discipline level. This has two advantages: first, it enables us to leverage an existing strength of the Press in the Modern Languages. We know who to market to, while librarians know that the LUP imprimatur is a guarantee of quality. OA may in time also serve as an editorial differentiator for prospective authors in our chosen field. Secondly, working at series level allows for more rapid progress than expensive larger collections but also avoids the inefficiency of title-by-title OA. Why are Presses Interested in Opening the Future? The benefits of open access for books are clear. In the spring of 2020, when CEU Press made its titles freely available on Project MUSE: the usage figures soared, 7 out of the 10 most downloaded books were over ten years old, and the most downloaded title in China was called Academic Freedom. As a case study, this clearly demonstrated the need to find a sustainable route to open access for books, in general, and for the Press in line with its mission. “ “ At the core, CEU Press aims to export to the rest of the world a body of knowledge that offers a path to a proper understanding of the region and the lessons learned that are of global relevance. Moreover, for us publishing is no longer simply about just producing content, but also about creating equitable opportunities for authors to make their research discoverable and accessible through open access and to create impact and engagement. We wanted to find a model that: 1) provided sustainable funding for publishing OA books; 2) allowed for a gradual transition to OA as we did not have a subsidy to support the change and; 3) did not charge authors Book Processing Charges (BPCs) to publish as most of our authors (like so many other HSS scholars) do not have access to funds to cover OA publishing costs. The Opening the Future (OtF) model allows for a gradual transformation in line with the pace of change in the library community, which minimises the risk a press might have to take. So as we increase our funding through this model, we can publish an increasing number of titles in OA. But its not an all or nothing approach. For the press, the challenges lie around the resources needed to launch a new business model and to manage three streams concurrently: print, online and open access, each of 7/34 which requires different know how and different workflows. This has an inherent level of complexity, which we hope we will reduce over time, for publishers as well as librarians.” which requires different know how and different workflows. – Anthony Cond, Liverpool University Press – Anthony Cond, Liverpool University Press Why are Presses Interested in Opening the Future? Funding levels for a series OtF are comfortably within library means, while for publishers it is a minor risk but a major opportunity, with the option to scale up if the model gains traction.” – Anthony Cond, Liverpool University Press 8/34 Financial Modelling and Scenario Planning The Membership Modeller The first step towards implementing an Opening the Future model is understanding your own press’s finances and what the transformation will look like. To that end, we have produced a membership modelling tool that will allow you to insert your own figures and then to model the resulting scenarios (see Appendix B). In the tool, boxes in yellow should be replaced with your own figures (and you should only type in the boxes that are highlighted in yellow). Boxes in green represent automatically calculated incoming finances while boxes in red represent calculated outgoings. Table 1: Description of variables in the spreadsheet modeller ‘Variables’ tab Variable Name Description Default Base cost per book The basic cost per book that the press needs to recover. €7,500.00 Approx. billing agent fee per membership transaction (presses may wish to add a slightly higher figure in this box to also account for sales agent fees, if applicable) The fee requested by a billing agent per membership transaction. €85.00 Bank fee per transaction The fee requested by the Press’s bank per transaction €15.00 Higher Band The fee rate for ‘large’ band (biggest) library subscribers. €1,200.00 Middle Band The fee rate for ‘middle’ band (middle size) library subscribers. €800.00 Lower Band The fee rate for ‘lower’ band (smallest) library subscribers. €350.00 Number of titles in backlist The number of titles that are offered in the ‘backlist’ package (i.e. how many books does the library get as an exclusive reward for joining?) 50 Number of yearly titles in frontlist The number of titles that the press aspires to make openly accessible every year under this scheme. 25 OA deposit fee per book (e.g. OAPEN per-title fee) The fee to deposit each OA book (for example, in OAPEN). €10.00 OA deposit annual membership fee (e.g. OAPEN annual membership fee) The annual membership fee to be able to deposit books (for example, the OAPEN annual €175.00 Table 1: Description of variables in the spreadsheet modeller ‘Variables’ tab 9/34 Variable Name Description Default fee). Secondary annual OA deposit location fee (e.g. JSTOR) The annual membership fee to be able to deposit books in a secondary location (for example, JSTOR). €100.00 One-time setup fee The one-off staff labour cost of sorting out the backlist titles into packages. €2,000.00 Approx. annual fee (based on 300 members) The annual fee from a platform provider to provide access to the backlist. €3,000.00 Approx. Financial Modelling and Scenario Planning The Membership Modeller annual deposit fee per backlist title The annual fee per backlist title book for a platform provider, to provide access to the title. €100.00 Creation of metadata records per backlist title The cost of creating metadata records for an item of backlist content. €50.00 Format-shift costs (e.g. improved digital copies, OCRing PDF text, creating epub) per title The costs of ensuring that the backlist is in a viable format for digital delivery. €100.00 Other tabs/sheets in the modeller also have configurable variables. For instance, the ‘Linear Data’ sheet describes what happens when members join at a rate of 84 per year, spread exactly evenly between the different banding levels. As a note: our experience with the Central European University Press and Liverpool University Press is that most memberships have originated from larger institutions, despite our attempt at fair pricing at the lower end. This sheet also allows you to enter the number of Book Processing Charges that are anticipated to be secured, independently, and so will not count towards the Opening the Future scheme (but will nonetheless enable OA). Different tabs/sheets depict different scenarios. For instance, the ‘Early Adopters’ sheets are designed to show a rapid uptake, followed by a longer tail. Each sheet shows the projections for revenue and the number of funded books in each year at this stage. However, the safety and beauty of the scheme is that, if any of these figures are not met, the book should simply be processed as a ‘sales-only’/‘toll-access’ title. The model does not require a press to make a book openly accessible until the revenue target to enable this has been met. When modelling costs, the crucial balance to strike is between value for money on the backlist and value for money on the OA frontlist. A good pricing level will yield a valuable return to libraries solely on the backlist (i.e. it will appear good value if they only received the backlist titles), solely on the frontlist (i.e. it will appear good value if they only received the OA frontlist titles), and it will appear very good value when the frontlist and backlist titles are combined. 10/34 Realistic Modelling There are many factors that will affect the accuracy provided by the financial modelling tool. The rate of growth, for instance, will be dependent on the success of the marketing strategy and the level of resource that is devoted to these activities. It is always better to plan for more conservative growth and then to exceed this target than to make unrealistic predictions. Our initial implementation of Opening the Future also took place against a number of significant world events and policy decisions that changed the landscape beneath our feet. First, the Covid-19 pandemic hit with associated fears for university library budgets. This caused a certain reticence among libraries amid budgetary constraint. Second, though, a number of policy announcements (such as the UKRI review) fuelled further OA book mandates. These look set to continue, with the review of the REF taking place in the UK. The US situation looks set never to be one of all- encompassing mandates, but it is true that various initiatives – such as Plan S – will steadily have influential knock-on effects. Understanding the types of institution who are willing to join an initiative is also important. In the first year of CEUP’s Opening the Future scheme, the Press achieved 41 signups across five packages (Figure 1). This amounted to £35,350 of OA funding or enough for five open-access monographs. Notably, all but one of the USA institutions was in the highest tier of subscriber fees. A similar pattern emerged for European libraries and almost all UK participants fell in the medium to high tiers. In the 6 month period beginning January 2022, CEUP’s membership increased by another 30 libraries, thereby accruing funding for further OA titles to be published. Realistic Modelling Possible results from these data include: • Future signups will occur faster than in this first year because the model is better understood; • Future signups will occur faster than in this first year because mandates place more pres on institutions to support OA monographs; • Future signups will occur more slowly than in this first year because library budgets are under increasing pressure, including from transformative agreements in the journal space; • Future signups will occur more slowly than in this first year because a proliferation of models dilutes library resources. Presses should, therefore, appraise their own brand strengths, including disciplinary placement, and make informed decisions about the likelihood of institutional support. For instance, if the press has authors who work at universities that have been known to support OA membership models in the past – such as Knowledge Unlatched, the Open Library of Humanities, arXiv, Subscribe to Open – then this may give confidence in that institution’s support. Creating an initial spreadsheet of authorial affiliations of the Press’s backlist and correlating these with institutions known to support OA initiatives may provide a benchmark of potential reach. Realistic Modelling Figure 1: CEUP signups from January to December 2021 While CEUP’s progress gives the best indicator that we have of signup rates, it is difficult to extrapolate from these data for a number of reasons: Figure 1: CEUP signups from January to December 2021 While CEUP’s progress gives the best indicator that we have of signup rates, it is difficult to extrapolate from these data for a number of reasons: Figure 1: CEUP signups from January to December 2021 While CEUP’s progress gives the best indicator that we have of signup rates, it is difficult to extrapolate from these data for a number of reasons: While CEUP’s progress gives the best indicator that we have of signup rates, it is difficult to extrapolate from these data for a number of reasons: 11/34 • This was the first year of operation, meaning that much of our time was spent explaining the model, asking librarians to understand the premises, and generally spreading the word; • This was the first year of operation, meaning that much of our time was spent explaining the model, asking librarians to understand the premises, and generally spreading the word; were operating during perhaps the most exceptional set of conditions in our lifetimes: C id 19 d i i h k k ff lib b d • We were operating during perhaps the most exceptional set of conditions in our lifetimes: the Covid-19 pandemic, with knock-on effects on library budgets; • Library budgetary planning takes a long time to go through – once a library has decided that it wishes to participate, it can take up to 12 months for the funding to be approved and to become available for expenditure; • Mandates for OA monographs are only now coming to prominence, with UKRI announcing their conditions mid-way through the year. • Mandates for OA monographs are only now coming to prominence, with UKRI announcing their conditions mid-way through the year. Tax Implications While this document cannot dispense and does not constitute formal accountancy or legal advice with respect to taxation, there are several considerations to bear in mind: • The United Kingdom’s HMRC (the formal tax body) previously advised the Open Library of Humanities (while the UK was still a member of the EU) that membership schemes with no direct benefit to the supporting organization (library) were exempt from VAT because there was no ‘supply’. • However, because Opening the Future provides subscription access to books, a supply does occur in this model. • However, because Opening the Future provides subscription access to books, a supply does occur in this model. • But the supply of e-books is a zero-rated activity for VAT purposes. 3 Frances Pinter and Lucy Montgomery, ‘Knowledge Unlatched: Toward an Open and Networked Future for Academic Publishing’, in Accentuate the Positive: Charleston Conference Proceedings, 2012, ed. by L. H. Hinds, K. P. Strauch, and B. R. Bernhardt (presented at the Annual Charleston Library Conference, United States of America: Purdue University Press, 2013), pp. 386–91 <http://docs.lib.purdue.edu/cgi/viewcontent.cgi?article=1387&context=charleston> [accessed 14 July 2020]; Frances Pinter and Christopher Kenneally, ‘Publishing Pioneer Seeks Knowledge Unlatched’, 2013 <http://beyondthebookcast.com/transcripts/publishing-pioneer-seeks-knowledge-unlatched/>; Lucy Montgomery and others, ‘Pilot Proof of Concept Progress Summary’ (Knowledge Unlatched, 2014) <http://collections.knowledgeunlatched.org/wp- content/uploads/2013/09/KU_Pilot_Progress_Summary_Report.pdf> [accessed 25 May 2014]. • But the supply of e-books is a zero-rated activity for VAT purposes. It is our belief, therefore, that Opening the Future should be treated as a zero-rated supply activity in regions that charge VAT. Supply of digital goods takes place in the country of the consumer. Hence, if you meet the VAT threshold in the country of supply, you may need to register and account for VAT, although you can charge a zero-rate. 12/34 Jisc Collections Jisc Collections works on behalf of Jisc members and customers in the United Kingdom, to negotiate and license the high-quality digital content agreements needed to support academic research, teaching and learning. Its strategic groups set the direction of national-level negotiations.2 Jisc Collections is the official intermediary for most content made available to UK libraries. Many UK libraries expect to be able to sign up through Jisc Collections. Jisc Collections have a template license developed in partnership with COPIM that can be used quickly to establish new Opening the Future programmes. Jisc Collections levy a transaction fee to publishers but do not otherwise take a cut. Jisc Collections is instead funded by its library member base. Contact: Caroline Mackay, Licensing manager (caroline.mackay@jisc.ac.uk) Knowledge Unlatched Knowledge Unlatched was originally a consortial model for open access to monographs, in which many libraries paid a small amount in order to ‘unlatch’ titles.3 Over time, it has transitioned into becoming a sales agent for open access initiatives and is now owned by Wiley. Its vision ‘is a sustainable market where scholarly books and journals are freely accessible for each and every reader around the world’. Knowledge Unlatched can be a useful sales agent for European regions in particular. They take a percentage fee per transaction. Contact: Catherine Anderson, Head of Sales (catherine@knowledgeunlatched.org) 2 Jisc Collections, ‘About Jisc Collections’, Jisc, 2021 <https://www.jisc.ac.uk/jisc-collections> [accessed 3 January 2022]. Organizational Partnerships Delivering a worldwide membership programme requires significant resources, which can be ameliorated by working with trusted partners. Here we list the partnerships that we developed for Opening the Future, their role in the delivery, pricing, and contact details. OAPEN OAPEN promotes and supports the transition to open access for academic books by providing open infrastructure services to stakeholders in scholarly communication. We work with publishers to build a quality-controlled collection of open access books and provide services for publishers, libraries, and research funders in the areas of hosting, deposit, quality assurance, dissemination, and digital preservation.5 We deposit all books published openly through the Opening the Future scheme in OAPEN to enhance discoverability and use of the open content. ontact: Tom Mosterd, Community Manager, DOAB / OAPEN (t.mosterd@oapen.org) Contact: Tom Mosterd, Community Manager, DOAB / OAPEN (t.mosterd@oapen.org LYRASIS LYRASIS is a library consortium that catalyzes and enables equitable access to the world’s knowledge and cultural heritage. LYRASIS helps its members succeed by working with them to identify their needs, issues and challenges and by providing products, services and learning 13/34 experiences to address them. LYRASIS helps its members succeed by providing leadership in and opportunities for innovation and sustainability and facilitating their pursuit of a preferred future.4 LYRASIS has long been a supporter of membership-based open access models in North America. Through their Open Access Community Investment Program (OACIP), LYRASIS has a broad marketing reach for open-access initiatives. LYRASIS is also usually already setup as a supplier in many North American institutional systems, thereby substantially lessening the overhead of payment processing for most presses. LYRASIS charge a percentage transaction fee, which can be included in the financial modelling tool. Contact: Sharla Lair, Senior Strategist, Open Access & Scholarly Communication Initiatives (sharla.lair@lyrasis.org) 3 January 2022]. Open Book Collective The Open Book Collective is a group of OA publishers, publishing service providers and research libraries who work together through an online platform to sustain an equitable infrastructure for OA Books. On the OBC platform, a forthcoming output of the COPIM project, libraries can easily support a diversity of publishers committed to promoting co-operation and communal values in a ‘one-stop shop’ environment. y ] 5 OAPEN, ‘Online Library and Publication Platform’, 2021 <https://oapen.org/> [accessed 3 January 4 LYRASIS, ‘Mission and Strategic Plan’, 2021 <https://www.lyrasis.org/about/Pages/Strategic-Plan.aspx> [accessed 3 January 2022]. 6 Project MUSE, ‘The MUSE Story’, 2021 <http://about.muse.jhu.edu/about/story/> [accessed 3 January 2022]. Contact: https://thoth.pub Project MUSE Project MUSE is a leading provider of digital humanities and social science content for the scholarly community around the world. For more than 25 years, Project MUSE has been the trusted and reliable source of complete, full- text versions of scholarly journals from many of the world's leading universities and scholarly societies. Currently, Project MUSE has over 700 journals from 125 publishers and offers nearly 14/34 70,000 books from more than 140 presses. All books are fully integrated with Project MUSE's scholarly journal content, with collection and single title purchasing, subscription, OA books/MUSE Open and evidence-based acquisition models available.6 70,000 books from more than 140 presses. All books are fully integrated with Project MUSE's scholarly journal content, with collection and single title purchasing, subscription, OA books/MUSE Open and evidence-based acquisition models available.6 Project MUSE is used, in Opening the Future, to deliver subscription and OA book content. Project MUSE offers robust metadata provision to participating libraries, many of whom are already using the platform. Of particular note, Project MUSE offers the requisite access control for the subscription portions of Opening the Future, thereby avoiding the need for presses to implement their own access control measures (see below on Technical Infrastructure). Contact: Nicole Kendzejeski, Associate Director (MUSE@jh.edu) • A WebAssembly GUI to manage metadata records. Thoth Thoth is an Open Dissemination System for Open Access books. It consists of: Thoth is an Open Dissemination System for Open Access books. It consists of: • A GraphQL API, implementing a data model specifically designed for OA books • A REST API to export metadata in formats like ONIX, MARC, etc. p • A WebAssembly GUI to manage metadata records. Presses who require a metadata management system can use Thoth to integrate with Open Book Collective. Presses who require a metadata management system can use Thoth to integrate with Open Book Collective. Contact: https://thoth.pub 15/34 Marketing and Outreach Marketing and outreach are the most crucial but also most frequently overlooked activities in the implementation of open-access business models. While there is a popular wisdom that claims that ‘if you build it, they will come’, this does not straightforwardly appear to be the case. Implementing a new model such as Opening the Future requires a rigorous outreach, marketing, and sales framework if it is to succeed. Emails to these individuals should: Emails to these individuals should: • Be addressed from a known and trusted figure, such as the acquisitions editor who worked with the author previously. • Be specifically actionable. The email should clearly ask the author to reach out to a particular librarian, if possible with a name and contact address. • Require as little work on the author’s part as possible. Use the below templates to attach the email that you would like the author to send to the library. • Be personalised so as not to come across as marketing spam. • Comply with all General Data Protection Regulation (GDPR) provisions for opt-out if an author does not wish to hear any more. • Comply with all General Data Protection Regulation (GDPR) provisions for opt-out if an author does not wish to hear any more. Outreach to Backlist Authors Authors who have published with the press are likely to be among the keenest to ensure its future prosperity. Authors who have previously published with the press are, therefore, a key outreach point for institutional contact. That said, it is by no means guaranteed that these authors will understand the Opening the Future model or, indeed, have any knowledge of open access publishing in general. There is, for example, no guarantee that authors have heard of book processing charges and it can be a dangerous field to open. Sample Template Email to Library from Author Dear [insert librarian name], Dear [insert librarian name], My publisher, [insert press name], has launched an innovative Open Access initiative called Opening the Future, which I would like you to consider supporting. With annual fees of between [€£$insert figure] and [€£$insert figure] per institution this is a low- risk way of supporting a model that can be scaled up throughout the world of smaller university presses. [insert press name] is offering collections of excellent titles in a model similar to the ‘subscribe to open’ scheme which you’ve already seen used successfully in the journal world. In essence [insert press name] has launched a collective subscription model that, through its membership scheme, makes library funds go further: achieving the dual objectives of increasing collections and supporting Open Access. The small annual fee would get the name of university term access to [insert number] titles, becoming perpetual access after three years; the membership revenue is used exclusively to publish new OA monographs. The initiative has the support of COPIM which is a reputable and well-funded driver of Open Access for monographs, dedicated to moving away from unaffordable BPCs and spreading costs more equitably. There are further details on the Opening the Future website and signing up takes only a few minutes through a simple form at [insert signup URL]. I would encourage you to consider membership and feel free to contact the press for further details. With many thanks for your consideration and with apologies if I have not addressed this email to exactly the right library colleague - I would be grateful if you could pass this on, Name of author Sample Email to Author Dear X, [insert your personal greeting] I am writing to let you know of an innovative Open Access scheme at [insert press name]. In order for this scheme to work, and for new scholarship to be well supported, I am asking for your help with contacting your library. Opening the Future is a membership programme for university libraries whereby they subscribe to a selection of our titles and in return we use their membership fees to publish new scholarship in Open Access format, freely available to all and with no author facing fees. You can find more details on the scheme at [insert web address]. 16/34 Our books will continue to be available in print, but the pandemic has shown that not only are digital editions essential for research and teaching, but that they must also be affordable. Our books will continue to be available in print, but the pandemic has shown that not only are digital editions essential for research and teaching, but that they must also be affordable. If you were able, please, to write to your library requesting that they sign up to support the initiative, it would be of great help. I attach a sample email you can use (copied below) and I believe the correct library contact is: [insert contact detail here] With your - and their - help we can begin to eliminate Book Processing Charges for scholars that do not have access to research funder budgets. And we can freely disseminate new scholarship far and wide. With many thanks for your consideration and best wishes, [insert your name] With many thanks for your consideration and best wishes, Outreach to Open Access or Scholarly Communications Librarians Open access librarians are often extremely savvy and well versed in new models for open access. Given the affordability of Opening the Future and the fact that scholarly communications budgets have been cut back in recent days, this initiative offers a powerful and affordable way for institutions to signal their support for open-access books, even when they do not have a great deal of available funding. One potential point of confusion for this group is that they may misconstrue Opening the Future as a read-and-publish or transformative agreement. That is, they may erroneously believe that they are paying for their authors to publish openly with the press. Communication with librarians in this group must stress the novelty of the approach and that the aim is to reach the entire frontlist becoming OA, thereby avoiding the need for hybrid interim measures. Key elements of this outreach include: • Stressing the affordability of the initiative, with particular reference comparison • Emphasizing the need to avoid Book Processing Charges. • Tying the request to authors from the institution without implying that this is a transformative agreement (i.e. showing that authors from the institution are publishing with the press). • Tying the request to subject-specific readership/usages (e.g. are there textbooks set on courses at the university that are published by the press?) • Personalisation so as not to come across as marketing spam. • Personalisation so as not to come across as marketing spam. • Complying with all General Data Protection Regulation (GDPR) provisions for opt-out if a librarian does not wish to hear any more. Outreach to Frontlist Authors The degree of enthusiasm for open access varies hugely between authors. Nonetheless, frontlist authors who are convinced of the merits of OA make for powerful allies. Writing to authors in this 17/34 category is particularly efficacious, when a work is in press or under contract, as they will often be keen to help the press to thrive. Many of the same principles apply as in the above outreach to backlist authors, except that the relationship here is likely to be warmer and more active. The key is to do as much work on behalf of the academic as possible so that it is easy for them to write to their librarian. Outreach to Library Consortia Approaching library consortia can be a powerful way to generate multi-site signups and quickly gain many members. The recent MIT Press report indicated substantial success working with large groups of libraries.7 That said, there are a number of aspects to bear in mind with consortial groups: • Consortial groupings can be slow in their decision making. There are often multiple layers of committees and budgets can be set up to 12 months in advance. Hence, while the rewards can be great, the lead time for consortial participation can be lengthy. • Consortial groupings can be slow in their decision making. There are often multiple layers of committees and budgets can be set up to 12 months in advance. Hence, while the rewards can be great, the lead time for consortial participation can be lengthy. • Consortial groups are more likely to respond to larger presses with more substantial asks. There is almost a sense that below a certain threshold, it is not worth the time/participation of the big groups. • Consortia can sometimes be distant from the purchasing process. Speaking directly on the ground with individual acquisitions and scholarly communications librarians can often yield more immediate direct results, although the rewards of landing a large-scale consortial group is key. • Some library systems act as mini-consortia. The California Digital Library (CDL), for instance, acts on behalf of the California system and is an extremely important player in the US market. Nine libraries of the CDL group are subscribing members of Opening the Future at both Liverpool University Press and CEU Press. • Some consortia have an extraordinarily large reach. The Center for Research Libraries (CRL) in the US is a powerful ally who has already endorsed the Opening the Future model at the Central European University Press. • Some consortia have an extraordinarily large reach. The Center for Research Libraries (CRL) in the US is a powerful ally who has already endorsed the Opening the Future model at the Central European University Press. • Some libraries are members of multiple consortial groups. Avoiding overlap in pricing calculations and factoring this in during growth predictions is important. Outreach to Acquisitions/Collection Librarians and Using Worldcat Data One of the core goals of the Opening the Future model is to shift from a bifurcated environment where acquisitions budgets are used to buy books, while scholarly communications budgets are used to fund OA, into a more holistic environment in which all acquisitions expenditure contributes to making content open for all. When selling the Opening the Future model to acquisitions librarians, the focus should be on the unique content that is delivered to each participating library (the backlist). Institutions in this situation will also usually focus on a teaching need for the title or whether faculty have requested a purchase, which can be challenging to demonstrate. Demand-driven acquisition paradigms have also instilled a false belief in the sustainability of on-demand access. 18/34 Various data sources, such as Worldcat, can help to make the case to acquisitions librarians, although the data can be read in different ways by different institutions. For instance, if Worldcat data indicate that a library has previously purchased every title from a press, some libraries will see this as a good indication that they should join the Opening the Future model as it represents a cheaper way to acquire all new books. Others, though, may question why they should subscribe when they already have all the titles. Hence, data are never neutral and require contextualisation for each library. 7 Raym Crow, ‘MIT Press Open Monograph Model (Direct to Open)’ (Chain Bridge Group and the MIT Press, 2022). Press Releases about New Signups The most effective marketing strategies present potential customers with multiple engagement opportunities. Because of the way that the timescales work in Opening the Future, there are many opportunities to bring the scheme to the attention of libraries. These points include: • Launch of the scheme/model • Every time a library signs up (a press release with a quote from the library in question gives the library the opportunity to signal their support for open publication practices and also allows you to express gratitude in public for the signup) • Every time a library signs up (a press release with a quote from the library in question gives the library the opportunity to signal their support for open publication practices and also allows you to express gratitude in public for the signup) 19/34 • Reaching the revenue threshold for the next book to become open access • Deciding which next new title will be open access (i.e. declaring the intent for a book to be published openly) h bli i f i l • Deciding which next new title will be open access (i.e. declaring the intent for a book to be published openly) • The publication of an open-access title • The publication of an open-access title Press releases that feature books and authors should be illustrated and eye catching. When launching these announcements via a social media presence, you should ‘tag’ the associated individuals – whether that is a library or an author – to ensure the broadest spread of material. All press releases should clearly indicate how libraries can support the model, with as few clicks as possible. All press releases should clearly indicate how libraries can support the model, with as few clicks as possible. Conferences and Events Examples of conferences at which it might be prudent to market the scheme are: Examples of conferences at which it might be prudent to market the scheme are: • • Advancing Research Communication and Scholarship (ARCS) Conference • • ALA Annual meeting • • ALA Midwinter meeting • • ALPSP conference • • American Historical Association • • American Theological Library Association (ATLA) • • Annual Library Leadership Institute (ALLI) conference • • Asian Conference on Literature and Librarianship (LIBRASIA) conference • • Asian Digital Library Conference (ADLC) • • Association of Asian Studies • • Association of College & Research Libraries (ACRL) conference(s). • • AUPresses Annual Meeting • • Australian Library and Information Association (ALIA) Information Online conference. • • Berlin Open Access Conference • • Canadian Association of Research Libraries • • Canadian Library Association (CLA) conference. • • Charleston Conference (also Preconference) • • Chartered Institute of Library and Information Professionals (CILIP) conference • • CNI / Jisc library leaders conference • • Conference of Directors of National Libraries of Asia and Oceania (CDNLAO) • • Congress of Southeast Asian Librarians (CONSAL) • • DCDC (RLUK, National Archives & Jisc) • • Electronic Resources and Libraries Conference (EL&R) • • Emerging Technologies in Academic Libraries (EmTAcL) conference. • • FORCE • • Frankfurt Book Fair • • Geneva Workshop on Innovations in Scholarly Communication • • ICOLC • • IFLA / WLIC International Federation of Library Associations • • Information Science in an Age of Change (ISAC) conference • • International Coalition of Library Consortia (ICOLC). • • International Conference on Asia-Pacific Digital Libraries (ICADL) • • Joint Conference on Digital Libraries (JCDL) • • Advancing Research Communication and Scholarship (ARCS) Conference • • ALA Annual meeting • • ALA Midwinter meeting • • ALPSP conference • • American Historical Association • • American Theological Library Association (ATLA) • • Annual Library Leadership Institute (ALLI) conference • • Asian Conference on Literature and Librarianship (LIBRASIA) conference • • Asian Digital Library Conference (ADLC) • • Association of Asian Studies • • Association of College & Research Libraries (ACRL) conference(s). • • AUPresses Annual Meeting • • Australian Library and Information Association (ALIA) Information Online conference. • • Berlin Open Access Conference • • Canadian Association of Research Libraries • • Canadian Library Association (CLA) conference. Conferences and Events • • Charleston Conference (also Preconference) • • Chartered Institute of Library and Information Professionals (CILIP) conference • • CNI / Jisc library leaders conference • • Conference of Directors of National Libraries of Asia and Oceania (CDNLAO) • • Congress of Southeast Asian Librarians (CONSAL) • • DCDC (RLUK, National Archives & Jisc) • • Electronic Resources and Libraries Conference (EL&R) • • Emerging Technologies in Academic Libraries (EmTAcL) conference. • • FORCE • • Frankfurt Book Fair • • Geneva Workshop on Innovations in Scholarly Communication • • ICOLC • • IFLA / WLIC International Federation of Library Associations • • Information Science in an Age of Change (ISAC) conference • • International Coalition of Library Consortia (ICOLC). Conferences and Events • • International Conference on Asia-Pacific Digital Libraries (ICADL) • • Joint Conference on Digital Libraries (JCDL) • ALA Annual meeting • • ALA Midwinter meeting 20/34 • • Joint University Librarians Advisory Committee (JULAC) conference • • LIBER - Association of European Research Libraries • • Library Association of Singapore (LAS) Libraries for Tomorrow conference • • Library Collective aka LibCon • • Library Publishing Forum • • London Book Fair • • Modern Language Association • • Munin Conference • • MUSE Meets conference • • Music Library Association/Theatre Library Association Joint Meeting • • National Acquisitions Group (NAG) • • NISO Plus • • North American Serials Interest Group (NASIG) • • North Carolina Serials Conference • • OA Tage/Open Access Days (Germany) • • OASPA conference • • Ontario Library Association OLA Super Conference • • Open Education Conference • • Open Science Fair • • Researcher 2 Reader (R2R) • • RLUK • • SCONUL - UK Society of College, National and University Libraries conference • • Society for Scholarly Publishing (SSP) • • The Higher Education Technology Agenda (THETA) • • UKSG • • WHEF conference • • Joint University Librarians Advisory Committee (JULAC) conference • • LIBER - Association of European Research Libraries • • Library Association of Singapore (LAS) Libraries for Tomorrow conference • • Library Collective aka LibCon • • Library Publishing Forum • • London Book Fair • • Modern Language Association • • Munin Conference • • MUSE Meets conference • • Music Library Association/Theatre Library Association Joint Meeting • • National Acquisitions Group (NAG) • • NISO Plus • • North American Serials Interest Group (NASIG) • • North Carolina Serials Conference • • OA Tage/Open Access Days (Germany) • • OASPA conference • • Ontario Library Association OLA Super Conference • • Open Education Conference • • Open Science Fair • • Researcher 2 Reader (R2R) • • RLUK • • SCONUL - UK Society of College, National and University Libraries conference • • Society for Scholarly Publishing (SSP) • • The Higher Education Technology Agenda (THETA) • • UKSG • • WHEF conference 21/34 Signup Workflow Despite its seeming simplicity, the workflow for Opening the Future is relatively complex, with multiple actors involved at different times. Coordination between different parties requires care and a technological infrastructure that can handle the effort. The workflow has to handle the following core activities: • Library sign up • Payment processing • Access control management • Library cancellation Process Diagram By default we recommend enabling access before payment is received and retracting it if not received within 3 months. Library Jisc Collections, Knowledge Unlatched, LYRASIS, Press Depending on the region, Jisc Collections, LYRASIS, Knowledge Unlatched or the Press itself must process the signup. In the case of Jisc Collections, the Library may also have to complete a signup on Jisc’s system instead. If a library signs up through Jisc Collections’s independent catalogue platform, it may be necessary to duplicate the signup data into the Press’s own CRM system and OtF site. Jisc Collections, Knowledge Unlatched, LYRASIS, Press, Project MUSE, Other Platform The Press or intermediary (Jisc Collections, Knowledge Unlatched, LYRASIS) informs the platform provider (Project MUSE as example) that access should be granted to the signup institution. Send welcome email to Library. Library Process Diagram Figure 2: The workflow for a generic/recommended Opening the Future programme Figure 2: The workflow for a generic/recommended Opening the Future programme 22/34 Action Completes form on website Action Completes form on website Notes Triggers email alerts to correct contacts at the Press and intermediaries Actors Actors and Actions Actors Library Notes Triggers email alerts to correct contacts at the Press and intermediaries Delivery of Subscription Content A core part of the Opening the Future model is the delivery of subscription content to participating libraries. This requires consideration of: 23/34 • The coherence of the subscription packages. It is vital that the collections of titles exhibit both academic and commercial appeal. • The coherence of the subscription packages. It is vital that the collections of titles exhibit both academic and commercial appeal. • The conditions of access. It is important that the content is hosted on a reputable platform that delivers appropriate metadata in standards-compliant formats and that the content is indexed in order to appear in library discoverability services. Some platform providers, such as Project MUSE, can help to fulfil these crtieria. • The conditions of access. It is important that the content is hosted on a reputable platform that delivers appropriate metadata in standards-compliant formats and that the content is indexed in order to appear in library discoverability services. Some platform providers, such as Project MUSE, can help to fulfil these crtieria. j p • The licensing. See below on SERU for the easiest path towards licensing of subscription content. j • The licensing. See below on SERU for the easiest path towards licensing of subscription content. In addition, there are a number of ‘gotchas’ that must be considered for the delivery of subscription content. In addition, there are a number of ‘gotchas’ that must be considered for the delivery of content. In addition, there are a number of ‘gotchas’ that must be considered for the delivery of subscription content. Fixed Packages Should Not Change While it is tempting to believe that packages can be reconfigured at will, the ability to deliver custom packages is beholden to the restrictions of external suppliers. For instance, ensuring consistent metadata is delivered to Project MUSE means that once a package has been setup by the remote supplier, it should be fixed permanently. This also allows libraries to ensure that once they have access to a package, it is never withdrawn, unless they cancel. 8 NISO, ‘Shared E-Resource Understanding (SERU)’, 2015 <http://www.niso.org/standards-committees/seru> [accessed 4 January 2022]. 9 NISO SERU Standing Committee, ‘NISO RP-7-2012, SERU: A Shared Electronic Resource Understanding’, 2012, 11. Licensing: Use SERU Where Possible While some institutions will require specific licenses, using the Shared E-Resource Understanding (SERU) eliminates much legal wrangling and overhead around licensing while working in a good faith capacity.8 Digital Preservation and Perpetual Access Depending on the specific delivery platform, it may be appropriate to deposit books in third-party platforms, held in trust in case of future Press insolvency. ‘Perpetual access’ as promised by this programme should also mean perpetual access beyond the lifespan of a press. By using robust, third-party digital preservation systems, presses can provide a higher level of guaranteed availability to libraries. As the NISO document on SERU describes itself: As the NISO document on SERU describes itself: As the NISO document on SERU describes itself: SERU embodies a desire by publishers and libraries for a cooperative and collaborative relationship that recognizes that the provision of timely, high-quality materials and their protection is in the mutual interests of all parties. SERU offers providers (such as publishers) and acquiring institutions (such as libraries) the opportunity to save both the time and the costs associated with a negotiated and signed license agreement by agreeing to operate within a framework of shared understanding and good faith. The statements below provide a set of common understandings for providers and acquiring institutions to reference as an alternative to a formal license when conducting business. Neither the statements of understanding nor this document constitute a license agreement. Because SERU is not a license, legal terms (such as jurisdiction, warranties, and liabilities) are not used. Rather, the SERU statements describe a set of commonly agreed-upon expectations for using and providing electronic resources. While licenses are appropriate in many situations, SERU offers an alternative when both the acquiring institution and the provider are satisfied with this approach.9 24/34 In cases where a formal license is required, the Press must take its own legal advice as to the suitability of any particular clause. However, we recommend that any license provides the following clauses: In cases where a formal license is required, the Press must take its own legal advice as to the suitability of any particular clause. However, we recommend that any license provides the following clauses: • Term access for the first three years; • Perpetual access after three years’ of subscription; • An open-access, Creative Commons license for front-list open-access content released under the scheme. • An open-access, Creative Commons license for front-list open-access content released under the scheme. Selection Procedures and Timescale It is imperative that the selection process for open-access books be transparent and trustworthy. Libraries fear that publishers will select books for the open-access route that they believe will not sell, thereby implicitly devaluing open access. We recommend that Opening the Future presses adopt a strict chronological hierarchy for the selection of the next open-access monograph. In other words, at the moment when sufficient funds have accumulated to make another book open access, the book that is next scheduled for publication in the current production schedule, but which hasn’t yet been announced as OA vs. non-OA should be the chosen volume. 25/34 Figure 3: As more members join, frontlist books can gradually be published OA Figure 3: As more members join, frontlist books can gradually be published OA If multiple books are on the same production schedule, the Press has latitude to select which book will be OA, but we would urge transparency around this process and for the Press to issue a justificatory statement of the title’s worth in such instances. If multiple books are on the same production schedule, the Press has latitude to select which book will be OA, but we would urge transparency around this process and for the Press to issue a justificatory statement of the title’s worth in such instances. All new frontlist titles therefore could be first planned as traditionally-sold ‘closed’ books and as soon as the press has accrued enough library support to fund a book, the metadata should be changed before any sales are made. The distributors should then move the book to an OA status. This should happen well before the book is published. This process, in which titles are selected prior to being sent to any third-party intermediaries, means that there is a long lead time for Opening the Future presses, before a book is made OA. However, by doing so, we completely avoid any allegations of double dipping through the provision of misleading data about title availability. Technical Infrastructures Two technical infrastructures exist, or will shortly exist, that can support the Opening the Future model. The first is the ‘signup’ system developed by the COPIM project and Birkbeck’s Centre for Technology and Publishing. The second is the Open Book Collective platform developed by COPIM. 26/34 ‘Signup’ Infrastructure For those who wish to run their own infrastructure, a signup platform is available at the Github repository of the Birkbeck Centre for Technology and Publishing. The platform is written in Django/python and is available under the GNU Affero General Public License v3.0. Features of this platform include: Features of this platform include: Features of this platform include: • CMS system • Ability to collect signup data • Access control management system • Multi-tenant hosting (more than one press per install) • News and updates system • Per user and per role access control Running the signup infrastructure requires an independent web host capable of serving wsgi applications in python. The overheads of running this system locally should be matched to the Press’s in-house technical capacity. As always, the cost of free software must be measured in the time that it takes for an in-house individual to maintain a site vs. the cost of externalising/outsourcing such provision. URL: https://github.com/BirkbeckCTP/signup Open Book Collective ‘Open Book Collective’ (OBC) is a platform and intermediary organisation that allows open-access academic publishers and infrastructure providers (‘initiatives’) to promote their publishing activities and to sell and manage their funding schemes. When launched, Open Book Collective will allow publishers to specify the data that they wish to collect and also to present Opening the Future-style initiatives on a central platform for open-access book funding models. Open Book Collective features advanced integration with the Thoth metadata system, allowing libraries to see links to their own institutions. Open Book Collective features advanced integration with the Thoth metadata system, allowing libraries to see links to their own institutions. Open Book Collective, when it launches, will be found at https://www.openbookcollective.org Open Book Collective, when it launches, will be found at https://www.openbookcollective.org 27/34 Appendix A: Jisc Collections Licensing Template See DOI 10.5281/zenodo.6907707 for a link to this appendix MS Word document. 28/34 Appendix B: Financial Modelling Spreadsheet 29/34 Appendix C: Frequently Asked Questions This section contains answers to the most frequently asked questions that we encountered in deploying Opening the Future. These FAQs can be used in response to queries about the model or simply to answer these questions if you have them yourself. The numbers in these FAQ responses pertain to the model at the Central European University Press’s implementation of Opening the Future. Why OA and Why Now? The Covid-19 pandemic has shown that open access in scholarly communications is the optimal solution in a time of severe societal restriction. It seems clear that there is an opportunity to reassess how academic books can reach the world. Even without the context of Covid-19, open access is a benefit to scholars and readers alike - the following list of positive benefits is taken from the OAPEN OA Books Toolkit: • Increased readership, usage and citation • Wider and more diverse audiences • Real-world impact and public engagement • Quicker and more lasting impact • More possibilities for readers to engage with and improve research • Greater author control • Compliance with funder mandates • Wider and more diverse audiences • Compliance with funder mandates During the first phase of Covid 19 many university and commercial publishers made their content open to the world as they rushed to assist students and researchers to gain access to content while libraries were closed. Over the months of mid-March until the end June 2020 CEU Press made 279 titles open to anyone with access to the Internet. Through the Project MUSE platform downloads exceeded 350,000 in 129 countries for books that under the closed model would have sold a couple of hundred copies in hardback. Growth in ebook usage was dramatic. What happens if a frontlist title has access to other funding as as BPC from a research grant? Our policy is to first seek funding from other sources and only if that is not available (which it is still not in most cases) would we apply the funds raised from this project to make books open. Why now when library budgets are under so much pressure? The Covid pandemic has at once exposed how vital open access is to the future of scholarly communications while also ripping the heart out of the library budgets that can make that transition possible. Opening the Future is designed to be affordable to yield excellent value per book. At an average projected cost of €16.00 per backlist title, €32.00 per frontlist title, or €10.67 per book on aggregate. Whichever way you look at it, Opening the Future provides a good return on library investment. Why would a research funder be interested in this programme and how can they get involved? Open access is of clear benefit to research funders, who can then ensure the maximum public impact of the work that they fund. Funders have, in the past, supported other consortial membership schemes in the journal space and we hope that this will translate to books as we seek a more open future. Is this program open to library consortia deals? Since success in opening all books depends on the income this will be considered but can reflect only the savings in administration and marketing costs. Please contact us to discuss: When will you make books open access? As soon as we have the revenue, the next book to be published will be OA, though with a gap of a few months to ensure the metadata that goes out about its OA status overrides any pre-publication publicity about the book. In this way we hope to avoid libraries buying books because their OA status was announced too late. ow can revenue targets be reached? And what happens if and when they are reache Revenue targets can be reached as more libraries join up. Once achieved we will reduce the membership fees, or with members’ consent, make more books open access. Can this model scale for use by other publishers without inundating libraries with lots of tiny deals? We hope that with the documented success of Opening the Future we will have a model that could lead to the widespread transition of university presses worldwide to OA. COPIM is also working on 30/34 producing something like a 'shopping mall' to facilitate support and enable library choice. Called the 'Open Book Collective' this non-profit platform will allow open-access academic publishers and infrastructure providers to promote their publishing activities, manage and sell their funding schemes to libraries. Is this like a ‘Read and Publish’ deal, but for books? No, the model is not based on the support of individual titles. If anything it’s more like a ‘Subscribe to Open’ offer. Participating libraries get unlimited access to curated selections of backlist eBooks at a much cheaper price than buying them in print one at a time. The subscription fees are then used to ‘unlock’ new OA books. It’s that simple. There are no BPCs charged, and authors at participating libraries do not get ‘preferential’ or ‘discounted’ publishing deals: OA books are chosen on merit, through the normal editorial proposal process and are peer reviewed. The cost of producing OA books is paid for by the collected library subscription fees: so the more libraries sign up, the more OA books can be published. What is the goal of this model? Nothing less than to show a route to sustainable OA for the foundational publications of the Humanities and Social Sciences. What do subscribing members get for their money? Members receive access to packages of 50 titles from CEU Press’s extensive backlist on Central and East Europe and the former Soviet Union - the history of the region dating back to the middle ages, communism and transitions to democracy. There are four packages to choose from but each 31/34 will contain titles proven by recent download figures to be popular and current according to data from Project MUSE. One package has been curated by an independent panel of subject expert library colleagues. While not a pick and mix model entirely there is sufficient choice for libraries to select what meets their collection and reader requirements best. Will member libraries/institutions have unlimited access to the books in the subscription packages? And what happens at the end of three years? Member libraries and institutions will have unlimited concurrent/simultaneous access to all titles in the package they’ve subscribed to during the term of their three year membership. They will be entitled to perpetual access to that package at the end of their three year membership. Membership is for a minimum of three years. Membership is for a minimum of three years. Member libraries and institutions will have unlimited concurrent/simultaneous access to all titles in the package they’ve subscribed to during the term of their three year membership. They will be entitled to perpetual access to that package at the end of their three year membership. You may sign up for access to a separate package at any time - this membership and package access will also be for a minimum of three years. How much do members pay per year? Library and institutional members are banded according to their size, as recognised by LYRASIS and Jisc. Based on this, the structure at Central European Press, as an example, is: • €1200 high tier, per year (approx $1,425 USD / £1,000 GBP) • €800 medium tier, per year (approx $950 USD / £700 GBP) • • €350 lower tier, per year (approx $425 USD / £300 GBP) Membership is for a minimum of three years. What will be the format of the book subscription package and what services will be available to members? The books in the subscription packages are hosted on Project MUSE in their standard DRM-free, unlimited-use model for ebooks. Content is delivered in chapter-based PDF format. Full-text searching is available across all books and within individual titles. MUSE supports authentication via IP, Shibboleth, and referring URL. Participating libraries will be able to make use of MUSE’s Library Dashboard to access MARC records and KBART files customised to their holdings, and to retrieve COUNTER 5-compliant usage statistics. MUSE collaborates with all major library discovery vendors and will ensure the packages are set up as collections to be activated in all pertinent discovery services. Books on the MUSE platform are preserved through participation in PORTICO’s E-Book Preservation service. Project MUSE is committed to the accessibility of content and complies with the Americans with Disabilities Act (ADA), in a manner consistent with the Web Accessibility Initiative Web Content Accessibility Guidelines 2.1 AA. 32/34 Will there be any DRM restrictions on the backlist? No, all backlist package titles that you are subscribed to will be DRM-free and accessible by multiple users simultaneously. How have the packages been composed? The four packages of 50 titles each have been assembled in different ways. The first covers History and is primarily made up of the most accessed titles on the Project MUSE platform. The second is Political Science, again selected by their ranking on Project MUSE. The third, selected by the editors at CEU Press, is wider in subject areas and includes gems in literature, such as the Classics list, gender studies, Roma, labour, public health, nationalities, Jewish studies, human rights and more. The fourth is a package made up of titles selected from the other three packages, by a small independent panel of librarians. If a library doesn't have a discretionary OA budget, can they still participate? You can sign up to as many packages as you like - simply fill in the form for each package you want access to, or you can always contact us if you want to discuss it first: Yes, absolutely. Funds from any budget are accepted. In fact, we are hoping that as libraries see this to be a cheaper way of building collections they will pay for this type of offer through their acquisitions budgets. What if I want access to more than one package? You can sign up to as many packages as you like - simply fill in the form for each package you want access to, or you can always contact us if you want to discuss it first. How will the OA books be made available? The new titles funded by the program to be published open access will be hosted on Project MUSE, and OAPEN and ORL. OA books will be available in PDF format with CC BY NC ND licences. Project MUSE supports open access books with MARC records, KBART files, and metadata sharing with major library vendors, to ensure that OA content is widely discoverable through library systems. The books will also be listed in DOAB. Press also sell print copies of the OA books? If so, where does the money from thes Yes. All OA titles will be available to purchase in print form. The revenue that we need to make books OA is already reduced by the amount that we hope to raise from continuing print sales. Print books can be bought through the normal channels. I already own the backlist titles/don't need access to the books but would still like to support the OA programme. Can I do this? p g Yes. We appreciate that some institutions may not wish to sign up to a book package, or may not be able to. However they might still want to support, and help to fund, the Open Access monographs that CEU Press will be publishing. For these institutions we have created an ‘OA Supporter 33/34 Membership’. It is simple and quick to join: just fill in the sign up form with the appropriate details and we’ll do the rest. No further action is required from you once we have processed the payment. Membership’. It is simple and quick to join: just fill in the sign up form with the appropriate details and we’ll do the rest. No further action is required from you once we have processed the payment. Why do we use sales agents and other organisations for outreach? Transitioning to Open Access is hard and we’re in the midst of a transition where we need to accommodate the needs of libraries with a model that is new. We understand that libraries often need to go through extensive deliberations amongst their own stakeholders to see if it is worth investing in our particular offer. As a small press we do not have the resources to talk to every librarian, so we rely on the non-profit membership organization LYRASIS in North America who are already communicating with librarians every day, and sales agents like Knowledge Unlatched elsewhere in the world. 34/34 Bibliography Crow, Raym, ‘MIT Press Open Monograph Model (Direct to Open)’ (Chain Bridge Group and the MIT Press, 2022) Jisc Collections, ‘About Jisc Collections’, Jisc, 2021 <https://www.jisc.ac.uk/jisc-collections> [accessed 3 January 2022] LYRASIS, ‘Mission and Strategic Plan’, 2021 <https://www.lyrasis.org/about/Pages/Strategic- Plan.aspx> [accessed 3 January 2022] Montgomery, Lucy, Christina Emery, Frances Pinter, and Leon Loberman, ‘Pilot Proof of Concept Progress Summary’ (Knowledge Unlatched, 2014) <http://collections.knowledgeunlatched.org/wp- content/uploads/2013/09/KU_Pilot_Progress_Summary_Report.pdf> [accessed 25 May 2014] NISO, ‘Shared E-Resource Understanding (SERU)’, 2015 <http://www.niso.org/standards- committees/seru> [accessed 4 January 2022] NISO SERU Standing Committee, ‘NISO RP-7-2012, SERU: A Shared Electronic Resource Understanding’, 2012, 11 OAPEN, ‘Online Library and Publication Platform’, 2021 <https://oapen.org/> [accessed 3 January 2022] Penier, Izabella, Martin Paul Eve, and Tom Grady, ‘Revenue Models for Open Access Monographs 2020’, Community-Led Open Publication Infrastructures for Monographs, 2020 <https://doi.org/10.5281/zenodo.4011836> Pinter, Frances, and Christopher Kenneally, ‘Publishing Pioneer Seeks Knowledge Unlatched’, 2013 <http://beyondthebookcast.com/transcripts/publishing-pioneer-seeks-knowledge- unlatched/> Pinter, Frances, and Lucy Montgomery, ‘Knowledge Unlatched: Toward an Open and Networked Future for Academic Publishing’, in Accentuate the Positive: Charleston Conference Proceedings, 2012, ed. by L. H. Hinds, K. P. Strauch, and B. R. Bernhardt (presented at the Annual Charleston Library Conference, United States of America: Purdue University Press, 2013), pp. 386–91 <http://docs.lib.purdue.edu/cgi/viewcontent.cgi?article=1387&context=charleston> [accessed 14 July 2020] Project MUSE, ‘The MUSE Story’, 2021 <http://about.muse.jhu.edu/about/story/> [accessed 3 January 2022]
https://openalex.org/W4242044471	http://www.scielo.br/pdf/mr/v18s2/1516-1439-mr-18-S02-1.pdf	English	null	Editorial	Materials research	2,015	cc-by	361	© 2015 © 2015 DOI: http://dx.doi.org/10.1590/1516-1439.18suppl2 Materials Research. 2015; 18(Suppl 2): 1 Editorial The Brazilian Conference on Materials Science and Engineering (CBECIMAT) is the most traditional meeting of the Materials Science and Engineering community in the country occurring since 1974. The Materials Societies that support this event are the Brazilian Ceramic Society (ABCeram), Brazilian Society of Metallurgy, Materials and Mining (ABM), and Brazilian Polymers Society (ABPol). For 2014, as an initiative to hold the event outside the most traditional regions with research activities in materials science, the event was held in Cuiaba (MT), organized by a group of researchers of the Universidade Estadual Paulista (UNESP), from November 9th to 13th. This was the 21th Edition of the event. ( ) The event was attended by 1025 participants, who come from 22 Brazilian states and also from other countries such as United States, Canada, Spain, England, Portugal, Chile, Uruguay, Argentina and Paraguay. In total, 1395 abstracts were submitted to be presented as posters and 95 as oral and keynotes talks, from renowned national and international experts, resulting in approximately 1500 technical and scientific presentations. The topics discussed during the congress included synthesis, processing and properties of traditional materials, as well as the development of new nanostructured materials, biomaterials, nanomedicine, interfaces and graphene. A lecture on the graduate courses in materials science and engineering in Brazil and a round table to discuss the prospects for the teaching of materials engineering were also realized. Four short courses on materials characterization techniques were offered. Finally, a meeting of the Materials Science and Engineering Nucleus (NUDCIMAT) from the Universities of the Montevideo Group Association (AUGM) was held during the conference. From the papers presented at the Conference, 128 were submitted to publication in Materials Research and after a rigorous peer review process, 49 were approved. The papers selected cover a wide range of topics in materials structure, processing, characterization and application. On behalf of the Organizing Committee, we would like to express our most sincere gratitude to all participants in the event, the scientific committee and to the support staff for their collaboration before, along and after this very successful conference. Carlos Roberto Grandini Guest Editor
https://openalex.org/W2103195519	https://academicjournals.org/journal/JABSD/article-full-text-pdf/8FC4C5F1913.pdf	English	null	Integration of social, ecological, political and technological issues into economic development programs: Key to sustainable development of human society	Journal of Agricultural Biotechnology and Sustainable Development	2,013	cc-by	14,714	Major environmental issues affecting human future and sustainability on earth decline, declining rain and depleting natural water resources, increasing soil salinity, increasing natural and man-made disasters, disposal of chemical and radioactive wastes, ocean oil spills and marine pollution, degrading coastal ecosystems, acid rains etc. The global environmental concerns are threat of global warming and climate change, stratospheric ozone depletion, deforestation and biodiversity erosion, overpopulation and resource depletion especially in poor developing countries (Sinha, 2006). Environmental problems of major concern are local, regional/national and global. The local concerns are air, water and noise pollution, declining groundwater table and increasing shortage of potable water, chemical spills and contaminated sites (land), acid sulfate soil, indoor air pollution and household hazardous wastes, urban sprawl and congestion, increasing health risks of urban residents, waste landfills and toxic emissions, solid waste (specially plastic waste) and sewage, green-land clearing and threatened species. The regional concerns are rural Integration of social, ecological, political and technological issues into economic development programs: Key to sustainable development of human society Rajiv K. Sinha1, Brijalkumar Soni1 and Binod Shankar2 1School of Engineering (Environment), Griffith University, Nathan Campus, Brisbane, QLD-4116, Australia. 2Department of Environment and Water Management, A.N. College, Patna, India. Accepted 6 February, 2013 Accepted 6 February, 2013 We need to plan for ‘Development without Destruction’ and manage our environment based on the ethical principle of social equality, economic prosperity with ecological security to achieve the goals of sustainable development. Environmental management and economic development programs should go hand in hand for achieving sustainability in global human society. Changes in the way we develop our economy, eat and drink, live and move, use and discard (as waste) the Earth’s resources, enjoy our lifestyle are needed to be changed quickly to bring us back into balance with our life support systems on Earth. We have also to overcome the ‘greed’ of having more and be ‘contented’ after our ‘needs’ are fulfilled. Greed based society can never be sustainable. The key to growing sustainably is not to produce less but to produce differently, in a way which is environmentally friendly and compatible, that is, by embracing the philosophy of ‘Cleaner Production’; not to consume less but to consume judiciously and efficiently within the regenerative capacity of the Earth ecosystems and with minimum waste generation. ogical, social, economic, ecological and political considerations, good health, social equity Key words: Technological, social, economic, ecological and political considerations, good health, social equity, eradication of poverty. Journal of Agricultural Biotechnology and Sustainable Development Vol. 5(2), pp. 12 –28, February 2013 Available online at http://www.academicjournals.org/JABSD DOI: 10.5897/JABSD10.015 ISSN 2141-2340 ©2013 Academic Journals Journal of Agricultural Biotechnology and Sustainable Development Vol. 5(2), pp. 12 –28, February 2013 Available online at http://www.academicjournals.org/JABSD DOI: 10.5897/JABSD10.015 ISSN 2141-2340 ©2013 Academic Journals Corresponding author. E-mail: Rajiv.Sinha@griffith.edu.au. Depleting forest and biodiversity: Destroying the protector of life on Earth Each year, around 17 to 18 million hectares of tropical forests rich in species diversity are cleared for timber, paper and fuel wood, and for fodder growing to export to meat producing industries in Europe and America. Every day a minimum of 3 (some put it 30) species of life is permanently disappearing from the face of Earth and at current rate of habitat destruction it can become 3 species every hour in coming years. Disappearance of 1 plant species may eventually lead to the extinction of up to 30 insect and animal species in the food chain as they depend upon plants for survival. Meat eating society is taking heavy toll on our biodiversity. The same amount of plant food that would feed 1.5 billion pure vegetarians would only feed 210 million meat eating people. Each time we take a medicine; there is one chance in two that our purchases owe its origin from wild species. A number of them are ‘life-saving’ medicine. Besides green plants are the main ‘source of life sustaining oxygen in air’ and ‘sink of greenhouse gas CO2’ on earth. Vanishing water resources on earth: Destroying the elixir of life Out of the total water on earth, only 2.5% are freshwater, 97.5% are seawater and only less than 1% is useful for *Corresponding author. E-mail: Rajiv.Sinha@griffith.edu.au. Sinha et al. 13 little more than 30 years it might no longer be able to feed its growing population. human use and consumption which are also being destroyed by human wastes and chemicals. Underground water table is rapidly falling throughout the world. Within 25 years, half of the world’s population could face hardship in finding enough freshwater for drinking, sanitation and food production. About 3 million litres of water is needed to produce 1 ha of corn; about 12 to 20 million litres to produce 1 ha of rice; about 250 L to produce 1 kg of wheat; about 25,000 L to produce 1 kg of meat; about 50 L to produce 1 L of whisky; about 250 L to produce 1 kg of steel and about 18,000 L to refine 1 ton of petroleum. About 45 industrial chemicals have been found in surface water. They are ‘Endocrine Distruptors’ in very low-parts-per billion. The cost of providing safe drinking water and sanitation to everyone in the world by 2025 will be US $ 180 billion per year. Piling toxic chemicals on earth: The demons of development and inducer of deadly human diseases Worldwide about 80 to 100,000 chemicals are in use every day in our industries and other developmental activities and some 1000 new chemicals are added each year. There are some 861 neuro-toxic chemicals in use in our cosmetics, perfumes and toiletries. The deadly chemicals DDT and PCBs have reached even up to the poles. A relationship between ‘chemicals and cancer’ is scientifically proved and women are more vulnerable. According to UNEP and WHO, some 25 million farmers and agricultural workers are poisoned by chemical pesticides every year and nearly 3 million people suffer from ‘acute pesticide poisoning’ and some 10 to 20 thousand people die every year from it in both the developed and the developing countries. Studies indicate that there is significant amount of ‘residual pesticides’ contaminating our food stuff long after they are taken away from farms for human consumption. Vegetable samples were contaminated 100% with HCH and 50% with DDT. US scientists predict that up to 20,000 Americans may die of cancer, each year, due to the low levels of ‘residual pesticides’ in the chemically grown food (UNEP Report, 1992). Exposures to chemical pesticides are linked with serious diseases and developmental dis- orders like ‘nervous system disorders’, ‘immune system suppression’, breast and other cancers’ ‘reproductive damages’, ‘impairment of brain development in children’ Chemicals in the breathing air: Destroying the source of life on earth Several millions tones of gases, carbon monoxide (about 106 million tonnes), carbon dioxide (4550 mt), sulfur dioxide (90 mt), oxides of nitrogen (60 mt), methane (84 mt), hydrocarbons (47 mt), CFC and halons (1 mt) and suspended particulate matter (30 mt) enter into our atmosphere every year from our industries and automobiles. Diesel vehicles emit over 100 micro- particles several of which are carcinogenic. Some 189 hazardous air pollutants (HAPs) have been identified in the atmosphere. UNEP estimates that over 3 million people in world die every year from air pollution related diseases and the non-cigarette smoker lung cancer rate due to outdoor air pollution in world today is estimated to about 2000 cases per year. According to WHO, coal power plants in the U.S. cuts short nearly 24,000 lives, including 2,800 from lung cancer and nearly 38,200 from heart attacks each year. VULNERABILITY OF THE HUMAN SOCIETY ON EARTH – HOW SUSTAINABLE IS OUR FUTURE? N-nitrosodimethylamine (NDMA), a principal ingredient in rocket fuel and methyl tertiary butyl ether (MTBE), a gasoline additive were discovered in surface and ground water sources; thousands of kilometers away from where they were used. DDT and PCBs were found on poles, thousands of kilometers away from where they were produced; radioactive isotopes strontium-90 (Sr90) and cesium-137 (Cs137) which are routine emissions from all nuclear power plants were found in the bone marrow of children in India far away from the sources of their emissions. All these chemical and radioactive contaminants followed the air routes to reach those distant destinations. From the atmosphere they precipitated down to Earth and entered into the human ecosystem. This shows how vulnerable we have become in the wake of material development and also pose a major question on how much sustainable is our future on Earth (Meadows et al., 1992). Depleting stratospheric ozone shield: Exposing mankind to radiation hazards It can cause non-melanoma skin cancers and blindness from juvenile cataracts worldwide; g g p Coal power plant gives out about 800 to 1000 g of CO2 for every unit (kWh) of electricity produced. In one year, an average car emits 4.3 tons of CO2. Agriculture has also been responsible for huge emissions of greenhouse gases and induction of global warming. Of the increase of atmospheric carbon over the last 150 years, about a third (33.3%) is thought to have come from agriculture. Over the last century, the world has warmed between 0.3 and 0.6°C. If emission of all the GHG are not arrested the world will on average be 1.3°C warmer by 2020. Global warming disturb rainfall patterns, ‘suppress human immune system’ and induce natural disasters. Mounting human wastes on earth: Poisoning the soil, air and water MSW is mounting on earth everywhere. In U.S., each sunset sees a new mountain of nearly 410,000 tons of garbage. The European Union throws away an estimated 2 billion tones of MSW each year. The character and composition of MSW are changing in modern techno- logical society with the non-biodegradable ‘synthetic material’ coming into existence. Biomedical waste constitute considerable portion of MSW, 10% is ‘infectious’ and 5% is non-infectious but potentially toxic and radioactive. Several toxic chemicals are used in modern homes, resulting into generation of household hazardous waste, which are mixed and disposed with the MSW ending up in landfills with serious consequences for environment and society. The ‘electronic waste’ has become serious problem. It is like ‘tsunami’ of ‘e-waste’ in world. It is virtually a chemical waste mixed with general MSW ending up in landfills. Waste landfills emit more powerful greenhouse gases (Methane and nitrous oxides) and toxic trace gases like ‘xylene’ and ‘toluene’ and toxic leachate that can contaminate groundwater. In 2005 waste landfills in Australia emitted some 17 million tons CO2-e (equivalent) of greenhouse gas equivalent to the and ‘disruption of hormonal systems’ and ‘disruption of hormonal systems’ emissions from 4 million cars. Every 1 kg of solid waste diverted from landfills prevents 1 kg of greenhouse gas emission equivalent to CO2. Depleting stratospheric ozone shield: Exposing mankind to radiation hazards CO2, the major greenhouse gas inducing global warming was 280 ppm in the pre-industrial times and now stands at 370 ppm, 30% higher. It is currently rising by 0.5% every year. Powerful greenhouse gases e.g. methane and nitrous oxide are emitted from solid waste management programs by composting or more from waste landfills. Molecule to molecule, CH4 is 20 to 25 times more powerful and N2O is 296 to 310 times more powerful GHG than carbon dioxide (CO2). Every 24 h, human activity on earth generates some 16 million tons of greenhouse gas CO2 and release into the atmosphere. Coal power plant gives out about 800 to 1000 g of CO2 for every unit (kWh) of electricity produced. In one year, an average car emits 4.3 tons of CO2. Agriculture has also been responsible for huge emissions of greenhouse gases and induction of global warming. Of the increase of atmospheric carbon over the last 150 years, about a third (33.3%) is thought to have come from agriculture. Over the last century, the world has warmed between 0.3 and 0.6°C. If emission of all the GHG are not arrested the world will on average be 1.3°C warmer by 2020. Global warming disturb rainfall patterns, ‘suppress human immune system’ and induce natural disasters. CO2, the major greenhouse gas inducing global warming was 280 ppm in the pre-industrial times and now stands at 370 ppm, 30% higher. It is currently rising by 0.5% every year. Powerful greenhouse gases e.g. methane and nitrous oxide are emitted from solid waste management programs by composting or more from waste landfills. Molecule to molecule, CH4 is 20 to 25 times more powerful and N2O is 296 to 310 times more powerful GHG than carbon dioxide (CO2). Every 24 h, human activity on earth generates some 16 million tons of greenhouse gas CO2 and release into the atmosphere. Hole in the ozone shield in the stratosphere over the Antarctica is growing larger and deeper, exposing man and other species to increased levels of harmful UB-V radiation. Some 66,000 tones of ozone depleting chemicals are released into the environment every year due to human activity. Increased UB-V radiation can cause suppression of human immune system making people vulnerable to infectious diseases. Degrading and eroding soil mantle: Destroying the food base on earth Billions of tones of top soil (the fertile layer) are eroded away every year, being washed away into the sea or carried away with wind converting into wasteland. Every year, nearly 27 million hectares or say, 47 ha min-1, of valuable fertile land are lost by desertification. Every minute a chunk of ‘fertile agricultural land’ of the size of tennis court is being converted into wasteland. Over the last two decades, the world has lost some 500 million metric tons of topsoil. Only the ‘top thin layer’ of the soil with humus is vital to grow food and is sustaining the entire civilization. Formation of just 1 cm of soil from parent rock take from 100 to 1,000 years and 2.5 cm of topsoil can take from 100 to 2,500 years. FAO has warned that the world's soil was degrading so fast that in J. Agric. Biotech. Sustain. Dev. 14 3rd World Summit on Sustainable Development (WSSD), Johannesburg, S.A., 2002 3rd World Summit on Sustainable Development (WSSD), Johannesburg, S.A., 2002 3. A system of socio-economic development which can provide good quality of life to all the people (rich and poor, men and women, adults and children) born on Earth, while protecting their basic life-support systems (air, water, soil, flora and fauna) and also safely disposing all the wastes (domestic, commercial and industrial) generated by them (Label et al., 1987, Goodland and Serafy, 1991). The WSSD (2002) was held at Johannesburg, South Africa to take stock of the situation about the achievements made towards attaining sustainability in the field of human development during the last 10 years since the 2nd Earth Summit in 1992. The task at Johannesburg was to recapture the momentum of the movement for sustainability begun at Stockholm (1972) and Rio (1992). ( ) In the 10 years between Rio (1992) and Johannesburg (2002), critical environmental problems such as greenhouse gas emission (global warming), loss of old growth and primary forests (biodiversity erosion), ocean plunder and waste of resources (piling waste), lurched from bad to worse. On the socio-economic front, the rich and the powerful got richer and more powerful, while the poor became poorer and weaker. The concept of ‘sustainable development’ and the much lauded movement for sustainability remained on paper. First UN Conference on Human Environment, Stockholm, Sweden, 1972 The first ever World Conference on Human Environment was held in Stockholm in June 1972 where representatives from more than 70 countries participated and pledged to save the environment. This may be regarded as the ‘1st Earth Summit’. The Conference was chaired by Mrs. Gro Harlem Bruntdland the then Prime Minister of Norway who was later entrusted with the task of forming the ‘World Commission on Environment and Development’ (WCED) in 1987 where she conceived the Sinha et al. Sinha et al. 15 term ‘sustainable development’. term ‘sustainable development’. confronted with a perpetuation of disparities between and within nations, a worsening of poverty, ill health and illiteracy, and the continuing deterioration of natural ecosystems on which we depend for our well-being”. However, integration of environment and development concerns and greater attention to them will lead to the fulfillment of basic needs, improved living standards for all, better protected and managed ecosystems and a safer more prosperous future. No nation can achieve this on its own but together we can in a global partnership for sustainable development’. At Stockholm, the world leaders declared – “Man is both creature and moulder of his environment, which gives him physical sustenance and affords him the opportunity for intellectual, moral, social and spiritual growth. Both aspects of man’s environment, the natural and the man-made, are essential to his well-being and to the enjoyment of basic human rights, the right to life itself”. Bruntdland commission report on sustainable development (1987) p Agenda 21 cautioned that the growing population and poverty in the developing countries and the unsustainable pattern of consumption in the developed industrialized countries would thwart the spirit and pace of sustainable development throughout the world. For ‘environmental sustainability’, ‘socio-economic prosperity’ and ‘environmental literacy’ of the global human society, and ‘political stability’ throughout the world should be a pre- condition. Poor and the deprived people, and the politically discriminated would violate all economic and ecological imperatives in order to survive (because survival is more important than sustainability) thus leading to unsustainability. Highly educated can be ‘environmentally illiterate’ and fail to realize the importance of sustainability. The report of the ‘World Commission on Environment and Development’ (WCED) was published as ‘Our Common Future’ (WCED, 1987). It redefined the concept of development which should encompass three components: 1. A systems of socio-economic development to meet the ‘needs’ (but not the ‘greed’) of the present generation without compromising with the abilities of the future generations to meet their own needs. 2. A system of stable socio-economic and ecological development that should improve the total quality of all life (human beings, plants and animals) on Earth now and in the future too, while maintaining the social and ecological integrity (natural and man-made ecosystems) of the earth upon which all life depends. 1. Two billion people currently do not have access to modern energy services; 2. About than 1.1 billion people globally do not have access to safe drinking water. It is predicted that water 1. Two billion people currently do not have access to modern energy services; UN Conference on Environment and Development, Rio de Janeiro, Brazil, 1992 The UN Conference on Environment and Development was held at Rio de Janeiro, Brazil, in 1992. This was the ‘2nd Earth Summit’ organized 20 years after Stockholm and was participated by 172 countries (more than twice that participated at Stockholm). ‘Environment and Sustainability’ and ‘Sustainable Development’ was the focus of discussion. The world leaders declared – “Human beings are the center of concerns for sustainable development. They are entitled to a healthy and productive life in harmony with nature”. t These are some of the disheartening episodes of the efforts made towards achieving global human sustainability: 1. Two billion people currently do not have access to modern energy services; The Agenda 21 (agenda for the 21st century) was adopted at UNCED. It was the blueprint for global sustainable development. Agenda 21, pronounced that “Humanity stands at defining moment in history. We are gy 2. About than 1.1 billion people globally do not have access to safe drinking water. It is predicted that water 16 J. Agric. Biotech. Sustain. Dev. supply will be the major constraints on sustainable development in the 21st century. sustainability built up since the Rio Earth Summit in 1992. Market and Opinion International (MORI) polled in 30 countries containing two third of world’s population and found that 91% people in Greece, above 60% in the U.S., Germany, Russia, France, Italy, Spain, Japan, Mexico, Brazil, Chile, the Republic of Korea and China, and about 54% in India wanted legal protection for the environment through legislative actions. Some 69% people in U.K. agreed that environmental degradation especially waste and pollution is affecting their daily life. 3. About 2.4 billion people lack adequate and improved sanitation; 4. More than 1.2 billion people still live on less than 1 dollar (IR 40.0) a day, that is, much below the poverty line; 5. More than 3 million people die of air pollution, and the same number dies from diseases caused by unsafe and polluted water. The target of ‘Health for All by the Year 2000’ never became a reality. p g y Opinion poll by Roper Worldwide found that 39% people in these 30 countries were of the view that environmental protection and sustainable development was of paramount importance today. The percentage of people expressing this view was significantly higher in India, Argentina, the Philippines and Germany. Gallup Polls made in the U.S. UN Conference on Environment and Development, Rio de Janeiro, Brazil, 1992 Around 2 billion hectares of soil, 15% of Earth’s land is now classed as degraded (WHO, 1997; UNDP, 2004; Steffen et al., 2004). Report of the Global Environmental Outlook (GEO)-3 on sustainability The planet is at ‘critical crossroads’ concluded the UNEP’s ‘Global Environmental Outlook 3’ report (GEO- 3). The study, collaboration between UNEP and some 1,000 individuals and 40 institutions around the world is the most authoritative assessment to date of where we (the Homo sapiens) have been, where we have reached, and where we are likely to go. GEO-3 shows how far the world has gone on the road to sustainable development since the Rio Earth Summit 1992. It said that though the world has made great strides in placing environment on the global developmental agenda, the twin evils of ‘poverty’ (in the developing world) and the ‘excessive consumption’ (in the developed world) due to too much of ‘prosperity’ continue to put enormous pressure upon it. Dire poverty and environmental degradation feed on each other. Ironically, growing poverty all over world is the main stumbling block on the road to environmental protection and sustainable development (UNEP-GEO, 2004-05). Opinion polls clearly show that people worldwide realize the importance of environmental protection and sustainable development for their existence and put the blame mainly on the corporate sectors specially the transnational corporations (TNCs) for all the environmental evils. THE STRATEGIES DEVELOPMENT Planning for sustainable development is like ‘preventing’ a disease to occur in the first place. As prevention reduces the chances of individual suffering from the disease, so does, environmental planning and management for the whole society. It would significantly reduce, if not completely prevent, the risk of environmental damage and the sufferings of the society caused due to it. At least, it will prevent any major ‘environmental disaster’ which struck the human society in the past (Sinha, 1994). UN Conference on Environment and Development, Rio de Janeiro, Brazil, 1992 found that 57% Americans were more concerned about pollution of rivers, lakes and streams while 29% cited fears about global warming. (Yet the U.S. has not signed the Kyoto Protocol). In another poll, 65% Americans saw erosion of biodiversity as the most serious problem facing the humanity. The MORI Millennium Poll wanted to know from people which section of the society was mainly responsible for the environmental degradation and the responsibility for its repair and restoration. Almost 6 in every 10 asserted that the ‘corporate sectors’ were mainly damaging the environment to make profit even at the cost of environment, and hence protection of environment was their moral responsibility. Indians also said that the company’s and industries must bear responsibility of environmental repair. But in the U.S., a majority of people asserted that environmental protection was government’s core responsibility and 90% of them wanted information on companies environmental and social records. 49% of the Europeans surveyed said that they would NOT buy a product made in a country with poor environmental and social standards (UNDP, 2003, 2004). 6. New highly toxic chemicals’ e.g. N- nitrosodimethylamine (a principal ingredient in rocket fuel), methyl tertiary butyl ether (a gasoline additive), and ‘acrylonitrile’ (used in textile industry) have appeared in surface and ground water; g 7. Half of the tropical rainforests and mangroves (treasure of pristine biodiversity) have already been lost; 8. About 75% of the marine fisheries have been fished to capacity; g 7. Half of the tropical rainforests and mangroves (treasure of pristine biodiversity) have already been lost; 8. About 75% of the marine fisheries have been fished to capacity; p y 9. In September 2000, the ozone hole over Antarctica covered more than 28 million square kilometers; 10. Some 75 billion tones of ‘top soil’ are eroding every year. Around 2 billion hectares of soil, 15% of Earth’s land is now classed as degraded (WHO, 1997; UNDP, 2004; Steffen et al., 2004). 9. In September 2000, the ozone hole over Antarctica covered more than 28 million square kilometers; 10. Some 75 billion tones of ‘top soil’ are eroding every year. Around 2 billion hectares of soil, 15% of Earth’s land is now classed as degraded (WHO, 1997; UNDP, 2004; Steffen et al., 2004). 10. Some 75 billion tones of ‘top soil’ are eroding every year. Seven pillars of sustainable development Science and technology came as a ‘mixed blessing’ for mankind. It brought peace and prosperity, comfort and facility, health and wealth for mankind through rapid utilization of the natural resources. It provided all the basic amenities of life and fulfilled all the human aspirations – copious amount of energy to drive all developmental activities and provides comforts of life, a good and secured dwellings to live and durable clothes to wear, a rapid means of transport and quick means of communication, nutritious foods to eat, clean water to drink, prompt healthcare and sanitation facilities, a system of weather forecast and advance warning system for coping with natural disasters and genetic engineering for improving the economic value of useful plants and animal species. The new paradigm of development is not the game of economics alone. All issues- economic, ecological, social (health), cultural (educational), legal (legislative), political and technological have to be merged into a collective decision making for sustainable development. They are mutually reinforcing. Technology plays greater role as they drive development (Axinn, 1996). y p ( ) Earlier economy was at the center of decision-making. This assumed that all environmental problems could be solved if economy was sound. This has now become an obsolete theory. Now ecology has to be at the center. We have to integrate ecological thinking into social and economic planning. The developmental activities have to be ‘economically viable’, ‘ecologically compatible’, ‘socially equitable’, ‘culturally acceptable’ and ‘politically justifiable’. Then only it can be environmentally sustainable. This will require giving up the ‘culture of consumerism’, ‘producing fewer consumers that the Earth can sustain and also achieving ‘equity in resource use and consumption’ across the world. Over-consumerism and too much of prosperity in one part of the world, or overpopulation and poverty in other part, would thwart the spirit of sustainable development. When used judiciously and legitimately for human development, science saved life and property, but when ‘misused’, it spelt disaster. Technology practically transformed the ‘traditional human society’ which was more vulnerable to the vagaries of nature, more dependent for survival on nature, less adapted to survive against odds, to ‘modern human society’ which is less vulnerable to the vagaries of nature, more fit to survive in difficult situations, less dependent on nature for survival and more competent to manage their affairs. p g Technology is the backbone and driving force of sustainable development. People’s mandate on sustainable development Several opinion polls were conducted on the eve of Johannesburg Summit (WSSD, 2002) by international agencies to know the views of people across world and assess their awareness on the question of environmental Sinha et al. 17 Sustainable development will deliver social, economic and ecological benefits, for it will conserve resources, reduce waste, prevent loss of energy and protect human health. The World Business Council for Sustainable Development (WBCSD) was formed in 1995. It is a coalition of 120 international companies that have a shared commitment to protect the environment through cleaner production and waste minimization in industries (Schmidheiny et al., 1997). 4. The social integrity of earth by maintaining an acceptable level of non-consumerist population (secondary consumers) within the ecological limits and the ‘carrying capacity’ of earth (IUCN, WWF, UNEP, 1991). Seven pillars of sustainable development However, unfortunately some of the technologies of the early 20th century proved to be a ‘curse in disguise’. They triggered the pace of economic development by indiscriminately using the scarce resources, and generated huge amount of waste and pollution undermining the very resource base upon which development was to be made. Towards the end of the last century when human environment specially the life- support systems air, water, soil and the natural ecosystems started showing signs of degradation that the scientific community realized that something wrong was going with the existing technologies used in the development process. This necessitated the development of more ‘appropriate technologies’ for human deve- lopment which has been termed as ‘green technologies’ or ‘sustainable technologies’ (Adams, 2001). The most important sustainability issue today is to save and protect the very life-support systems (air, water, food and soil) and the biodiversity, protect human health, arrest global warming and depletion of stratospheric ozone shield, arrest desertification, augment clean energy and material resources to continue and maintain the pace of development, and to safely dispose all the wastes generated by the society. What we need to sustain Hybrid-vehicles are also quiet and much less polluting. Hydrogen fuel which is 4.5 times powerful than petrol with ‘zero emission’ (only water vapor) makes a big promise for a sustainable future. U.S., Germany and China are ahead. BMW, Germany has several fleets operated on hydrogen. Iceland also has buses running on hydrogen fuel. 7. Natural gas, micro-hydel, solar, wind, tidal and geothermal power projects for electricity generation has also come up successfully in world and significantly reduced pollution and greenhouse gas emission. More efficient WIND TURBINES has been developed and they perform very high along the seashore of world. Denmark is generating over 90% of its electricity need through seashore wind turbines. The ‘solar photo voltaic cell technology’ has brought a revolution and a new era in economic ‘lighting and heating’ devices and reduction in greenhouse gas (CO2). PV system is now used on large scales in domestic, industrial and commercial sectors especially in both urban and rural areas. China is leading in solar power generation. Australia is also coming up fast in a revolutionary way supported by Govt. 8. Development of technologies for recycling of metals, glasses, papers and cardboards, and tough plastics to get back those materials for reuse in society. It avoids mining of ores of metals and the associated environmental hazards, protects trees from cutting to make ‘pulp’ and saves huge amount of freshwater and energy in their production from virgin materials while also reducing emissions of greenhouse gases. Production of plastics from ‘synthetic organic chemicals’ is also hazardous process. The ‘solar photo voltaic cell technology’ has brought a revolution and a new era in economic ‘lighting and heating’ devices and reduction in greenhouse gas (CO2). PV system is now used on large scales in domestic, industrial and commercial sectors especially in both urban and rural areas. China is leading in solar power generation. Australia is also coming up fast in a revolutionary way supported by Govt. 4. Development of more energy efficient electrical appliances and instruments such as automatic lighting control system (ALCS), compact fluorescent light bulbs, low voltage tungsten halogen and sodium lamps etc. They save electricity from 70 to 80%. Modern ACs, fridges, washing machines, dishwashers use 75% less energy than older models. Reverse cycle AC (for cooling and heating) use 67% less electricity, rheem pump hot water system (using air to heat water) use only 60% energy. What we need to sustain 1. The ecosystems and the accompanying biodiversity with the variety of species and their habitats; 2. The natural capital of earth e.g. the stock of productive soils, forest and the fresh water and all the non- renewable resources by preventing their unsustainable use; 1. The ecosystems and the accompanying biodiversity with the variety of species and their habitats; 2. The natural capital of earth e.g. the stock of productive soils, forest and the fresh water and all the non- renewable resources by preventing their unsustainable use; g Technological developments (environmental technologies) in the past decades and especially after the1980s have delivered spectacular improvements in the environmental quality, cleaner air and water, low 3. The ecological integrity of earth and the resilience of natural life-support systems by maintaining an adequate number of primary producers; J. Agric. Biotech. Sustain. Dev. 18 combustion on coal beds and reaction with hydrogen is a brilliant new idea to utilize the huge coal reserves of world. It is like ‘using coal without mining’. Australia is pioneering in the technology. It also utilizes 90% of coal as compared to hazardous conventional mining which only utilize 60%. This has led to significant reduction in the emission of greenhouse gas carbon dioxide (CO2) and other toxic pollutants. Methane is piped out and supplied to power plants for electricity generation. emission automobiles, less toxic and hazardous wastes, water and energy efficient homes and appliances etc. Some of the remarkable technological achievements of the 20th and 21st century marching towards sustainability are: 1. A significant reduction in the air borne lead (Pb) has been achieved by developing ‘Lead Free Petrol’ and use of Natural Gas and Ethanol as auto-fuel. 2. A considerable reduction in the emission of CO, HC and NOx by modification in the IC engines, by installation of ‘catalytic converters’, and introduction of direct fuel injection and ‘lean-burn combustion technologies’. The ‘flue-gas desulfurization technology’ in conventional coal power plants has also led to significant reduction in sulfur dioxide (SO2) emission which causes acid rains. More efficient air pollution control equipment and devices such as ‘electrostatic precipitators’, ‘bag filters’ have also been developed for conventional coal power plants. 3. More fuel efficient, quieter and less polluting automobiles driven on environmentally benign auto-fuels like compressed natural gas (CNG), liquidified petroleum gas (LPG) and ethanol, less body and wheel weight and aerodynamic bodies to induce less resistance to wind. What we need to sustain Electric stove with fan forced oven are 30% more efficient than conventional electric stove. Gas stoves are much more energy efficient and microwaves uses 55% less electricity. Compact fluorescent lamps use 70 to 80% less electricity for same light with 8 times longer life and prevent emission of nearly a ton of GHG in its lifetime (Energy efficient appliances and instruments have reduced the emission of greenhouse gas carbon dioxide (CO2) in the same proportion). 8. Development of technologies for recycling of metals, glasses, papers and cardboards, and tough plastics to get back those materials for reuse in society. It avoids mining of ores of metals and the associated environmental hazards, protects trees from cutting to make ‘pulp’ and saves huge amount of freshwater and energy in their production from virgin materials while also reducing emissions of greenhouse gases. Production of plastics from ‘synthetic organic chemicals’ is also hazardous process. 9. Partial plugging of the ‘ozone hole’ in the stratosphere has been achieved by developing lesser evil substitute hydrochloroflurocarbon (HCFC) which has 30% less ODP than CFC, and large part of it is destroyed in the atmosphere before reaching the stratosphere. ( ) p p ) 5. Development of eco-efficient water appliance. A significant reduction in the waste of potable water has been achieved by developing ‘automatic closing taps’, ‘water efficient showers’ and ‘dual-flushing cisterns’ in the bathrooms and toilets and development of ‘sprinkler and drip irrigation’ systems in agriculture. New bathing shower uses 9 to 16 L of water per minute as compared to 20 to 30 L used by inefficient showers. The dual-flush cistern saves 6 L of water each time in urinal flushing. New cloth washing machines and dishwashers are very efficient in water use. Drip and sprinkler systems saves40 to 60% of fresh water in farming. p g p 10. In the polystyrene foam manufacturing industry ‘carbon dioxide’ is being used as the ‘blowing agent’ replacing CFC. The new technology has eliminated 3.5 million pounds of the dangerous ozone depleting and global warming chemical chlorofluorocarbon (CFC) every year. y 11. In the paper pulp industry an environmentally benign oxidant ‘hydrogen peroxide’ is now being used as the bleaching agent replacing the dangerous ‘chlorine’. g 6. Development of ‘clean coal technology’ by generation of clean coal seam gas (methane) through underground 6. What we need to sustain ‘Sodium silicate’ is now used as an environmentally benign alternative to the phosphorus-containing additives in washing powder. 21. Eco-designing of products to reduce material use and improve efficiency. Ferrocement technology’ was developed in India. It use much less cement and steel, has higher degree of toughness, durability, ductibility, tensile strength and crack resistance in less than 25 mm of thickness, which is found in 100 mm thick wall of conventional RCC Technology. 14. Hybridization technology and ‘agri-biotechnology’ (ABT) has produced high yielding varieties (HYVs) of crops (miracle maize, miracle rice and miracle wheat) which tripled and even quadrupled food production on the same available land area thus increasing agricultural sustainability. 14. Hybridization technology and ‘agri-biotechnology’ (ABT) has produced high yielding varieties (HYVs) of crops (miracle maize, miracle rice and miracle wheat) which tripled and even quadrupled food production on the same available land area thus increasing agricultural sustainability. gy 22. Development of sustainable environmental biotechnologies for municipal and industrial waste treatment, waste reduction, reuse and recycling (converting waste into resource), diverting huge volumes of solid wastes from going to the landfills every year and saving land, cost of transport and landfill construction, and emission of greenhouse gas methane (CH4) from landfills. The most significant development is the global revival of ‘vermiculture biotechnology’ with scientific use of some versatile waste eater and chemically tolerant earthworms species like Eisenia fetida, Perionyx excavatus and Eudrillus euginae whom the great visionary scientist Sir Charles Darwin called as ‘unheralded soldiers of mankind and friends of farmers’. They are ‘protective’, ‘productive’, ‘disinfecting’, ‘detox- ifying’ and ‘neutralizing’ (Sinha and Greenway, 2004). 15. A new environmentally friendly technology called ‘catalytic dehydrogenation of diethanolamine’ has been developed in herbicide producing industries that avoids the use of toxic chemicals cyanide and formaldehyde. 16. Biological control of pests and diseases in agriculture has become more effective after scientific improvement of the herbal pesticides like ‘azadirachtin’ and ‘pyrethrin’, and development of ‘transgenic crops’. Use of earthworms vermicompost also repels and suppresses ‘pests’ and ‘diseases’ by over 80% in crops due to the presence of ‘chitin’ and cellulose degraders microbes. The fermented solution of vermi-compost called ‘vermicompost tea’ and the liquid filtered through the body of earthworms called ‘vermiwash’ is an effective biopesticides. 17. Development of biodegradable ‘agro-plastic’. It carries great significance for the environment and society. It is the cheapest and most convenient material ever known to mankind. What we need to sustain Development of ‘clean coal technology’ by generation of clean coal seam gas (methane) through underground non-ionic and 2. An environmentally benign non-ionic and Sinha et al. Sinha et al. Sinha et al. 19 materials with superior technical properties; ultra strong and light weight. Automobile and aircraft industries are using carbon fibres instead of steel to reduce weight and improve fuel efficiency. Use of steel is reduced by 25%. 21. Eco-designing of products to reduce material use and improve efficiency. Ferrocement technology’ was developed in India. It use much less cement and steel, has higher degree of toughness, durability, ductibility, tensile strength and crack resistance in less than 25 mm of thickness, which is found in 100 mm thick wall of conventional RCC Technology. 22. Development of sustainable environmental biotechnologies for municipal and industrial waste treatment, waste reduction, reuse and recycling (converting waste into resource), diverting huge volumes of solid wastes from going to the landfills every year and saving land, cost of transport and landfill construction, and emission of greenhouse gas methane (CH4) from landfills. The most significant development is the global revival of ‘vermiculture biotechnology’ with scientific use of some versatile waste eater and chemically tolerant earthworms species like Eisenia fetida, Perionyx excavatus and Eudrillus euginae whom the great visionary scientist Sir Charles Darwin called as ‘unheralded soldiers of mankind and friends of farmers’. They are ‘protective’, ‘productive’, ‘disinfecting’, ‘detox- ifying’ and ‘neutralizing’ (Sinha and Greenway, 2004). materials with superior technical properties; ultra strong and light weight. Automobile and aircraft industries are using carbon fibres instead of steel to reduce weight and improve fuel efficiency. Use of steel is reduced by 25%. 21. Eco-designing of products to reduce material use and improve efficiency. Ferrocement technology’ was developed in India. It use much less cement and steel, has higher degree of toughness, durability, ductibility, tensile strength and crack resistance in less than 25 mm of thickness, which is found in 100 mm thick wall of conventional RCC Technology. materials with superior technical properties; ultra strong and light weight. Automobile and aircraft industries are using carbon fibres instead of steel to reduce weight and improve fuel efficiency. Use of steel is reduced by 25%. biodegradable surfactants called ‘alkyl glycoside’ made from saccharide has been developed to replace the non- biodegradable anionic ‘alkylaryl sulphonate’ used in shampoos. p 13. The sustainable vermiculture biotechnology All the end-products (detoxified and disinfected nutritive water, vermicomposted sludge and huge earthworm biomass) are useful in agriculture. materials. All species earthworms are potential source of ‘biological raw materials’ for production of useful ‘biodegradable’ industrial products in rubber, lubricant, soaps, detergent and cosmetics industries and also as valuable source of ‘proteins’ for production of ‘nutritive feeds’ for promoting allied ‘food industries’ like fishery, dairy, poultry and piggery for meat and milk production. and huge earthworm biomass) are useful in agriculture. (3) The vermi-remediation technology for land remediation by removing chemical contaminants from soils and reducing soil salinity while also improving the total physical, chemical and biological properties of soil and its nutritive value. Earthworms have been found to bio-accumulate heavy metals, pesticides and lipophilic organic micro-pollutants like the polycyclic aromatic hydrocarbons (PAH) from the soil. Significantly, vermiremediation leads to total improvement in the quality of soil and land where the worms inhabit and make them highly productive. They swallow large amount of soil every day, grind them in their gizzard and digest them in their intestine with aid of enzymes. Only 5 to 10% of the digested and ingested material is absorbed into the body and the rest is excreted out in soil in the form of fine mucus coated granular aggregates called ‘vermicastings’ which are rich in NKP (nitrates, phosphates and potash), micronutrients and beneficial soil microbes including the ‘nitrogen fixers’, ‘phosphate solubilizers’ and ‘mycorrhizal fungus’. Hence the polluted land is not only ‘cleaned-up’ but also ‘improved in quality and fertility’. The soil becomes lighter and porous biochemically active and the productivity is increased to several times. During the vermi-remediation process of soil, the population of earthworms increases significantly benefiting the soil in several ways. A ‘wasteland’ is transformed into ‘wonderland’. We have successfully experimented on vermi- composting, vermi-filtration, vermi-remediation and vermi- agro-production technologies at Griffith University, Australia with excellent results. They are proving to be most cost-effective and cheap environmental biotech- nologies for ‘environmental management and sustainable development’. Huge earthworm biomass which is finding new uses in industries and agriculture comes as byproduct in all above technologies. They all have significant contribution towards ‘sustainable agriculture’. Vermifiltration of wastewater was our innovative studies done in 2005 and the technology has now been commercialized in India by Transchem Agritech in Gujarat. The sustainable vermiculture biotechnology They are treating 400 kl of sewage everyday and the treated water is being supplied to farmers for use in agriculture saving huge amount of potable water. Nearly 80% water is used in global agriculture. Several Vermi- filtration plants are also operating in Chile, Mexico and Venezuela for treatment of municipal and industrial wastewater (This was based on the works done by Prof. Toha and Dr. Soto of University of Chile).(masoto@cec.uchile.cl). y University of (4) The vermi-agro-production technology for restoring and improving soil fertility and significantly boosting crop productivity. Vermicompost is a highly nutritive ‘miracle growth promoter’. Growths and yield of crop plants are enhanced by 30 to 40% higher over chemical fertilizers by worms and its vermicast. Studies at CSIRO Australia found that the earthworms and vermicompost can increase growth of wheat crops by 39%, grain yield by 35%, lift protein value of the grain by 12% and fight crop diseases. Vermicompost also increases ‘biological resistance’ in plants and protect them against pest and diseases either by repelling or by suppressing them. Organically grown fruits and vegetables on vermicompost have also been found to be highly nutritious, rich in ‘antioxidants’ and can be highly beneficial for human health even for protecting against ‘cancers’ and ‘cardiovascular’ diseases. The sustainable vermiculture biotechnology Globally six (6) environmental biotechnologies have been identified for sustainable development by scientific use of earthworms: 18. The ‘membrane filtration technology’ by ‘reverse osmosis’ is most significant new development of water and wastewater treatment without the use of chemicals. It can produce ‘high-quality disinfected water’. It can remove several chemicals (VOCs, MTBE, NDMA, several endocrine disrupters) and endospores and cysts of pathogenic bacteria and protozoa not removed by conventional methods. (1) The vermi-composting technology for efficient management of most municipal and industrial organic wastes including sewage sludge by biodegradation and stabilization and converting them into vermicompost (nutritive organic fertilizer). Earthworm participation enhances natural biodegradation and decomposition of organic waste from 60 to 80% given the optimum conditions of temperature (20 to 30°C) and moisture (60 to 70%). It takes nearly half the time to convert waste into compost and the process becomes faster with time as the army of degrader worms grows. 19. The ‘dematerialization technology’ is a significant new development. It leads to reduction in the use of materials (in weight) and energy (in MW) over in all industrial goods and products while fulfilling the same services. It has reduced the use of environmental resources (metals and glasses) and synthetic plastics by more than 20 to 25% in the manufacture of containers, cans and bottles and consequently the energy used and greenhouse gas emitted in their manufacture, and also the quantity of wastes generated after their use and discard. Steel beverage cans have been downsized by 40% since 1970. It saves materials and reduce mining activities which has severe impact on environment. (2) The vermi-filtration technology for treatment of municipal and industrial wastewater, purification, detoxification and disinfection for their reuse. Earthworms body work as a ‘bio-filter’. They can remove the BOD5 by over 90%, COD by 80 to 90%, TDS by 90 to 92%, the TSS by 90 to 95% and the total coliforms by over 99% from wastewater. There is no sludge formation which plagues all the conventional treatment plants. Worms also remove the dangerous ‘endocrine disrupting chemicals’ from sewage which cannot be done in the p 20. Development of eco-efficient non-mineral materials which can replace natural resources of earth in development and reduce mining. Ceramics, carbon and glass fibres and alloys of aluminium and lithium are new J. Agric. Biotech. Sustain. Dev. 20 conventional systems. Development of more green technologies for sustainable development al., 1999). Technology has provided efficient low-emission vehicles, but what if people continue to drive unjudiciously and unethically; technology has provided energy efficient bulbs, air-conditioners and appliances, but what if people do not buy them as they are more costly or carelessly leave them switched on even during day time and when not needed; technology has provided water efficient taps and showers, but what if people carelessly take longer shower than actually needed, fails to close the tap in-between brushing or shaving, or leave the leaking taps unattended; technology has provided solution to complete waste treatment, waste reduction, reuse and recycling, but what if the industries choose to dump the untreated wastes because treatment or recycling may be more costly than disposal? Similarly what if the society does not cooperate in separating the recyclable wastes from the non-recyclables at source? Environmental technologies have to find solutions for both ‘preventive’ as well as ‘curative’ actions for the environmental degradation. It has to protect the vital life support systems of the planet Earth – the air, water, soil and the ecosystems, and also manage all the wastes generated by the civilization. It must promise to deliver clean air, safe food, safe water, clean energy, sustainable dwelling and a sustainable transport system for the civilization. More appropriate and sustainable green technologies need to be developed in future for environmental protection and conservation, restoration and repair in the following priority areas of human development to achieve complete sustainability for the human society: 1. Commercialization of clean hydrogen fuel for automobiles, and solar, wind, geothermal and oceanic energy for utility power generation. 1. Commercialization of clean hydrogen fuel for automobiles, and solar, wind, geothermal and oceanic energy for utility power generation. Technological process and products that promote sustainability are often costly than the contemporary articles that serve the same purpose and provide the same service. Energy efficient fluorescent lights are much more costly than the ordinary filament bulbs; the fuel efficient and reduced emission hybrid cars are more costly than several other models with same facilities. gy y p g 2. Making the conventional energy (fossil fuels and hydropower) sources more clean and green and thus more sustainable. 3. More efficient energy use and conservation in homes, institutions and industries. Guarantee for human rights 7. More recycling technologies for all municipal and industrial wastes, for their safe disposal and conversion into valuable resources. 7. More recycling technologies for all municipal and industrial wastes, for their safe disposal and conversion into valuable resources. Economic development and environmental protection require social development – efforts to promote and protect internationally guaranteed civil, political, economic, social and cultural human rights. Neither economic development nor environmental protection can be fully assured in the absence of respect for fundamental rights and freedoms. 8. Methods for ‘safe disposal’ of existing non- biodegradable plastic wastes, and development and commercialization of ‘biodegradable plastics’. 9. More dematerialization technology to increase the efficiency of natural resource (metals and glasses) use and plastic use and reduce their waste. Certain human rights- such as access to health and environmental information about the diverse consumer products being used in day to day living (especially about the chemicals used in their production process), information about the environmentally safe quality of land, air and water being used by the society, raising voice against any form of environmental degradation (e.g. tree logging, air, water and noise pollution) in the locality, public participation in governance and redress for any environmental harm- may be very important in achieving sustainable development. 10. Environmental biotechnology and bioremediation technologies. They are emerging as most cost-effective technologies for environmental management- solid waste and wastewater treatment, conversion of organic wastes into resource (biofertilizers and biopolymers), stabilization of mined wasteland, decontamination of chemically contaminated sites, safe disposal of hazardous wastes, and soil conservation and erosion prevention etc. 10. Environmental biotechnology and bioremediation technologies. They are emerging as most cost-effective technologies for environmental management- solid waste and wastewater treatment, conversion of organic wastes into resource (biofertilizers and biopolymers), stabilization of mined wasteland, decontamination of chemically contaminated sites, safe disposal of hazardous wastes, and soil conservation and erosion prevention etc. . Telecommunication and information technology contributes to sustainability Telecommuting is a new concept of working from home using PCs and electronic links. It reduces the need to drive to work preventing emissions and congestion in cities, the need to heat or cool and light big offices which again saves energy and cuts emissions. More than half of the managers of AT & T Telecom Company in the U.S. telecommute one day a week, reducing 80,000 tons of carbon dioxide annually due to reduced travel and transport (UNEP Report, 2002). Several official works/business including banking and money transfers can now be done over telephone or through the internet saving travel distance, time and resources. Similarly live ‘videoconferencing’ instead of face-to-face talk in official meetings is another emerging environmentally friendly idea. It reduces the need of transport, often long travel distances and times (even visit to overseas) and significantly cuts pollution and carbon emissions while saving resources. However, the boom is communication and information technology has also brought in large amount of electronic products whose generation (version) is fast changing, resulting into their rapid use and discard as electronic wastes. (5) ‘The vermi-health protection technology’ by the use of ‘bioactive compounds’ from earthworms to develop ‘potential modern medicines’ to combat some chronic and deadly diseases like ‘cancers’, cure ‘heart diseases’ and protect human health. The earthworm’s ‘anti-oxidant’, ‘anti-microbial’, ‘anti-cancerous’, ‘immune-boosting’ and ‘clot dissolving’ medicine chest is so powerful as that of any plant and even many pharmaceuticals. y p y p (6) ‘The vermi - industrial production technology’ for use of earthworms to produce some valuable industrial raw y p y p (6) ‘The vermi - industrial production technology’ for use of earthworms to produce some valuable industrial raw Sinha et al. 21 Development of more green technologies for sustainable development Development of more green technologies for sustainable development General human nature is to buy cheaper products even if it is harmful to the environment and as long as it meets the social and cultural objectives nicely. Hence not only the green technologies have to be cost-effective, their products have to be cheaper too, so that everyone can afford easily and reasonably. 4. Cost-effective technology for water and wastewater (both municipal and industrial) treatment. 5. More efficient water use and conservation in homes, industries and agriculture. 6. Cleaner production in all mining, manufacturing and consumer industries to reduce and even prevent the use of toxic chemicals in production process and eliminate their emissions in wastes. Educating for environmental responsibilities of society: Sustainable consumption by rich society can lead to universal sustainable development Companies in developed nations are also beginning to take notice of the growing numbers of ‘green consumers’ in society as awareness about environmental health grew worldwide (Eklington and Hailes, 1989). Global consumer opinion seems heavily weighted towards a growing interest in what lies behind today’s product and services that they buy. Apart from price and quality, they want to know how, where and who has produced the product. They want to know what chemicals are there in the product, which can have even a suspected impact on health. This increasing awareness about environment and health in human society is a sign of hope for a sustainable future. Government and industry must build on that. At the root of the entire environmental problem is the unethical increase in the ‘culture of consumerism’ in the developed nations, while the unethical increase in the ‘number of consumers’ (population) in the developing nations. There is also a growing tendency for increasing consumerism in the handful of elitist societies in the developing nations. The environmental impact of both of this cultures, are exceeding the ‘ecological limits’ and the ‘carrying capacity’ (regeneration of renewable resources and assimilation of all wastes) of the Earth ecosystems with severe consequences for the future. Global consumption expenditure, private and public, has increased at an unprecedented rate worldwide but more in the rich developed nations, since the 1970s reaching some US $ 24 trillion in 1998 (UNEP-DTIE, 2002). This unprecedented growth in consumption (rather over-consumption) in affluent societies of developed nations (and handful of elitist people in the developing nations) has had positive impact as it promoted the growth of consumer industries, but highly negative impact on the environment as more consumption meant more extraction of environmental resources leading to more deforestation, soil degradation, waste and pollution, biodiversity erosion and social inequality. The adverse impacts were felt more in the developing nations where a vast majority cannot afford and suffers from ‘under- consumption’ and inequality. They provided some of their precious developmental resources (geological and biological for dollar earnings) and were also made the Sustainability education for the modern technological society The social components of the environmental management and sustainable development is as important as the economic and technological (Lovins et Environmental education for sustainability is fundamental J. Agric. Biotech. Sustain. Dev. 22 ‘dumping grounds’ of the by-products (often hazardous wastes) of over consumption in developed nations. to the success of sustainable development programs (Fien, 2001). Societal response to environmental issues is often slow as compared to the responses to economic issues such as price rise or health issues such as spread of disease. The society has to be educated to ‘demand’ and also ‘accept’ a particular technology and the technological product which is ‘environmentally friendly’, or ‘reject’ all those products which are ‘environmentally unfriendly’, and then only the industries will be encouraged or even compelled to produce environmentally friendly products. Society has to be educated about the judicious and sustainable use of the 5 Ps (paper, petrol, potable water, power (electricity) and plastics) in their daily- life. These are the most commonly and most frequently used resources by the society everyday and their production and use has considerable environmental impact. Society has also to be made aware about the hidden dangers of environmentally unfriendly products and the adverse impact of their use on human health. They must know their ‘environmental rights and responsibilities’, what is good or bad about environment, which sustain their life on Earth. ) p p Governments in developing nations nurse serious misgivings that the developed nations having followed the culture of ‘over-consumerism’ for several decades, and now wants to deprive the developing nations from their legitimate right to develop and consume in the name of environmental protection and sustainable development. In 1998, UNEP’s Division of Technology, Industry and Economics (DTIE) launched a ‘Sustainable Consumption Program’ and tried to dispel the misgivings about reducing consumption worldwide. It emphasized “Sustainable consumption is not about consuming less but it is about consuming differently, consuming efficiently, consuming judiciously, and having an improved quality of life for all, (not for handful of few) both in the developed and the developing countries. It also means sharing between the rich and the poor” (UNEP-CDG, 2000). THE ECONOMIC PERSPECTIVES FOR SUSTAINABLE DEVELOPMENT The classical economics has failed to guide true human development. Current GNP (gross national product) of nations is a false indicator of true economic prosperity. It does not distinguish between resource uses that sustain development and those that undermine it. It is like a ‘malfunctioning strand’ of our ‘cultural DNA code’ – carrying erroneous information and signaling to the body- politic a form of economic growth analogous to that of cancers cells that consume the host body. All basic biological and physical systems of earth (the true capital resources e.g. soil, water and biodiversity) that sustains life are under severe stress and in fact ‘exhausting’, as ecological destruction continues unabated, yet the key economic indicators shows that the world is prospering (Ekins, 1992). The goals of sustainable development cannot be achieved unless we have a new world order with equity at all levels; social, economic, political and cultural. Till the evils of ‘poverty and hunger’, ‘inequities and ‘deficiencies’ exist in any part of world, there can be no sustainability in development. Social and economic equity is a necessary condition for sustainable human development. If the rich get richer and the poor becomes more in number, that development will not be sustainable over time. If development discriminates against the women and children, that development will also not be sustainable. , p There is mounting evidence that environmental degradation and economic decline (aggravating poverty) feed on each other, and the fate of the poor and the fate of the planet (Earth) have become tightly entwined “The poor are both the victims and the agents of environmental damage. They are forced to consume the environmental resources (even from the very fragile ecosystems) in order to survive, and the impoverishment of the environment leads to worsening poverty and unsustainablity”. Economic deprivation and environmental degradation have thus come to reinforce each other in a vicious cycle that perpetuates poverty and destitution in many developing countries and thwarts all efforts towards sustainable development (UNDP, 2003, 2004). ( , ) Conventional economists and the ignorant politicians are not bothering to deduct the cost of environmental destruction (e.g. waste and pollution creation and their health impact, loss of forest and species, degradation of land and soil etc.) and the cost of environmental repair and restoration (e.g. Social equity, reduction of population and eradication of poverty: A necessary condition for sustainability Sustainability refers not only to the natural environment but also to the social environment because social and environmental issues are necessarily inter-wined. Some societies have benefited most by new technological development and unsustainable use of resources while others have suffered. There is a nexus between ‘Poverty and Pollution’, between ‘Population and Poverty’ and between ‘Population and Pollution’. Nearly 930 millions of people (consumers) are being added every year to the earth demanding more of all resources, more forest to produce food and fuel, and more land to live. If this consuming population is affluent it would further aggravate the problem of unsustainability. Hence we must have a sustainable level of population Sinha et al. 23 to environmental factors; piling waste and poor sanitary conditions (WHO, 1997). that the earth ecosystem can reasonably support. Technology used to harness the resources from the biosphere plays a critical role in resource use and sustainable development. More appropriate and efficient a technology, more efficiently the resource will be used, lesser will be the waste and pollution generation and greater will be the sustainability. THE ECONOMIC PERSPECTIVES FOR SUSTAINABLE DEVELOPMENT afforestation, waste management and pollution control, soil regeneration and wasteland management, waste water recycling etc.) from the GNP or the rate of economic growth. All economic development programs are implemented through political decisions. The developers and decision makers have to understand that all economic development programs involves the systematic transformation of the world of ‘living things’ (the biosphere – or the natural world which is a product of centuries of evolutionary process) into a world of ‘human artifacts’ (the technosphere or the surrogate world). This means all human development process is necessarily ‘anti-evolutionary’, and therefore, ‘anti-nature’ and it amounts to gross interference into the nature to achieve the goals of economic development. It is imperative that we have to change the strategies of development in order to minimize our interference into the nature. The new economic theory is ‘Environmental Economics’ which advocates for judicious balance between ‘economy and ecology’ and amalgamation of ‘economic development’ programs with ‘ecological conservation’ strategies to usher in the era of sustainable development. The new economic philosophy of development also stresses mankind to switch over from the ‘fossil fuel based economy’ to ‘renewable and clean energy based economy’. Good health: A necessary condition for human sustainability There can be no sustainable development without ensuring basic health services for all in the society. The World Health Organization (WHO) reports that 25% of all preventable illness are directly caused by environmental factors. Modern health hazards, caused by development that lacks environmental safeguards, such as urban air and water pollution, exposure to ago-industrial chemicals and toxic wastes is becoming common. Communicable diseases, especially HIV/AIDS, cholera and other water borne diseases, tuberculosis and malaria, pose serious obstacle on the road to sustainable development. p It is estimated that in this modern technological world 3 million people die every year of water borne diseases or from use of unsafe water. More than 1 billion people globally breathe unhealthy air, and 3 million people die each year from air pollution; two thirds of them poor people, mostly women and children. Two million people die every year as a result of slow exposure to indoor pollution caused by burning of wood and dung cake. Around 90% of malaria cases in the world are attributable Economic planners have to understand that every natural resource, commodity, goods and services that we use from the environment has an ‘environmental cost’ (the hidden cost of environmental damage and repair J. Agric. Biotech. Sustain. Dev. 24 the net value added (Goodland and Serafy,1991). the net value added (Goodland and Serafy,1991). while the raw material is procured from the earthly resources) other than its ‘economic cost’ (the cost of processing, manufacturing and trading) and only after adding the two costs, we arrive at the true cost of the product. There may be ‘social cost’ as well in the form of impaired human health and quality of life. The concept of circular economy: Recycling and reuse of waste and resources Final industrial solid waste (ISW) disposal limited to about 4,500 million tones. Classical economics only accounts for the electricity generation and irrigation potential of any hydro-power project but never takes into account the losses which incur by way of land submergence and destruction of trees, forest and biodiversity by constructing huge dams. They are not bothered to add the value of the life sustaining oxygen produced, carbon dioxide (greenhouse gas) absorbed, and biodiversity protected by the trees and forest, if they were alive? Environmental economics would account for all these. In classical economics a tree has a value only when it is dead and become a ‘timber’, while in environmental economics a live tree has more value as it do more services to mankind by providing food, fodder, fuel and the vital oxygen. It is important that the cost of ‘environmental damage’ and the cost of ‘environmental repair’ must be included in the economic calculations for GNP of every nation (Tietenberg, 1988). If all nations both developed and the developing, follows the philosophy of ‘circular economy’ with some set targets for 2010 or 2015 it will great leap forward for the global sustainable development. The concept of circular economy: Recycling and reuse of waste and resources The new economic philosophy is to cut the use of basic (virgin) materials from the environment dramatically, by boosting ‘recycling and re-use’ the waste including water, energy and materials from one facility/industries/organizations becoming an input in another facility. In circular economy, all economic activities pursue low resource exploitation, maximum efficiency in using materials and energy, and low waste generation. China, the largest growing economy in world has adopted the concept of ‘circular economy’. It has set the following targets for 2010 using 2003 as baseline (UNEP-DTIE, 2002): p q y Environmental damage can occur at both ends of the cycle - the ‘production cycle’ as well as at the ‘consumption cycle’. In the production cycle environmental damage is caused by way of deforestation, earth cutting, soil erosion and pollution etc. to obtain the raw materials by mining the Earth and process it in the industries, while in the consumption cycle, the damage is caused because of waste generation once the product is consumed and discarded after use. Fossil fuels, nuclear fuels, several metals, papers and plastics and even procurement of human food and drinking water from the nature cause environmental damage at both ends of the cycle. 1. Resource productivity per ton of energy, iron and other resources increased by 25%; 1. Resource productivity per ton of energy, iron and other resources increased by 25%; y We only pay for the economic cost of the water (cost of harnessing and supply) when we use it in our homes. We do not pay for the treatment of the wastewater when we have used it and converted into sewage. We only pay for the cost of food grown in farms and its processing and transport. We never pay for the damage done to the environment due to production and use of chemical fertilizers and pesticides. We pay for the electricity we consume, but not for the environmental damage that occur by operation of coal power or nuclear power plants and by the construction of huge dams for hydro-power. y 2. Energy consumption per unit of GDP decreased by 18%; 3. Average water use efficiency for agricultural irrigation improved by up to 50%; p y p ; 4. Reuse rate of industrial solid waste (ISW) raised above 60%; 5. Recycle and reuse rate for major renewable resources increased by 65% y 6. Making the profit making companies more accountable for sustainable development p g 7. They deliberately promote ‘unsustainable consumption patterns’ in both North and South nations through electronic and print media. Opinion polls made on the eve of Johannesburg Summit (2002) clearly show that people worldwide want the companies in both private and public sectors to bear the responsibility of environmental degradation and act with greater responsibility towards its repair and restoration. Opinion polls made on the eve of Johannesburg Summit (2002) clearly show that people worldwide want the companies in both private and public sectors to bear the responsibility of environmental degradation and act with greater responsibility towards its repair and restoration. The behavior of big companies and corporate sectors, and the need to make them more responsible and accountable, has captured public attention and interest in all discussions relating to environmental protection and sustainable development. Maximizing ‘short-term profit’ even at the cost of environment and society had been the key principle of operation of almost all companies in world whether in the developed or in the developing countries. Burning examples are the ‘Petrochemical and Agrochemical Industries’ of world. One has deliberately thwarted the development of ‘Hydrogen Fuel Technology’ and the other has been conspiring against the development of ‘sustainable alternatives’ to the ‘deadly agrochemicals’ for their own vested interest. UNEP report says 3 Republican Senators were seen secretly attending a meeting of Oil Producing Nations in Saudi Arabia. Recently the Prince of Saudi Arabia has realized it and has requested U.S. to give them the technology for development of ‘Hydrogen Fuel’ in the interest of humanity. In the U.S. farmers practicing ‘vermiculture’ in agriculture had to face problems with the USEPA. We have also faced problems in India, while educating farmers about use of vermicompost in farming. Agents of agrochemical industries operate in every country misleading the farmers about adverse effects of earthworms on farm soil. Perhaps the biggest error of the Rio Earth Summit (1992) was its decision not to create a mechanism to regulate the economic activities of ‘big companies and corporations’. Although, the NGOs like Greenpeace and the Third World Network who dominated at the Rio Summit had identified the primary role of the transnational corporations (TNCs) in damaging the environment. The economic record of the last decades show that the cause sustainable development loses when governments give up their task of regulating companies. Making the profit making companies more accountable for sustainable development TNCs should also be made liable for economic compensation for the harmful effects of their operation on the environment, safety of workers and ill effects on the health of workers and the residents in the area. In the U.S., Louisiana’s Lower Mississippi River Industrial Corridor has been dubbed by the environmentalists and the local residents as ‘Cancer Alley’. It has over 125 companies manufacturing a range of products including fertilizers, paints, plastics and gasoline. These polluting industries were given a ‘tax break’ of U.S. $ 111 million and the Louisiana government wiped out $ 3.1 billion off the books in property taxes. It was an economic incentive given to the industries to create more jobs even at the cost of human health and the environment. Ecology was sacrificed at the altar of economy. It was an incentive to pollute more. Sustainability for all’ was compromised with ‘prosperity for few’ (WB, 2004). Environmental auditing and accounting of developmental activities There has to be EAA and environmental cost-benefit analysis (ECBA) of all those developmental activities and their associated technologies which sustains growth and development- such as management of domestic and industrial wastes; tree planting and afforestation activities; soil conservation, remediation and wasteland development. EAA and ECBA are needed for those developmental activities and technologies, too, which otherwise undermine growth and sustainable development- such as deforestation for timber mining and agriculture; encroachment of farmlands and old growth forest for urban development; extraction and processing of ores and minerals from the earth crust in mining and metallurgical industries; production and use of fossil fuels and nuclear fuels; construction of huge dams for hydropower generation; and production and use of agro- In a pilot study in Mexico, the planners tried to measure the cost of environmental damage and resource depletion due to development. When an adjustment was made for the depletion of oil, forests, and groundwater, Mexico’s net national product was almost 7% lower. A further adjustment for the costs of avoiding environmental degradation, particularly air and water pollution and soil erosion, brought the national product down to another 7%. In the agriculture and animal husbandry sector adjustment for the cost of soil erosion sharply reduced Sinha et al. Sinha et al. 25 chemicals for agriculture etc., so as to make these developmental activities environmentally more sustainable chemicals for agriculture etc., so as to make these developmental activities environmentally more sustainable unsound technologies and production systems and hazardous materials like chemical pesticides (in the name of pest and disease control and crop protection) and hazardous wastes (in the name of recyclables) to the developing countries in the South. CONCLUSION It is impossible to separate economic development issues from environmental issues. Almost all forms of development for human welfare erode the environmental resources upon which they must be based, and environmental degradation would necessarily undermine economic development. It is a vicious circle. In the end, the solution to the problem is sustainable development, a process of change in which the exploitation of environmental resources, the direction of economic investments, the orientation of technological innovations and institutional changes are made consistent with the present and well as future needs. Environmental policy decisions would require imposition of some new ‘environmental taxes’ like pollution tax, waste tax, tree tax and logging tax etc. and reduction in the regular income taxes. It will require to enhance the prices of environmental goods and services (such as water and energy sources) in order to force the society for their judicious use and consumption. Economic incentives have to be given to recycling industries and those saving water and energy in industrial operations by way of reduction in taxation and reduced cost of inputs (raw materials, water and electricity). Sustainability in human society with good quality of life for all can be achieved in two ways: 1. By persuading the people to ‘behave ethically’ towards environment, ‘reduce consumption’, and have a ‘simpler life-style’; 1. By persuading the people to ‘behave ethically’ towards environment, ‘reduce consumption’, and have a ‘simpler life-style’; 2. By embracing the philosophy of ‘sustainable development’ with appropriate technologies that allow people to enjoy the same good quality of life with high standard of living, but at a significantly lower ‘environmental cost’ (Schumacher, 1973). p ( y) Some pragmatic policy decision has to be taken by the government of all nations with regard to water and energy pricing in society and production of consumer goods by the industries. The short-term objectives of reducing the water and power prices may give fillip to high water and energy consumption with severe environmental consequences, waste and pollution. Developed nations are paying high environmental cost (by way of greenhouse gas emission) for the cheap auto fuel, which is in fact at the cost of the whole society. ‘Polluter-Pay- Principle’ has to be enforced in the matters of water and energy pricing and in the matters of waste generation. There has to be a ‘Waste and Pollution Tax’ added up with the consumer products, water and the auto fuel at source. THE POLITICAL PERSPECTIVES FOR SUSTAINABLE DEVELOPMENT The recent trend of ‘deregulation and liberalization’ in all countries (even in nations with communist philosophy) have increasingly allowed companies to do much as they like: All economic development plans and projects are executed and implemented through some political decision at local, regional or federal level and political decisions are often made to meet the societal goals. In democracy ‘eco-vote’ plays critical role in planning for development. Sometimes a political decision is taken to fulfill the election promises to a particular section of the society even at the cost of environment. When it comes to setting priorities- whether society is to be benefited or the environment, or both, obviously societal needs come first. The fact remains that if environment suffer, society can never remain apart and is bound to suffer. Political maturity requires a perfect planning for striking a judicious balance between the two. 1. Their activities generate more than half the greenhouse gases emitted by industrial sectors. 2. They dominate in both mining and trade of natural resources and commodities, thus affecting forests, soils, water and marine ecosystems. 2. They dominate in both mining and trade of natural resources and commodities, thus affecting forests, soils, water and marine ecosystems. y 3. They control about four fifths of the land cultivated worldwide for export of crops. y 3. They control about four fifths of the land cultivated worldwide for export of crops. 4. They dominate global and national industry and transport, and these developmental activities are major sources of waste and pollution, including toxic and hazardous wastes, unsafe consumer products and occupational health hazards. p 6. They are major transmitters of environmentally j At the Rio Earth Summit, Brazil, 1992, the whole world J. Agric. Biotech. Sustain. Dev. 26 was seen to be divided into two eco-political blocks of North and South on the issues of ‘carbon emission’ and over the ‘rights and access to the genetic resources of the tropical forests’. The ‘arrogance and ignorance’ of the politicians of some developed nations refused to meet the obligations of Rio Convention and which still continues today. The arrogant statement of the then U.S. President George Bush in 1992, that- “conventions like these are not going to compel the U.S. citizens to give up their current life-style” expose the political immaturity of a head of state in planning for sustainable development. THE POLITICAL PERSPECTIVES FOR SUSTAINABLE DEVELOPMENT Greening of politics is an urgent requirement today and it must be incorporated in the national and international agenda of every nation for appropriate planning and decision making on vital issues of development and environment. take a hard decision over cutting in the production of ‘private cars’ and ‘subsidize’ the cost of public transport instead of the petrol. Government should force the industries to produce and manufacture ‘durable’ goods and not ‘disposable ’ones. All planning and policies for development of appropriate environmental technologies for sustainable development have to be economically viable and socially acceptable. It is also the responsibility of the academia to educate the politicians and decision makers about the benefits of sustainable development technologies. They have to be educated about the ‘hidden cost’ of a particular developmental activity and product such as the ‘fossil fuels’ and ‘plastic bags’ which is apparently cheap at its face value, but are proving very costly in terms of human sustainability. The convenient but ‘non-biodegradable’ plastic bags used in the groceries, cause health hazard during manufacture and also recycling. They have to be ‘banned’ all over the world. Wrong political decisions made for rapid resource exploitation and economic development in some Asian and African nations e.g. for construction of huge dams for hydro-power, clearing of ‘tropical forest’ for fodder plantation and export earnings, installation of nuclear power plants and dumping of nuclear and toxic wastes has adversely affected the ecology of the country, irreversibly damaged the ecosystems and biodiversity, displaced and uprooted large sections of human societies from their homes as ‘ecological refugees’ for narrow socio-political and socio-economic gains. Damages done to the human environment due to the political conflicts and tensions, terrorist activities, wars and battles resulting from international politics unleashed by arrogant political decisions is also of great concern. REFERENCES Adams W (2001). Green Development; 2 nd Edition, Routledge Pub., London, U.K. Axinn GH (1996). Philosophy of Sustainable Development; In Rajiv K. Sinha (ed.) Environmental Crisis and Human’s at Risk; INA Shree Publications, Jaipur, India. pp. 137-142. Eklington J, Julia H (1989). The Green Consumer Guide – From Shampoo to Champagne: High Street Shopping for Better Environment; Victor Gollancz Ltd. London. Ekins P (1992). The Gaia Atlas of Green Economics; Doubleday / Anchor Pubublication. Fien J (2001): Education for Sustainability; TELA Series, Australian Conservation Foundation; Centre for Innovation and Research in Environmental Education, Griffith Eco-Centre, Griffith University, Nathan Campus, Brisbane. Making economic development programs less dependent upon the ‘fossil fuels’ and more dependent on renewable energy sources, less energy and material intensive and more equitable in its social and economic impact on global level is the need of the hour. A ‘sustainable hydrogen fuel economy’ in place of ‘fossil fuel economy’ must be established if an environmentally and economically sustainable world is to be left to our children and grandchildren. p Goodland D, Serafy D (1991). Environmentally Sustainable Economic Development: Building on Brundtland, UNESCO, Geneva. IUCN WWF, UNEP (1991): Caring for the Earth: A Strategy for Sustainable Living; Earthscan, U.K. Lovins A, Brown L, Flavin C, Postal S (1999). Picturing a Sustainable Society; In L. Starke (ed.) ‘State of the World (1999)’, World Watch Institute, Washington. g Label G, Gregory, Hal K (1987) Will We Survive ? Sustainable Development: A Guide to Our Common Future; The Report of World Commission on Environment and Development (WECD). p ( ) Myers N (ed.) (1994). The GAIA Atlas of Planet Management; GAIA Books, Ltd., London. p. 272. g A sustainable future for the human society will be determined: Meadows DH, Meadows DL, Randers J (1992). Beyond the Limits: Confronting Global Collapse: Envisioning a Sustainable Future; Chelsea Green Publication; Post Mills., VT. 1. Not by the megawatt of energy which will be produced, but by the fact that how they will be produced and from what source? Milbrath LW (1989). Envisioning a Sustainable Society; State University of New York Press. Sinha RK (1994). Development Without Destruction; Environmentalist Publishers, Jaipur, India. 2. Not by the quantity of food that will be produced, but by the quality of the food, and that how they will be produced? Sinha RK, Greenway M (2004). CONCLUSION Even the government of nations may have to Given the difficulty of changing people, the second option appears to be more pragmatic. However, technology should never be seen as a ‘silver bullet’ solving all the world’s environmental ills. Environmental education for sustainability can definitely change people in course of time and correct human behavior. Technology has provided environmentally sound alternatives for sustainable development in many cases but there is a sad lack of necessary ‘political will’ to implement it because there is fear of resistance from the Sinha et al. Sinha et al. 27 mighty industrial lobby and their political friends who want to continue with the obsolete and destructive technologies for their own political and economic gains. Is it not ironic that man stepped on moon in 1969, but has not been able to commercialize the use of solar, wind and hydrogen power as a viable alternative to destructive oil, and manufacture automobiles run on hydrogen fuels? It is perhaps not favorable to the oil rich nations of world and their ‘purchased politicians’ in the decision making bodies of west. The ‘addiction’ to the ‘cheap oil’ stopped the technologically capable western world to go for sustainable alternative. materials and technologies whose production, application and utilization entails heavy environmental damage from beginning to end of the cycle; 4. By how soon we are able to ‘reduce’ and ‘recycle’ all those ‘human wastes’ successfully and develop safe and benign alternatives to those thousands of ‘toxic chemicals’ used in various developmental activities; chemicals used in various developmental activities; 5. By how soon we are able to commercialize the ‘hydrogen fuel technology’ for our automobiles and all other renewable sources of energy for power generation; 6. By how soon we switch over completely to ‘Organic Farming’ to produce ‘chemical-free and health protective organic foods’ for civilization; 5. By how soon we are able to commercialize the ‘hydrogen fuel technology’ for our automobiles and all other renewable sources of energy for power generation; gy p g 6. By how soon we switch over completely to ‘Organic Farming’ to produce ‘chemical-free and health protective organic foods’ for civilization; gy p g 6. CONCLUSION By how soon we switch over completely to ‘Organic Farming’ to produce ‘chemical-free and health protective organic foods’ for civilization; In the words of Gerald Durrel (1984), ‘At the present rate of progress and unless something is done quickly, disaster stares us in the face. Erosion, desertification and pollution have become our lot. It is a weird form of suicide, for we are bleeding our planet to death. We are led by’ sabrerattling politicians’ who are ignorant of biology, beset by sectarian groups noted for their narrow- mindness and intolerance, surrounded by powerful commercial interests whose only interest in nature is often to rape it’ (Myers, 1994). 7. By how soon we are able to arrest the dangerously growing menace of soil and biodiversity erosion, desertification and land degradation; 8. By how soon we are able to arrest the exploding ‘human population’ (consumers on Earth) and the ‘culture of consumerism’ and learn to live a ‘sustainable life-style’ by embracing the philosophy of 5 P’s (judicious use of paper, petrol, power, potable water and plastics) and 5 r’s (refuse, reduce, reuse, recycle and responsibility about waste) in daily life (Seymour and Girardet, 1987; Milbrath, 1989). p y There is sufficient evidence to indicate that the world is going on the wrong path of development. It is leading to economic, ecological, social and political deterioration and the worst ‘moral degradation’ in the name of development. It has given birth to violent societies, violence against both man and nature. There are more accidents, crime, delinquency, drugs, alcoholism and deadly diseases than ever before. Mankind will perish if the protection of the environment does not become an integral part of all technological development, planning and management. We must seek lessons from history. The flourishing civilizations of the past perished not at the hands of any cruel enemy, but due to human’s own activity, clearing of forest, overuse of soil and lack of awareness for development compatible with the environment (Meadows et al., 1992). REFERENCES Green Technologies for Environmental Management and Sustainable Development: Giving Better Quality of Life at Lower Environmental Cost: Pointer Publishers, India. p. 432. ISBN 81-7132-375-8. 3. By how soon we get rid of those developmental J. Agric. Biotech. Sustain. Dev. 28 Better Quality of Life at Lower Environmental Cost: Pointer Publishers, India. p. 432. UNDP (2004). Human Development Report 2004; United Nation Development Program; NY. p Sinha RK (2006). Development, Environment, Human Health and Sustainability: Vol. I :The Price of Unsustainable Development: (p.324) ISBN 81-7132-480-0; Vol. II :The Wisdom of Sustainable Development: (p.342) ISBN 81-7132-481-9. Pointer Publishers, Jaipur, India p g UNEP-CDG (2000). Sustainable Consumption and Production: Creating Opportunities in a Changing World; Report of the 4 th International Business Forum, Berlin, 1999. UNEP-DTIE (2002). Sustainable Consumption and Cleaner Production: Global Status 2002; United Nation Environment Program & Division of Technology, Industry and Economics, Paris, Cedex, France. Steffen W, Sanderson A,Tyson PD,Jäger J, Matson PA, Moore III B, Oldfield F, Richardson K, Schellnhuber HJ, Turner II BL, Wasson RJ, (2004). Global Change and the Earth System: A Planet Under Pressure; IGBP Global Change Series; Berlin-NY, Springer-Verlag. gy y UNEP-GEO (2004-05): Global Environment Outlook (GEO) Year Book 2004-05; UNEP Publication, Nairobi, Kenya. WCED (1987): Our Common Future; World Commission on Environment and Development; Oxford University Press. Seymour J, Girardet H (1987). Blueprint for Green Planet; Dorling Kindersley, London. WB (2004). World Development Indicators; World Bank Report, Washington, D.C. y Schumacher EF (1973). Small is Beautiful. Harper and Row; UK. g WHO (1997). Health and Environment in Sustainable Development; Five Years After the Rio Earth Summit; World Health Organization Publication, Geneva. p. 242. Schmidheiny S, Chase R DeSimone L (1997): Signals of Change: Business Progress Towards Sustainable Development; The World Business Council for Sustainable Development (WBCSD); p ( ) Tietenberg T (1988). Environment and natural Resource Economics; Glenview, III; Scott, Forseman & Company. UNDP (2003). Human Development Report 2003: Millennium Development Goals; United Nation Development Program; Oxford University Press.
https://openalex.org/W4379208040	https://journal.formosapublisher.org/index.php/ajabm/article/download/4237/4139	Indonesian	null	Procedure for Establishing an Individual Limited Liability Company	Asian Journal of Applied Business and Management	2,023	cc-by	3,926	Asian Journal of Applied Business and Management (AJABM) Vol. 2, No. 2, 2023: 285-294 A B S T R A C T A B S T R A C T In business world, many are experiencing quite a very rapid development. Many entrepreneurs choose individual limited liability company in running their business. The research method used is normative juridical which is qualitative in nature based on literature research. Data sources come from secondary data sources and tertiary data. The data was analyzed using a deductive mindset with a descriptive method and data analysis using a conceptual approach and a statute approach. Based on the results of the study, it was found that the concept of liability at PT Perorangan UMK is limited liability and protection for creditors against the risk of default by PT Perorangan UMK is through the enforcement of non-litigation and litigation legal remedies. The main conclusion is the importance of reviewing the risks that arise from the addition of Article 153J which has been amended in Article 109 of the Job Creation Law regarding the limitation of MSEs' liability which has not further regulated the protection of PT Perorangan UMK creditors. Procedure for Establishing an Individual Limited Liability Company Fauziah Agustini1, Nursaiba Ritonga2, Indah Purnama Sari3, Devi Marsella Br Barus4, Rifqa Khairunnisa5 Fakultas Ekonomi, Universitas Negeri Medan Corresponding Author: Indah Purnama Sari indahprnm54@gmail.com Fauziah Agustini1, Nursaiba Ritonga2, Indah Purnama Sari3, Devi Marsella Br Barus4, Rifqa Khairunnisa5 Fakultas Ekonomi, Universitas Negeri Medan Corresponding Author: Indah Purnama Sari indahprnm54@gmail.com A R T I C L E I N F O Keywords: Establishment, Individual Limited Liability Company, Procedure, Company Received : 18, March Revised : 20, April Accepted: 21, May ©2023 Agustini, Ritonga, Sari, Barus, Khairunnisa: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. A R T I C L E I N F O Keywords: Establishment, Individual Limited Liability Company, Procedure, Company Received : 18, March Revised : 20, April Accepted: 21, May ©2023 Agustini, Ritonga, Sari, Barus, Khairunnisa: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. A R T I C L E I N F O Keywords: Establishment, Individual Limited Liability Company, Procedure, Company ( DOI: https://doi.org/10.55927/ajabm.v2i2.4237 ISSN-E: 2964-7932 https://journal.formosapublisher.org/index.php/ajabm 285 Agustini, Ritonga, Sari, Barus, Khairunnisa PENDAHULUAN Industri ialah salah satu sendi utama dalam kehidupan warga modern, salah satu pusat aktivitas manusia untuk memenuhi kehidupan kesehariannya. Kegiatan industri merupakan suatu bentuk transaksi yang ekonomis. Perseroan Terbatas ialah wujud usaha aktivitas ekonomi yang sangat disukai dikala ini, Perseroan Terbatas merupakan bentuk usaha kegiatan ekonomi yang paling disukai saat ini, karena disamping pertanggungjawabannya yang bersifat terbatas, Perseroan Terbatas juga memberi kemudahan bagi pemilik (pemegang saham) untuk mengalihkan perusahaannya (kepada setiap orang) dengan menjual seluruh saham yang dimilikinya pada perusahaan tersebut, serta keuntungan-keuntungan lainnya. Perseroan Terbatas sebagaimana sesuai dengan ketentuan Pasal 1 ayat (1) Undang-Undang Nomor 40 Tahun 2007 adalah: “badan hukum yang merupakan persekutuan modal, didirikan berdasar pada perjanjian, melakukan kegiatan usaha dengan modal dasar yang seluruhnya terbagi dalam saham, dan memenuhi persyaratan yang ditetapkan dalam undang-undang ini serta peraturan pelaksanaannya. Dengan demikian, jelas bahwa setiap perseroan terbatas adalah badan hukum persekutuan modal yang menjalankan perusahaan.” Apabila seseorang telah mendirikan Perseroan Terbatas untuk bisnis yang akan jalankan biasanya memerlukan penyesuaian mulai dari penambahan modal, perubahan pemegang saham, maksud dan tujuan, susunan pengurusan dan lain-lain, maka perubahan yang berkaitan dengan identitas atau informasi perusahaan, apapun bentuknya, harus memperhatikan aturan yang berlaku. Informasi yang dimaksud tertulis di anggaran dasar perusahaan kalau informasi tersebut berubah artinya Perseroan Terbatas tersebut harus melakukan perubahan anggaran dasar. Perubahan terhadap Perseroan Terbatas tersebut wajib membuat akta perubahan anggaran dasar yang dimana anggaran dasar tersebut dibuat dihadapan notaris. Anggaran dasar merupakan bagian dari akta pendirian yang memuat aturan main dalam Perseroan Terbatas yang menentukan setiap hak dan kewajiban dari pihak-pihak dalam anggaran dasar, baik itu Perseroan Terbatas sendiri, pemegang saham, maupun pengurus. Berdasarkan yang tertulis di atas, maka adapun yang akan dibahas mengenai permasalahannya yaitu sebagai berikut: Bagaimana Pendirian PT Perseroan Terbatas Perorangan ini terbentuk?, Bagaimanakah proses penyelesaian pendirian PT Perseroan Terbatas Perorangan ini dari awal hingga ke tahap akhir pengesahan nya?, Adakah kekurangan atau kelebihan dalam mendirikan PT Perseroam Terbatas Perorangan ini?. Serta Adapun tujuan dari penelitian ini untuk: mengetahui bagaimana proses pendirian PT Perseroan Terbatas Perorangan ini berlangsung, dan ntuk mengetahui apa apa saja syarat syarat atau pun langkah langkah yang harus di ambil dalam mendirikan PT Perseroan Terbatas Perorangan ini. Prosedur Pendirian Perseroan Terbatas Perorangan 2, No. 2, 2023: 285-294 Asian Journal of Applied Business and Management (AJABM) Vol. 2, No. 2, 2023: 285-294 Prosedur Pendirian Perseroan Terbatas Perorangan Fauziah Agustini1, Nursaiba Ritonga2, Indah Purnama Sari3*, Devi Marsella Br Barus4, Rifqa Khairunnisa5 Fakultas Ekonomi, Universitas Negeri Medan Corresponding Author: Indah Purnama Sari indahprnm54@gmail.com A B S T R A K Dalam dunia bisnis saat ini sangat banyak mengalami perkembangan yang cukup sangat pesat. Para pengusaha banyak memilih PT Perseroan Terbatas perorangan dalam menjalankan usahanya. Metode penelitian yang digunakan ialah yuridis normatif yang bersifat kualitatif yang didasarkan pada penelitian pustaka. Sumber data berasal dari sumber data sekunder dan data tersier. Data tersebut dianalisis menggunakan pola pikir deduktif dengan metode deskriptif dan analisi data menggunakan conceptual approach dan statute approach. Berdasarkan hasil penelitian ditemukan bahwa konsep pertanggungjawaban pada PT Perorangan UMK adalah pertanggungjawaban terbatas dan pelindungan bagi kreditur terhadap risiko gagal bayar PT Peroranagan UMK adalah melalui penegakan upaya hukum non-litigasi dan litigasi. Kesimpulan utama adalah pentingnya peninjauan kembali risiko yang muncul terhadap penambahan Pasal 153J yang telah diatur perubahannya dalam Pasal 109 UU Cipta Kerja mengenai pembatasan pertanggungjawaban UMK yang belum mengatur lebih lanjut mengenai perlindungan kreditur PT Perorangan UMK. A R T I C L E I N F O A B S T R A K Kata Kunci: Pendirian, Perseroan Terbatas Perorangan, Tata Cara, Perusahaan Received : 18, March Revised : 20, April Accepted: 21, May ©2023 Agustini, Ritonga, Sari, Barus, Khairunnisa: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. Dalam dunia bisnis saat ini sangat banyak mengalami perkembangan yang cukup sangat pesat. Para pengusaha banyak memilih PT Perseroan Terbatas perorangan dalam menjalankan usahanya. Metode penelitian yang digunakan ialah yuridis normatif yang bersifat kualitatif yang didasarkan pada penelitian pustaka. Sumber data berasal dari sumber data sekunder dan data tersier. Data tersebut dianalisis menggunakan pola pikir deduktif dengan metode deskriptif dan analisi data menggunakan conceptual approach dan statute approach. Berdasarkan hasil penelitian ditemukan bahwa konsep pertanggungjawaban pada PT Perorangan UMK adalah pertanggungjawaban terbatas dan pelindungan bagi kreditur terhadap risiko gagal bayar PT Peroranagan UMK adalah melalui penegakan upaya hukum non-litigasi dan litigasi. Kesimpulan utama adalah pentingnya peninjauan kembali risiko yang muncul terhadap penambahan Pasal 153J yang telah diatur perubahannya dalam Pasal 109 UU Cipta Kerja mengenai pembatasan pertanggungjawaban UMK yang belum mengatur lebih lanjut mengenai perlindungan kreditur PT Perorangan UMK. A R T I C L E I N F O Kata Kunci: Pendirian, Perseroan Terbatas Perorangan, Tata Cara, Perusahaan 286 Asian Journal of Applied Business and Management (AJABM) Vol. TINJAUAN PUSTAKA Menurut Soedjono Dirjosisworo Osakeyhtiö atau PT kegiatan dilakukan oleh badan hukum yang didirikan atas dasar kontrak bisnis yang modal 287 Agustini, Ritonga, Sari, Barus, Khairunnisa sahamnya terbagi seluruhnya menjadi saham dan memenuhi UU no. 40 2007 sebagaimana telah diubah dengan Peraturan penerapan. Di samping H.M.N. Purwosutjipto, perusahaan saham gabungan tersebut perkumpulan yang berbentuk badan hukum. Nama badan hukum ini belum ditentukan “persekutuan” tetapi “perseroan” karena modal badan hukumnya terdiri dari saham atau saham milik mereka. Menurut Zaen Ashhadie, joint stock company adalah sebuah bentuk melakukan bisnis dengan badan hukum yang awalnya dikenal sebagai nama Naamloze Vennootschap (NV). Definisi "terbatas" dalam suatu perusahaan Tanggung jawab pemegang saham terbatas, yang hanya terbatas pada nilai nominal seluruh saham yang dimilikinya. Menurut Abdulkadir Muhammad, yang dimaksud dengan “usaha” adalah tentang penentuan modal yaitu saham dan syarat-syaratnya "terbatas" berarti batas tanggung jawab pemegang saham, yaitu dibatasi oleh jumlah nominal saham yang dimiliki. Perusahaan terbatas adalah persekutuan badan hukum. R. Ali Rido juga berpendapat bahwa perusahaan saham gabungan itu bentuk perusahaan dibuat yang mengatur perusahaan dalam kegiatan Bersama beberapa orang modal tertentu dibagi menjadi saham, yang anggotanya dapat memiliki satu atau lebih saham dan memiliki tanggung jawab terbatas sampai dengan jumlah saham yang dimiliki. Ada pengaturan dalam sejarah perkembangan perusahaan saham gabungan dalam fase stagnan setelah pengenalan KUHD di Indonesia (Hindia Belanda pada saat itu) tahun 1848 dengan prinsip konsistensi/ prinsip kerukunan. Amandemen pertama peraturan Pada tahun 1995, sebuah perusahaan saham gabungan baru didirikan berdasarkan undang-undang UU Perseroan Terbatas No. 1 dan 12 tahun 1995 (dua belas) tahun kemudian, pemerintah melakukan perubahan lagi UU No 40 Tahun 2007 Perusahaan saham gabungan menggantikan undang-undang sebelumnya. Dua kali perubahan peraturan kelembagaan pada perusahaan saham gabungan dapat menggambarkan sifat yang berlawanan ketika menghadapi kegiatan ekonomi yang biasanya cair dan dinamis. METODOLOGI Artikel ini disusun dengan menggunakan metode penelitian kepustakaan, berdasarkan hasil penelitian atau kajian dalam ilmu hukum normatif, penelitian ini bersifat deskriptif, kualitatif, legislasi analitis, bahan sekunder berupa buku, jurnal, berita dan laporan resmi. , jenis data yang digunakan data sekunder diperoleh secara tidak langsung atau melalui media perantara dan data sekunder lainnya yang berasal dari jurnal, skripsi dan tesis. HASIL PENELITIAN Contoh PT Perorangan Terbatas g PT ASI Pudjiastuti Aviation (Susi Air) Susi Air adalah maskapai penerbangan yang berada di bawah perusahaan PT ASI Pudjiastuti Aviation. Pemilik dan CEO nya adalah Susi Pudjiastuti. Berpusat dan berkantor di Pangandaran, Jawa Barat, Susi Air memegang Sertifikat Operator Udara AOC 135-028. 288 Asian Journal of Applied Business and Management (AJABM) Vol. 2, No. 2, 2023: 285-294 PT ASI Pudjiastuti Aviation didirikan pada tahun 2004 dan Susi Air mulai beroperasi dengan 2 pesawat pada bulan Desember 2004. Operasi pertama dilakukan di Medan pada tanggal 27 Desember 2004. Tujuannya adalah kemanusiaan, sebagai tanggapan terhadap gempa bumi dan tsunami. Sejak itu, 2 pesawat Susi Air telah secara permanen berbasis di Medan. Pada tahun 2006 Susi Air mulai mengembangkan rute terjadwal yang berbasis di luar Medan dan telah berkembang secara signifikan. Hingga saat ini Susi Air mengoperasikan 49 armada pesawat yang terdiri atas32 Cessna Grand Caravan C208B, 9 Pilatus PC-6 Turbo Porter, 3 Piaggio P180 Avanti II, 1 Air Tractor AT802 “Fuel Hauler”, 1 Piper Archer PA-28 dan 1 LET 410 untuk pesawat sayap tetap. Operasi helikopter dimulai pada akhir 2009 dengan 1 Agusta Westland Grand A109S dan 1 Agusta Westland Koala A119Ke bergabung dengan armada pada Maret 2010. g p Susi Air mulai membuka penerbangan perdana untuk 6 wilayah, yakni Dabo, Singkep, Gunung Sitoli, Sumenep, Ternate, dan Masamba pada 4 Januari 2021. Kemudian pada 6 Januari 2021 untuk wilayah Manokwari, Merauke, dan Wamena. Perusahaan Susi Air telah memiliki 20 basis operasional utama di Medan, Banda Aceh, Padang, Dabo, Bengkulu, Jakarta, Pangandaran, Palangkaraya, Samarinda, Tarakan, Malinau, Kupang, Masamba, Manokwari, Biak, Nabire, Timika, Jayapura, Wamena, dan Merauke. Operasi didukung lebih dari 140 pesawat, 75 insinyur dan mekanik pesawat, serta 650 staf darat dan pendukung lainnya. Hingga kini,Susi Air sukses mencapai 150-225 penerbangan per harinya. Sebanyak 40.800 jam penerbangan telah ditempuh. Kemudian telah melayani 196 rute domestik, 164 tujuan domestik, dan 32 rute komersial. a. Oktober 2008: Pesawat Susi Air jenis Diamond DA-40 melakukan pendaratan darurat di Lapangan tembak Pusat Pendidikan Infanteri, Kabupaten Bandung Barat. Penyelidikan kecelakaan dilakukan oleh Indonesia Komite Nasional Keselamatan Transportasi. Mereka menemukan bahwa pilot tidak berlisensi di Indonesia. Kecelakaan itu disebabkan oleh kelaparan bahan bakar karena kegagalan pompa bahan bakar. b. 9 September 2011: Pesawat Susi Air jenis Caravan C 208 B pk-VVE, dari Wamena, Papua, menuju Kenyem, jatuh di Distrik Pasema Kabupaten Yahukimo, sekitar pukul 12.20 WIT.Kedua pilot tewas dan puing-puing ditemukan di daerah pegunungan. HASIL PENELITIAN c. 9 September 2011: Pesawat Susi Air Cessna 208B Grand Caravan (PK BVQ) meluncur dari landasan pacu di Kupang bandara El Tari. Insiden ini mengakibatkan pesawat menghalangi landasan pacu selama 50 menit dan menyebabkan dua pesawat Boeing 737 penerbangan komersial untuk mengalihkan ke Makassar, Sulawesi. c. 9 September 2011: Pesawat Susi Air Cessna 208B Grand Caravan (PK BVQ) meluncur dari landasan pacu di Kupang bandara El Tari. Insiden ini mengakibatkan pesawat menghalangi landasan pacu selama 50 menit dan menyebabkan dua pesawat Boeing 737 penerbangan komersial untuk mengalihkan ke Makassar, Sulawesi. d. 23 November 2011: Pesawat Cessna 208B Grand Caravan (PK VVG) hancur setelah lepas landas di Bandara Sugapa, Nabire, Papua. Kecelakaan pesawat terjadi setelah menghindari landasan pacu di sebuah landasan pacu kurang dikelola di Pegunungan Bintang di Papua Barat. Insiden tersebut menewaskan seorang kopilot asal Spanyol, Albert d. 23 November 2011: Pesawat Cessna 208B Grand Caravan (PK VVG) hancur setelah lepas landas di Bandara Sugapa, Nabire, Papua. Kecelakaan pesawat terjadi setelah menghindari landasan pacu di sebuah landasan pacu kurang dikelola di Pegunungan Bintang di Papua Barat. Insiden tersebut menewaskan seorang kopilot asal Spanyol, Albert 289 Agustini, Ritonga, Sari, Barus, Khairunnisa Citores. Sedangkan pilot Jesse Becker serta dua awak yang mengoperasikan penerbangan kargo dalam keadaan luka parah. Citores. Sedangkan pilot Jesse Becker serta dua awak yang mengoperasikan penerbangan kargo dalam keadaan luka parah. Citores. Sedangkan pilot Jesse Becker serta dua awak yang mengoperasikan penerbangan kargo dalam keadaan luka parah. g g g e. Maret 2021: Pesawat Susi Air sempat disandera Kelompok Kriminal Bersenjata (KKB) Papua selama dua jam di lapangan terbang Wangbe, Kabupaten Puncak pada Maret 2021. e. Maret 2021: Pesawat Susi Air sempat disandera Kelompok Kriminal Bersenjata (KKB) Papua selama dua jam di lapangan terbang Wangbe, Kabupaten Puncak pada Maret 2021. f. 23 Juni 2022: Pesawat Susi Air mengalami kecelakaan saat terbang dari rute Timika, Kabupaten Mimika, Papua ke Kampung Duma, Kabupaten Nduga. Pesawat Susi Air dilaporkan membawa 7 orang penumpang termasuk pilot, Capt Doyle Peter PEMBAHASAN Prosedur Pendirian Perseroan Terbatas Perorangan Prosedur Pendirian Perseroan Terbatas Perorangan Pengajuan Nama Perseroan Terbatas. Adapun persyaratan yang dibutuhkan sebagai berikut: melampirkan asli formulir dan pendirian surat kuasa, melampirkan photocopy Kartu Identitas Penduduk (KTP) para pendirinya dan para pengurus perusahaan, melampirkan photocopy Kartu Keluarga (KK) pimpinan/pendiri PT. Proses ini bertujuan untuk melakukan pengecekan nama PT, dimana pemakaian PT tidak boleh sama atau mirip sekali dengan nama PT yang sudah ada maka yang perlu siapkan adalah dua atau tiga pilihan nama PT, usahakan nama PT mencerminkan kegiatan usaha anda. Disamping itu, pendaftaran nama PT ini bertujuan untuk mendapatkan persetujuan dari instansi terkait (Kemenkumham) sesuai dengan UUPT dan Peraturan Pemerintah Republik Indonesia Nomor 43 Tahun 2011 Tentang Tata Cara Pengajuan dan Pemakaian Nama Perseroan Terbatas. g j Pembuatan Akta Pendirian. Hal-hal yang perlu diperhatikan dalam pembuatan akta ini, yaitu: Kedudukan PT, yang mana PT harus berada di wilayah Republik Indonesia dengan menyebutkan nama Kota dimana PT melakukan kegiatan usaha sebagai Kantor Pusat, Pendiri PT minimal 2 orang atau lebih, menetapkan jangka waktu berdirinya PT selama 10 tahun, 20 tahun atau lebih atau bahkan tidak perlu ditentukan lamanya artinya berlaku seumur hidup, menetapkan maksud dan tujuan serta kegiatan usaha PT, akta notaris yang berbahasa Indonesia, setiap pendiri harus mengambil bagian atas saham, kecuali dalam rangka peleburan, modal dasar minimal Rp50.000.000,00 (lima puluh juta Rupiah) dan modal disetor minimal 25% (duapuluh lima persen) dari modal dasar, minimal 1 orang Direktur dan pemegang saham harus WNI atau Badan Hukum yang didirikan menurut hukum Indonesia, kecuali PT dengan Modal Asing atau biasa disebut PT PMA. Pembuatan SKDP. Permohonan SKDP (Surat Keterangan Domisili Perusahaan) diajukan kepada kantor kelurahan setempat sesuai dengan alamat kantor PT Anda berada, yang mana sebagai bukti keterangan alamat perusahaan (domisili gedung, jika di gedung). Persyaratan lain yang dibutuhkan adalah: photocopy Pajak Bumi dan Bangunan (PBB) tahun terakhir, perjanjian sewa atau kontrak tempat usaha bagi yang berdomisili bukan di gedung perkantoran, Kartu Tanda Penduduk (KTP) Direktur, Izin Mendirikan Bangun (IMB) jika PT tidak berada di gedung perkantoran. 290 Asian Journal of Applied Business and Management (AJABM) Vol. 2, No. 2, 2023: 285-294 Asian Journal of Applied Business and Management (AJABM) Vol. 2, No. 2, 2023: 285-294 Pembuatan NPWP. Permohonan pendaftaran NPWP diajukan kepada Kepala Kantor Pelayanan Pajak sesuai dengan keberadaan domisili PT. Persyaratan lain yang dibutuhkan, adalah: NPWP pribadi Direktur PT, photocopy KTP Direktur (atau photocopy Paspor bagi WNA, khusus PT PMA), SKDP, dan akta pendirian PT. p Pembuatan Anggaran Dasar Perseroan. Dasar Hukum Pendirian PT Perseroan Terbatas Perorangan Undang-Undang Republik Indonesia Nomor 11 Tahun 2020 Tentang Cipta Kerja, Peraturan Pemerintah Republik Indonesia Nomor 8 Tahun 2021 Tentang Modal Dasar Perseroan Serta Pendaftaran Pendirian, Perubahan, dan Pembubaran Perseroan Yang Memenuhi Kriteria Untuk Usaha Mikro dan Kecil dan Peraturan Pemerintah Republik Indonesia Nomor 7 Tahun 2021 Tentang Kemudahan, Pelindungan, Dan Pemberdayaan Koperasi dan Usaha Mikro, Kecil, dan Menengah. Kelebihan PT Perseroan Terbatas Perorangan Keuntungan Usaha Sepenuhnya Di Tangan Pemilik Usaha. Pada dasarnya, berbentuk PT perorangan. Perorangan dibentuk hanya satu orang saja. Semua resiko, kendala, kerugian, dan juga keuntungan akan ditanggung dan dihadapi oleh pemilik usaha itu sendiri. Dengan demikian keuntungan usaha sepenuhnya bisa berada pada tangan si pemilik usaha itu sendiri. Pemilik Usaha Memegang Kekuasaan Penuh. Pada PT perorangan usaha keseluruhannya akan dikelola oleh manajemen perusahaannya seorang diri, seperti didalam hal jabatan direktur, manajer, dan pelaksanaan harian perusahaan juga ditanganinya sendiri. Dengan adanya pemilik usaha akan menjadi pemeran utama dalam mengambil dan mengatur kebijakan pada setiap aspek dalam perusahaannya, seperti dalam aktivitas penjualan seharihari, komunikasi dengan pihak pembeli, pihak ketiga yang mempunyai kepentingan dalam perusahaan, serta dalam pengolahan keuangan dan data perusahaan. p Memiliki Persyaratan Yang Mudah. Syarat Hal ini tentu saja berbeda dengan syarat pendirian CV dan PT. Dalam mendirikan PT perorangan tidak memerlukan akta yang dibuat khusus oleh notaris, dan juga tidak memerlukan pendaftaran pada pengesahan dan pengadilan oleh kementrian Hukum dan Hak Asasi Manusia (HAM), dan juga pelaku usaha bisa bebas dalam menggunakan nama perusahaan tidak ada yang mengaturnya. Memiliki Modal Pendirian Kecil. Pada hakikatnya, PT Perorangan atau Perusahaan Perorangan memang ditujukan bagi para pelaku usaha UMKM sehingga modal dalam pendirian usahanya pun terbilang cukup minim. Kategori UMKM yang bisa melakukan pendaftaran dan pendirian PT (Perseroan Terbatas). Prosedur Pendirian Perseroan Terbatas Perorangan Permohonan ini diajukan kepada Menteri Kemenkumham untuk mendapatkan pengesahan Anggaran Dasar Perseroan (akta pendirian) sebagai badan hukum PT sesuai dengan UUPT. Persyaratan yang dibutuhkan antara lain: bukti setor bank senilai modal disetor dalam akta pendirian, Bukti Penerimaan Negara Bukan Pajak (PNBP) sebagai pembayaran berita acara negara dan akta asli pendirian. p y g p Mengajukan Surat Izin Usaha Perdagangan (SIUP). SIUP ini berguna agar PT dapat menjalankan kegiatan usahanya. Namun perlu untuk diperhatikan bahwa setiap perusahaan patut membuat SIUP, selama kegiatan usaha yang dijalankannya termasuk dalam Klasifikasi Baku Lapangan Usaha Indonesia (KBLUI) sebagaimana Peraturan Kepala Badan Pusat Statistik Nomor 57 Tahun 2009 Tentang Klasifikasi Baku Lapangan Usaha Indonesia. Permohonan pendaftaran SIUP diajukan kepada Kepala Suku Dinas Perindustrian dan Perdagangan atau Koperasi Usaha Mikro Kecil Menengah dan Perdagangan kota atau kabupaten terkait sesuai dengan domisili PT. Adapun klasifikasi dari SIUP berdasarkan Peraturan Menteri Perdagangan No.39/M- DAG/PER/12/2011 Tentang Perubahan Kedua Atas Peraturan Menteri Perdagangan No.36/M-DAG/PER/9/2007 tentang Penerbitan Surat Izin Usaha Perdagangan adalah sebagai berikut: SIUP Kecil, wajib dimiliki oleh perusahaan perdagangan yang kekayaan bersihnya lebih dari Rp50.000.000,00 (lima puluh juta rupiah) sampai dengan paling banyak Rp500.000.000,00(lima ratus juta rupiah) tidak termasuk tanah dan bangunan tempat usaha; SIUP Menengah wajib dimiliki oleh perusahaan perdagangan yang kekayaan bersihnya lebih dari Rp500.000.000,00 (lima ratus juta rupiah) sampai dengan paling banyak Rp10.000.000.000,00 (sepuluh milyar Rupiah) tidak termasuk tanah dan bangunan tempat Usaha; SIUP Besar, wajib dimiliki oleh perusahaan perdagangan yang kekayaan bersihnya lebih dari Rp10.000.000.000,00(sepuluh milyar Rupiah) tidak termasuk tanah dan bangunan tempat usaha. y p ) g p Mengajukan Tanda Daftar Perusahaan (TDP). Permohonan pendaftaran diajukan kepada Kepala Suku Dinas Perindustrian dan Perdagangan dan/atau Koperasi Usaha Mikro Kecil Menengah dan Perdagangan kota atau kabupaten terkait sesuai dengan domisili perusahaan. Bagi perusahaan yang telah terdaftar akan diberikan sertifikat TDP sebagai bukti bahwa perusahaan/badan usaha telah melakukan wajib daftar perusahaan sesuai dengan Peraturan Menteri Perdagangan Republik Indonesia No.37/MDAG/PER/9/2007 tentang Penyelenggaraan Pendaftaran Perusahaan. Berita Acara Negara Republik Indonesia (BNRI). Setelah perusahaan melakukan wajib daftar perusahaan dan telah mendapatkan pengesahan dari Menteri Kemenkumham, maka harus di umumkan dalam BNRI dari perusahaan yang telah diumumkan dalam BNRI, maka PT telah sempurna statusnya sebagai badan hukum. Demikianlah prosedur pendirian perusahaan 291 Agustini, Ritonga, Sari, Barus, Khairunnisa terbatas perorangan. Sebagai pemilik perusahaan, pastikan Anda memahami dan memenuhi semua persyaratan hukum yang terkait dengan pendirian perusahaan. Dasar Hukum Pendirian PT Perseroan Terbatas Perorangan KESIMPULAN DAN REKOMENDASI Meski pendirinya hanya satu orang, akan tetapi perlu ditegaskan bahwa PT (Perseroan Terbatas) Perorangan statusnya tetap badan hukum sama seperti PT (Perseroan Terbatas) yang selama ini kita kenal dengan adanya minimal 2 Pemegang saham dan pendiri (selanjutnya disebut PT biasa). Status PT Perorangan atau Perseroan Perorangan sebagai badan hukum ditegaskan di dalam Pasal 1 PP Nomor.8 Tahun 2021 yang menyebutkan Perseroan Terbatas, yang selanjutnya disebut Perseroan adalah badan hukum yang merupakan persekutuan modal, didirikan berdasarkan perjanjian, melakukan kegiatan usaha dengan modal dasar yang seluruhnya terbagi dalam saham atau badan hukum perorangan yang memenuhi kriteria usaha mikro dan kecil sebagaimana diatur dalam peraturan perundang-undangan mengenai usaha mikro dan kecil. Dalam penelitian ini masih perlu dilanjutkan dengan proses yang lebih mendalam kaitannya tentang prosedur pendirian PT Perorangan baik dengan membandingkan dengan beberapa PT Perorangan lainnya tentang prosedur PT Perorangan. p g PP No 8 tahun 2021 selanjutnya, menentukan bahwa kriteria modal mikro adalah usaha dengan modal di bawah Rp1.000.000.000,00 (satu miliar rupiah) dan usaha kecil dengan modal anatara Rp1.000.000.000,00 (satu miliar rupiah) sampai dengan Rp5.000.000.000,00 (lima miliar rupiah). Asian Journal of Applied Business and Management (AJABM) Vol. 2, No. 2, 2023: 285-294 perkembangan usaha dikarenakan biasanya jika ada suatu usaha ingin mengikuti tawaran atau tender resmi dari mitra lain pelaku usaha wajib memenuhi terlebih dahulu persyaratan yaitu minimal berbadan hukum PT atau minimal CV. Resiko Kerugian Di Tangan Pribadi. Pendirian PT Perorangan seperti yang sudah dijelaskan diatas bahwa PT Perorangan atau Perusahaan Perorangan dibentuk hanya oleh satu orang saja dan semua resiko, kendala, kerugian akan ditanggung dan dihadapi oleh pemilik usaha itu sendiri. Kelangsungan Usaha Relatif Singkat. PT perorangan atau perusahaan perorangan yang didirikan hanya satu orang pendiri saja dan tidak selalu baik dalam sistem manajemen perusahaan perorangan karena terkadang pelaku usaha hanya cenderung kewalahan dalam mengambil dan mengatur keputusannya sendiri sehingga bisa menimbulkan masalah dalam mengembangkan kemajuan usahanya. Potensi Manajemen Administrasi Perusahaan Lemah. Perusahaan Perorangan yang didirikan oleh satu orang pendiri saja, tentunya akan membawa resiko bagi sistem manajemen administrasi atau juga tata kelola perusahaannya. Dimana dalam hal ini, data pengelolaan keuangan dan perusahaan dapat terkelola secara kurang maksimal. Kekurangan PT Perseroan Terbatas Perorangan Kelonggaran Perizinan Dapat Menghambat Perkembangan Usaha. PT Perorangan bisa dikategorikan atau dikatakan sebagai usaha skala mikro, maka merek hanya cukup dengan mengurus surat izin usaha mikro dan kecil saja dan tidak memerlukan pembuatan serta mempunyai surat izin usaha perdagangan dan tanda daftar perusahaan. Akan tetapi dengan ketiadaan SIUP (Surat Izin Usaha Perdagangan) ini justru bisa menghambat dalam 292 Asian Journal of Applied Business and Management (AJABM) Vol. 2, No. 2, 2023: 285-294 PENELITIAN LANJUTAN Penelitian ini masih memiliki keterbatasan, maka perlu dilakukan penelitian lanjutan mengenai topik “Prosedur Pendirian Perseroan Terbatas Perorangan” untuk menyempurnakan penelitian ini, serta memberi informasi bagi pembaca. 293 Agustini, Ritonga, Sari, Barus, Khairunnisa UCAPAN TERIMA KASIH Puji dan syukur kami panjatkan kepada Tuhan Yang Maha Esa, karena atas berkat dan rahmat-Nya, kami dapat menyelesaikan jurnal ini. Penulisan jurnal ini dilakukan dalam rangka untuk memenuhi salah satu tugas mata kuliah regulasi bisnis, Universitas Negeri Medan. Kami menyadari bahwa tanpa bantuan dan bimbingan dari berbagai pihak, cukup sulit bagi kami untuk menyelesaikan karya tulis ilmiah ini. Oleh sebab itu kami mengucapkan terima kasih kepada Ibu Sabda Dian Nurani Siahaan S.Pd., M.B.A selaku sebagai Dosen pengampu mata kuliah Regulasi Bisnis, serta teman sekelompok yaitu Nursaiba Ritonga, Devi Marsella Br Barus, Rifqa Khairunnisa dan Indah Purnama Sari. DAFTAR PUSTAKA Perkembangan Hukum PT Perorangan (One Person Company) http://digilib.iblam.ac.id/140/1/TESIS%20AISHA%20IBLAM_merged.pdf http://openjournal.unpam.ac.id/index.php/rjih/article/view/12669 https://ejournal.undip.ac.id/index.php/notarius/article/download/43800/28 813 https://jurnal.uisu.ac.id/index.php/alhikmah/article/download/5951/4399 http://jurnal.unissula.ac.id/index.php/JIMU/article/download/26780/7461 https://profilpelajar.com/Susi_Air https://jurnal.usu.ac.id/index.php/jmim/article/view/2303/0 https://eprints.umk.ac.id/4533/3/BAB_II.pdf https://rechtsvinding.bphn.go.id/ejournal/index.php/jrv/article/download/ 405/237 294
https://openalex.org/W4250376219	http://oceanrep.geomar.de/8498/2/essd-2-79-2010.pdf	English	null	Nordic Seas and Arctic Ocean CFC data in CARINA	null	2,009	cc-by	10,903	Open Access Earth System Science Data Open Access Earth System Science Data Open Access Earth System Science Data Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. Nordic Seas and Arctic Ocean CFC data in CARINA Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson1, K. A. Olsson2,, T. Tanhua3, and J. L. Bullister4 1Bjerknes Centre for Climate Research, Uni Research, Bergen, Norway 2Department of Chemistry, University of Gothenburg, G¨oteborg, Sweden 3Leibniz-Institut f¨ur Meereswissenshaften, Marine Biogeochemie, Kiel, Germany 4Paciﬁc Marine Environmental Laboratory, NOAA, Seattle, Washington, USA present address: Volvo Technology Corporation, Environment & Chemistry, Gothenburg, Sweden Received: 22 September 2009 – Published in Earth Syst. Sci. Data Discuss.: 16 October 2009 Revised: 1 February 2010 – Accepted: 9 February 2010 – Published: 15 February 2010 Abstract. Water column data of carbon and carbon relevant hydrographic and hydrochemical parameters have been retrieved from a large number of cruises and collected into a new database called CARINA (CARbon IN the Atlantic). These data have been merged into three sets of ﬁles, one for each of the three CARINA regions; the Arctic Mediterranean Seas (AMS), the Atlantic (ATL) and the Southern Ocean (SO). The ﬁrst part of the CARINA database consists of three ﬁles, one for each CARINA region, containing the original, non- adjusted cruise data sets, including data quality ﬂags for each measurement. These data have then been subject to rigorous quality control (QC) in order to ensure highest possible quality and consistency. The data for most of the parameters included were examined in order to quantify systematic biases in the reported values, i.e. secondary quality control. Signiﬁcant biases have been corrected for in the second part of the CARINA data product. This consists of three ﬁles, one for each CARINA region, which contain adjustments to the original data values based on recommendations from the CARINA QC procedures, along with calculated and interpolated values for some missing parameters. Here we present an overview of the QC of the CFC data for the AMS region, including the chloroﬂuorocarbons CFC-11, CFC-12 and CFC-113, as well as carbon tetrachloride (CCl4). The Arctic Mediterranean Seas is comprised of the Arctic Ocean and the Nordic Seas, and the quality control was carried out separately in these two areas. For the secondary QC of the CFCs we used a combination of tools, including the evaluation of depth proﬁles and CFC ratios, surface saturations and a crossover analysis. This resulted in a multiplicative adjustment of data from some cruises, while other data were ﬂagged to be of questionable quality, which excluded them from the ﬁnal data product. Data coverage and parameter measured Repository-Reference: doi: CARINA.AMS.V1.2, doi:10.3334/CDIAC/otg.CARINA.AMS.V1.2 Available at: http://cdiac.ornl.gov/oceans/CARINA/Carina inv.html Coverage: Nordic Seas: 59.60–82.35◦N, 35.23◦W–28◦E; Arctic Ocean: 60.7–90◦N, circumpolar Location Name: Nordic Seas; Arctic Ocean Correspondence to: E. Jeansson (emil.jeansson@uni.no) Nordic Seas: Date/Time Start: 1982-02-28; Nordic Seas: Date/Time End: 2003-10-13 Arctic Ocean: Date/Time Start: 1991-07-26; Arctic Ocean: Date/Time End: 1997-10-15 Published by Copernicus Publications. Data coverage and parameter measured Nordic Seas: Date/Time Start: 1982-02-28; Nordic Seas: Date/Time End: 2003-10-13 Arctic Ocean: Date/Time Start: 1991-07-26; Arctic Ocean: Date/Time End: 1997-10-15 Nordic Seas: Date/Time Start: 1982-02-28; Nordic Seas: Date/Time End: 2003-10-13 Arctic Ocean: Date/Time Start: 1991-07-26; Arctic Ocean: Date/Time End: 1997-10-15 Available at: http://cdiac.ornl.gov/oceans/CARINA/Carina inv.html Coverage: Nordic Seas: 59.60–82.35◦N, 35.23◦W–28◦E; Arctic Ocean: 60.7–90◦N, circumpolar Location Name: Nordic Seas; Arctic Ocean Correspondence to: E. Jeansson (emil.jeansson@uni.no) Correspondence to: E. Jeansson (emil.jeansson@uni.no) Published by Copernicus Publications. 80 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA Data Product Data Product Exchange File Exchange File Units Parameter Name Flag name Parameter Name Flag Name station STANBR day DATE month DATE year DATE latitude LATITUDE decimal degrees longitude LONGITUDE decimal degrees cruiseno depth Meters temperature CTDTMP ◦C salinity sf SALNTY SALNTY FLAG W ctdsal ctdsf CTDSAL CTDSAL FLAG W pressure CTDPRS decibars cfc11 cfc11f picomole kg−1 cfc12 cfc12f picomole kg−1 cfc113 cfc113f picomole kg−1 ccl4 ccl4f picomole kg−1 pf11 ppt pf12 ppt pf113 ppt pccl4 ppt pf11, pf12, pf113 and pccl4 are the partial pressures of the compounds, expressed in ppt (10−12 atmospheres). For a complete list of all parameters available in CARINA see Key et al. (2009). Note the diﬀerent names for the parameters in the Exchange ﬁles (the individual cruise ﬁles) and the merged data product. pf11, pf12, pf113 and pccl4 are the partial pressures of the compounds, expressed in ppt (10−12 atmospheres). For a complete list of all parameters available in CARINA see Key et al (2009) Note the diﬀerent names for the parameters in the Exch For a complete list of all parameters available in CARINA see Key et al. (2009). Note the diﬀerent names for the parameters in the Exchange ﬁles (the individual cruise ﬁles) and the merged data product. (such as 14C, 13C and SF6) reported in the individual cruise ﬁles than has been included in the secondary QC. The second part of the CARINA database consists of three merged data ﬁles, one for each region, and contains only the data consid- ered being of “good” quality. Data coverage and parameter measured Repository-Reference: doi: CARINA.AMS.V1.2, doi:10.3334/CDIAC/otg.CARINA.AMS.V1.2 Available at: http://cdiac.ornl.gov/oceans/CARINA/Carina inv.html Coverage: Nordic Seas: 59.60–82.35◦N, 35.23◦W–28◦E; Arctic Ocean: 60.7–90◦N, circumpolar Location Name: Nordic Seas; Arctic Ocean Correspondence to: E. Jeansson (emil.jeansson@uni.no) Nordic Seas: Date/Time Start: 1982-02-28; Nordic Seas: Date/Time End: 2003-10-13 Arctic Ocean: Date/Time Start: 1991-07-26; Arctic Ocean: Date/Time End: 1997-10-15 Published by Copernicus Publications. These ﬁles include: data values from the original cruise ﬁles, some of which have been ad- justed based on the secondary QC processes as part of CA- RINA (see Key et al., 2009); interpolated values for nutri- ents, oxygen and salinity if those data were missing for indi- vidual samples and if interpolation could be made according to criteria described in Key et al. (2009). It also contains calculated carbon parameters when applicable, e.g., if total dissolved inorganic carbon (TCO2) and alkalinity (TA) were measured, pH was calculated from these. E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA the parameter was not measured; Adjustments for the CFCs are multiplicative. Flag 3 means that the data are of questionable quality and are not included in the merged data product. Table 2. CARINA cruises with CFC data in the Arctic Ocean. ND denotes “no data”, i.e. the parameter was the CFCs are multiplicative. Flag 3 means that the data are of questionable quality and are not included in th Table 2. CARINA cruises with CFC data in the Arctic Ocean. ND denotes “no data”, i.e. the parameter was not measured; Adjustments for the CFCs are multiplicative. Flag 3 means that the data are of questionable quality and are not included in the merged data product. Cruise No. EXPOCODE Year Country/Institute P.I. Sampled area CFC11 CFC12 CFC113 CCl4 2 06AQ19930806 1993 USA/LDEO W. Smethie Laptev Sea 1 1 1 ND 3 06AQ19960712 1996 Canada/BIO;Germany/IfMK P. Jones/M. Rhein Laptev and Kara Seas 1 1 ND 1 47 18SN19940724 1994 Canada/BIO P. Jones Canadian Basin 1.05 1 Flag 3 1 48 18SN19970803a 1997 Canada/BIO P. Jones/K. Azetsu-Scott Baﬃn Bay 1 1 1 1 49 18SN19970831a 1997 Canada/BIO P. Jones/K. Azetsu-Scott Canadian archipelago 1 1 1 1 50 18SN19970924a 1997 Canada/BIO P. Jones/K. Azetsu-Scott Canada Basin 1 1 1 1 177 77DN19910726 1991 Canada/BIO P. Jones Eurasian Basin 1 1 ND 1 a The three 1997 cruises is really one cruise, divided in three parts. Abbreviations: BIO=Bedford Institute of Oceanography (Canada); IfMK=Institut f¨ur Meereskunde, Kiel (Germany); LDEO=Lamont Do- herty Earth Observatory (USA). Abbreviations: BIO=Bedford Institute of Oceanography (Canada); IfMK=Institut f¨ur Meereskunde, Kiel (Germany); LDEO=Lamont Do- herty Earth Observatory (USA). process can be found on the CARINA website at CDIAC, http://cdiac.ornl.gov/oceans/CARINA/Carina inv.html. Norwegian Seas (Bullister and Weiss, 1983). After that a decade passed without any CFC sampling in the AMS re- gion, i.e. the Arctic Ocean and the Nordic Seas. In 1991 annual measurements of transient tracers commenced in the Nordic Seas as a part of the Atlantic Climate Change Pro- gram (ACCP) and later by CLIVAR (Climate Variability and Prediction) Atlantic activities. These measurements went on 1 Introduction CARINA (CARbon IN the Atlantic) is a database of carbon relevant data from hydrographic cruises in the Arctic, At- lantic and Southern Oceans. The database was initiated as an essentially informal, unfunded project in Kiel, Germany in 1999, with the main goal to create a database of carbon relevant variables in the ocean to be used for accurate assess- ments of oceanic carbon inventories and uptake rates. Not only the collection of data, but also the quality control of the data has been a main focus of the project. During the project, both primary and secondary quality control (QC) of the data has been performed. The CARINA database con- sists of essentially two parts: The ﬁrst part is the individual cruise ﬁles containing all the data that were reported for dis- crete water samples collected during the cruise, along with the corresponding data quality ﬂag for each measurement. In most cases, the groups directly involved in making the mea- surements provided the data values contained in these ﬁles. These ﬁles are in WOCE Hydrographic Program (WHP) ex- change format where the ﬁrst lines consist of the condensed metadata. Information on these ﬁle formats can be found in the WHP manuals at http://whpo.ucsd.edu/manuals.html. The values in these ﬁles are based on the direct measure- ments made on the individual water samples and are not cal- culated from other parameters or interpolated from surround- ing measurements; i.e. no adjustments have been applied to the reported values. In many cases there are more parameters Here we describe the data and present an overview of the secondary QC of the chloroﬂuorocarbon (CFC) com- pounds CFC-11, CFC-12 and CFC-113, and carbon tetra- chloride (CCl4). In this study we treat CCl4 as a member of the CFC family of chemical compounds, despite not be- ing a “true” CFC. All the Arctic Mediterranean Seas (AMS) CFC values in the merged data ﬁle have been adjusted ac- cording to the values in Table 1 (Nordic Seas) and Table 2 (Arctic Ocean). For an overview of all parameters and ad- justments of the AMS data the reader is referred to Olsen et al. (2009) and Jutterstr¨om et al. (2010). Information on the secondary QC of oxygen, nutrients, TCO2 and TA data is found in Falck and Olsen (2009), Olafsson and Olsen (2010), Olsen (2009a and b), respectively. More details to the Earth Syst. Sci. 1 Introduction Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ 81 Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA Table 1. CARINA cruises with CFC data in the Nordic Seas. ND denotes “no data”, i.e. the parameter was not measured. Adjustments for the CFCs are multiplicative and the presented numbers been applied in the merged data product. Flag 3 means that the data are of questionable quality and are not included in the merged data product. Cruise No.a EXPOCODE Year Country/Institute P.I. CFC11 CFC12 CFC113 CCl4 36 18HU19820228 1982 USA/SIO R. Weiss/D. Wallace 1.05 1 ND ND 67 316N20020530 2002 USA/LDEO W. Smethie 1 1 0.9 ND 91 34AR19970805 1997 Sweden/UGOT T. Tanhua/A. Olsson 0.95b 1 1 1 117 58AA19940224 1994 Sweden/UGOT E. Fogelqvist 1 Flag 3 ND 0.9 119 58AA19950217 1995 Sweden/UGOT A. Olsson/T. Tanhua 1.1 Flag 3 Flag 3 ND 120 58AA19961121 1996 Sweden/UGOT E. Fogelqvist/T. Tanhua/A. Olsson 1 1 ND ND 125 58AA20010527 2001 Sweden/UGOT A. Olsson/E. Jeansson 1 0.95 ND ND 128 58GS20030922 2003 Sweden/UGOT A. Olsson/E. Jeansson 1 0.95 ND ND 129 58JH19911105 1991 USA/BNL; USA/PMEL D. Wallace/J. Bullister 1 ND ND 1 131 58JH19921105 1992 USA/BNL; USA/PMEL D. Wallace/J. Bullister 0.95 1 1 1.05 133 58JH19931106 1993 USA/BNL; USA/PMEL D. Wallace/J. Bullister 1 1 0.95 ND 135 58JH19940723 1994 Sweden/UGOT E. Fogelqvist/T. Tanhua Flag 3 Flag 3 Flag 3 Flag 3 136 58JH19941028 1994 USA/BNL; USA/PMEL D. Wallace/J. Bullister 1 1 1 1 138 58JH19951108 1995 USA/BNL; USA/PMEL D. Wallace/J. Bullister 1 0.95 1 1 140 58JH19961030 1996 USA/PMEL; USA/BNL J. Bullister/D. Wallace 1 1 1.05 1 141 58JH19970414 1997 USA/PMEL; USA/BNL J. Bullister/D. Wallace 1 1 1 1 142 58JH19980801 1998 USA/PMEL; USA/BNL J. Bullister/D. Wallace 1 1.05 1.1 1 143 58JH19990615 1999 USA/PMEL; USA/BNL J. Bullister/D. Wallace 1 1 Flag 3 1 144 58JH20000527 2000 USA/PMEL; USA/BNL J. Bullister/D. Wallace 1 1.05 Flag 3 0.95 176 74JC19960720 1996 Sweden/UGOT E. Fogelqvist/A. Olsson 1 1 1 Flag 3 179 77DN20020420 2002 Canada/BIO P. Jones 1 1 Flag 3c Flag 3c Abbreviations: BIO=Bedford Institute of Oceanography (Canada); BNL=Brookhaven National Laboratory (USA); LDEO=Lamont Doherty Earth Observatory (USA); PMEL=Paciﬁc Marine Environmental Laboratory, NOAA (USA); SIO=Scripps Institution of Oceanography (USA); UGOT=University of Gothenburg (Sweden). Abbreviations: BIO=Bedford Institute of Oceanography (Canada); BNL=Brookhaven National Laboratory (USA); LDEO=Lamont Doherty Earth Observatory (USA); PMEL=Paciﬁc Marine Environmental Laboratory, NOAA (USA); SIO=Scripps Institution of Oceanography (USA); UGOT=University of Gothenburg (Sweden). Table 2. CARINA cruises with CFC data in the Arctic Ocean. ND denotes “no data”, i.e. 2.0.1 CFC partial pressure In addition to the concentration values, the CARINA data product also includes calculated partial pressures for the dis- solved CFC (and for CCl4) samples; the pCFC data, ex- pressed with units of pico (10−12) atmospheres or parts-per- trillion (ppt). The pCFC of a water sample is computed from the expression pCFC=CSW/F(Θ, S) (Doney and Bullis- ter, 1992), where CSW is the concentration (in pmol kg−1) of the dissolved CFC in the seawater sample and F(Θ, S) is the solubility of the CFC as a function of potential temper- ature and salinity (Warner and Weiss, 1985; Bu and Warner, 1995; Bullister and Wisegarver, 1998). This conversion of dissolved CFC concentrations to pCFCs removes the strong dependency on the CFC concentration on the temperature and salinity of the seawater sample and makes it possible to directly compare the levels of dissolved CFCs in the ocean with the equivalent equilibrium atmospheric partial pressures or atmospheric mixing ratios. Figure 1. Map of the Arctic Mediterranean Seas showing all sta- tions with CFC data in the CARINA data collection. until 2000 within the repeated hydrographic surveys con- ducted by the Institute of Marine Research (IMR) in Bergen, Norway (B¨onisch et al., 1997; Karstensen et al., 2005; see also Olsen et al., 2009). The CFC measurement program was headed by Brookhaven National Laboratory (BNL) be- tween 1991 and 1995 and by NOAA’s Paciﬁc Marine Envi- ronmental Laboratory (PMEL) between 1996 and 2000 (e.g., Karstensen et al., 2005). Between 1995 and 2003 consider- able CFC sampling was also conducted during the EU funded projects ESOP, ESOP II and TRACTOR, mostly by Univer- sity of Gothenburg (UGOT). The data from these projects are all included in the Nordic Seas part of the CARINA database, where 21 out of the total 35 cruises contain CFC data. In addition the CARINA data set includes 26 cruises from the Arctic Ocean, and seven of those contain CFC data, collected between 1991 and 1997. These cruises sampled the diﬀerent basins of the Arctic Ocean, but also the Baﬃn Bay and the Canadian archipelago. The Arctic Ocean data are described in the end of this paper (Sect. 7). 2 Data Provenance and Structure The cruise on C.S.S. Hudson in the winter of 1982 col- lected an extensive set of CFC samples in the Greenland and Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 82 Figure 1. Map of the Arctic Mediterranean Seas showing all sta- tions with CFC data in the CARINA data collection. ibration scale (http://bluemoon.ucsd.edu/pub/cfchist/). More information of the CFC data from each individual cruise can be found in the metadata accompanying the individual cruise ﬁles, http://cdiac.ornl.gov/oceans/CARINA/Carina inv.html. Depth proﬁles of all CFCs for the Nordic Seas cruises are presented in Figs. 2–5 and relationships between CFC-11 and the other CFCs can be found in Figs. 6–8. These ﬁgures have served a valuable tool in the QC and will be referred to when appropriate. The cruise ID (EXPOCODE) is com- posed of the ships code and the starting date of the cruise and is given in Tables 1 and 2. More information of the EX- POCODES and the ships included in the Nordic Seas Carina can be found in Olsen et al. (2009). ibration scale (http://bluemoon.ucsd.edu/pub/cfchist/). More information of the CFC data from each individual cruise can be found in the metadata accompanying the individual cruise ﬁles, http://cdiac.ornl.gov/oceans/CARINA/Carina inv.html. 2.1 Analysis approach As with the other parameters of CARINA, also the CFC data were subject to secondary quality control (QC). However, the transient behaviour of these compounds present some inher- ent problems with identifying and quantifying oﬀsets in a straightforward way, as can be done for most of the other parameters. Therefore, a number of tools are used for the consistency control of the data and adjustments are recom- mended if there is consensus in the results from the diﬀer- ent methods. For the quality evaluation of the Nordic Seas CFC data we used depth proﬁles of the CFCs, relationships between CFC-11 and the other CFCs and the surface satu- rations of the CFCs. The CFC data also underwent a con- sistency analysis using crossover and inversion approaches (Johnson et al., 2001; Tanhua et al., 2010), customised for the Nordic Seas (Olsen et al., 2009). An overview of the Nordic Seas data included in the sec- ondary QC can be found in Table 1, and the station positions in Fig. 1. The CFC data in the CARINA data set have all been determined by a similar analytical method, based on a purge and trap pre-treatment technique in combination with gas chromatography with electron capture detection (e.g., Bullis- ter and Weiss, 1983; Fogelqvist, 1999). The values have been calibrated against gas-phase standards. The precision of the analysis is typically between 1 and 2%, and the over- all accuracy before secondary QC is typically estimated to be roughly twice that. All results are reported on the SIO-98 cal- A useful tool for quality control of CFCs is the sur- face saturation, which is the ratio of the measured surface (<20 dbar) concentration to the calculated equilibrium con- centration of the CFC. The equilibrium concentration of a Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 83 CFC-11 vs. pressure (dbar) for all Nordic Seas cruises in CARINA. The red dots mark data from each speciﬁc cruise, an the CFC-11 data from all Nordic Seas CARINA cruises. The inserted map shows the speciﬁc cruise track. www.earth-syst-sci-data.net/2/79/2010/ 2.1 Analysis approach function of time is calculated from the solubility mpound and the atmospheric history of the CFC t al., 2000; Bullister 2008, http://cdiac.ornl.gov/ w atmCFC html) In much of the surface layer CFC-11 and CFC-12 are close to equilibrium with lying atmosphere. Consistently, most cruises in th Seas CARINA show surface saturations for CFC CFC 12 between 90 and 100% of equilibrium w Figure 2. CFC-11 vs. pressure (dbar) for all Nordic Seas cruises in CARINA. The red dots mark data from each speciﬁc cruise, and the grey dots shows the CFC-11 data from all Nordic Seas CARINA cruises. The inserted map shows the speciﬁc cruise track. CFC-11 and CFC-12 are close to equilibrium with the over- lying atmosphere. Consistently, most cruises in the Nordic Seas CARINA show surface saturations for CFC-11 and CFC-12 between 90 and 100% of equilibrium, with some clear exceptions (see Fig. 9). However, saturations in the CFC as a function of time is calculated from the solubility of the compound and the atmospheric history of the CFC (Walker et al., 2000; Bullister 2008, http://cdiac.ornl.gov/ oceans/new atmCFC.html). In much of the surface layer of the world ocean the observed concentrations of dissolved www.earth-syst-sci-data.net/2/79/2010/ Earth Syst. Sci. Data, 2, 79–97, 2010 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 84 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CFC-12 vs. pressure (dbar). See Fig. 2 for speciﬁcations. Figure 3. CFC-12 vs. pressure (dbar). See Fig. 2 for speciﬁcations. surface waters of the Greenland Sea of ∼80% have been re- ported by several authors (Bullister and Weiss, 1983; Rhein, 1991; Anderson et al., 2000). This low saturation is gen- erally considered to be a feature of deep-water formation regions where gas exchange rates are not rapid enough to bring the exceptionally deep winter mixed layers into equi- librium with the overlying atmosphere. Thus, low saturation levels do not prove that there is an oﬀset in the data. Further- more, the CFC saturations are possibly time-dependent in the North Atlantic, with a lower degree of saturation prior to the 1990s when the atmospheric increase was faster (Tanhua et al., 2008), and this might also be expected for the Nordic Seas. 2.1 Analysis approach 2 for speciﬁcations. E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 85 Figure 4. CFC-113 vs. pressure (dbar). See Fig. 2 for speciﬁcations. al., 2001; Roether et al., 2001), which makes comparisons with the other CFCs more diﬃcult. However, these non- stable behaviours appear to be in part temperature dependent, with more rapid removal rates in warmer water (≥13 ◦C). Since the surface waters of the Nordic Seas generally are sig- niﬁcantly colder than 13 ◦C, average removal rates, although still signiﬁcant, may be slower in the AMS than in some other regions. ferent cruises, within 300 km from each other. Since two cruises can cross at several points, in diﬀerent hydrographic regimes, each cruise pair’s crossover stations were divided into groups of stations, or clusters; up to 6 clusters were de- ﬁned, each within a circle of maximum radius of 175 km. Proﬁles of the cruises in each cluster were interpolated us- ing a piecewise cubic hermite interpolating approach, which minimizes extrapolation. The interpolated proﬁles of each cruise were then averaged and an oﬀset and standard devi- ation proﬁle for each cluster was calculated from data be- low 1900 dbar. From these proﬁles a weighted mean oﬀset and weighted mean standard deviation were determined for each cruise pair and used for the inversion that calculated the corrections. The inversion method closely followed that of Johnson et al. (2001). They presented three models of diﬀerent complexity to adjust a number of parameters and found that a model of intermediate complexity, the Weighted Least Square (WLSQ), showed the most satisfactory results. In the WLSQ model the standard deviation of each crossover is included in the calculation, but no a priori assumptions are made about the quality of the measurements. When this assumption is made a maximum allowed range of adjust- ments is set for each cruise, which tends to decrease the ad- justments of individual cruises on cost of the overall perfor- mance. Models that do not include the uncertainty of the oﬀset values are considered too simple (Johnson et al., 2001; Tanhua et al., 2010). From Fig. 9 it can be seen that the saturation of CFC-11 and CFC-12 in general follow each other very well, and the same is true for their uncertainty. They are also in agree- ment with the values for CCl4. 2.1 Analysis approach CFC-113, on the other hand, frequently shows saturation as low as 70–80% (Fig. 9), consistent with the slower gas exchange and perhaps non- stable behaviour, and the variability of the saturation is larger. Some cruises deviate from the assumption that the saturation of CFC-11 and CFC-12 should be consistent to each other (Fig. 9). This will be taken into account when summarising the results from the diﬀerent quality controls. www.earth-syst-sci-data.net/2/79/2010/ 2.1 Analysis approach CFC-113 and CCl4 have a slower gas transfer velocity than CFC-12, 20% slower for CFC-113 (e.g., Roether et al., 2001) and 12% for CCl4 (Huhn et al., 2001), so in some cir- cumstances a lower degree of saturation for these compounds might be expected. Another issue of importance is the non- stable behaviour of these two species in seawater (Huhn et Figure 3. CFC-12 vs. pressure (dbar). See Fig. 2 for speciﬁcations. North Atlantic, with a lower degree of saturation prior to the 1990s when the atmospheric increase was faster (Tanhua et al., 2008), and this might also be expected for the Nordic Seas. CFC-113 and CCl4 have a slower gas transfer velocity than CFC-12, 20% slower for CFC-113 (e.g., Roether et al., 2001) and 12% for CCl4 (Huhn et al., 2001), so in some cir- cumstances a lower degree of saturation for these compounds might be expected. Another issue of importance is the non- stable behaviour of these two species in seawater (Huhn et North Atlantic, with a lower degree of saturation prior to the 1990s when the atmospheric increase was faster (Tanhua et al., 2008), and this might also be expected for the Nordic Seas. CFC-113 and CCl4 have a slower gas transfer velocity than CFC-12, 20% slower for CFC-113 (e.g., Roether et al., 2001) and 12% for CCl4 (Huhn et al., 2001), so in some cir- cumstances a lower degree of saturation for these compounds might be expected. Another issue of importance is the non- stable behaviour of these two species in seawater (Huhn et surface waters of the Greenland Sea of ∼80% have been re- ported by several authors (Bullister and Weiss, 1983; Rhein, 1991; Anderson et al., 2000). This low saturation is gen- erally considered to be a feature of deep-water formation regions where gas exchange rates are not rapid enough to bring the exceptionally deep winter mixed layers into equi- librium with the overlying atmosphere. Thus, low saturation levels do not prove that there is an oﬀset in the data. Further- more, the CFC saturations are possibly time-dependent in the Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA Figure 4. CFC-113 vs. pressure (dbar). See Fig. 2 for speciﬁcations. E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA Figure 4. CFC-113 vs. pressure (dbar). See Fig. 2.2 Crossover and inversion In accordance with most of the CARINA data also the Nordic Seas CFCs underwent consistency evaluation with a crossover analysis, using the cnaX scripts described by Tan- hua et al. (2010); with some minor modiﬁcations applied in the Nordic Seas (Olsen et al., 2009). The cnaX crossover routine deﬁnes crossover points from station pairs, from dif- Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ 86 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARIN Figure 5. CCl4 vs. pressure (dbar). See Fig. 2 for speciﬁcations. E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 86 86 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CA Figure 5. CCl4 vs. pressure (dbar). See Fig. 2 for speciﬁcations. Figure 5. CCl4 vs. pressure (dbar). See Fig. 2 for speciﬁcations. As mentioned in Sect. 3 the results can not be applied in a straight-forward way due to the transient nature of the CFCs, and the known transient behaviour of the Nordic Seas (e.g., Bullister and Weiss, 1983; Blindheim and Rey, 2004; Karstensen et al., 2005). The increase of the atmospheric CFC levels until, at least, the early 1990s (Walker et al., 2000) is likely to result in increased deep-water concentra- tions with time, and it is therefore very important to assess any oﬀsets from the inversions together with the other tools used in the secondary QC; most importantly the surface sat- uration, but also depth proﬁles and ratios between the CFCs. The result of the crossover analysis is thus a tool to iden- tify cruises where an oﬀset is possibly present, which is then conﬁrmed or rejected by other methods. For the crossovers in the Nordic Seas only data from depths below 1900 m have been used in order to minimize the eﬀect of vertical mix- ing, which may result in higher CFC concentrations; deep convection in the Greenland Sea down to 1600 m have been reported in 2002 (Ronski and Bud´eus, 2005). the results from the inversions with the surface saturations. If any oﬀset is found for both these indicators, and are con- sistent in direction and magnitude, an adjustment is recom- mended. The CFC ratios and depth proﬁles are also consid- ered to support the decision. 2.2 Crossover and inversion The applied adjustments were never smaller than ±5%, or larger than ±10%. The lower limit was set to avoid adjust- ing biases that are either the result of natural variability or not signiﬁcant in comparison to the precision of the mea- surements. The upper limit of adjustment was adopted both to minimise the risk of erroneously reducing diﬀerences due the transient behaviour of the CFCs, and also to be con- sistent with the saturation diﬀerence between CFC-11 and CFC-12, which did not exceed 10% for any cruise except one (58AA20010527). Earth Syst. Sci. Data, 2, 79–97, 2010 2.3 Adjusted cruise data sets Here we will discuss the cruises that have to be considered further after the diﬀerent quality controls, and motivate any adjustments made to the data. As mentioned above any ad- justment is based on the combined results of the crossover analyses and the surface saturations, but the CFC ratios and depth proﬁles are also taken into account. We have The results of the WLSQ inversion of cnaX crossovers are shown in Fig. 10, which shows the correction factors of the diﬀerent CFCs that were suggested by the analyses. These suggestions will be viewed in the light of the previously de- scribed quality controls. The overall strategy is to compare Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 87 FC-11 vs. CFC-12 for the Nordic Seas data. See Fig. 2 for speciﬁcations. Figure 6. CFC-11 vs. CFC-12 for the Nordic Seas data. See Fig. 2 for speciﬁcations. in some of the cruise data the surface saturations have been very high, which may have been caused by a contamination, or co-eluting chromatographic peak near CFC-113, while the surface saturation is very low at times, which could be due to unknown removal processes. Therefore we want to recom- mend caution with using the CFC-113 data, and make sure that these potential problems with this compound are appre- ciated. determined correction factors in steps of 5% since the applied methods do not make a more accurate determination possi- ble. This is consistent with the analyses of the North Atlantic CFCs (Steinfeldt et al., 2010). This is equivalent to stating that most data are accurate to within 5%, and only obvious deviations from this could be unambiguously detected and adjusted. In contrary to the NA group we have, in some occa- sions, applied corrections also for CFC-113 and CCl4. How- ever, especially for CFC-113 this may be somewhat prob- lematic due to analytical problems connected to this CFC; determined correction factors in steps of 5% since the applied methods do not make a more accurate determination possi- ble. This is consistent with the analyses of the North Atlantic CFCs (Steinfeldt et al., 2010). This is equivalent to stating that most data are accurate to within 5%, and only obvious deviations from this could be unambiguously detected and adjusted. 2.3 Adjusted cruise data sets In contrary to the NA group we have, in some occa- sions, applied corrections also for CFC-113 and CCl4. How- ever, especially for CFC-113 this may be somewhat prob- lematic due to analytical problems connected to this CFC; Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ 88 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA Figure 7. CFC-11 vs. CFC-113. See Fig. 2 for speciﬁcations. E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 88 Figure 7. CFC-11 vs. CFC-113. See Fig. 2 for speciﬁcations. In the following sub-sections (each header) we refer to the cruises by their respective EXPOCODES while the numbers in the parenthesis corresponds to the cruise numbers they have in the merged CARINA data ﬁle (see Table 1). been larger based on the surface saturation, however, due to the potential problems with this CFC (see above) we prefer to be more careful when applying a correction of CFC-113 data. As stated above caution is advised for the usage of these data. 2.3.3 34AR19970805 (91) This 1982 cruise had a suggested correction factor of 1.25 for CFC-11, the largest oﬀset of all cruises for CFC-11, and a suggested correction factor of >1.1 for CFC-12. However, since this is the oldest cruise in the dataset, these large oﬀsets are not un-expected considering the transient nature of the CFCs and the rapidly increasing atmospheric levels of CFCs at the time of the cruise. The saturation of CFC-11 was in the order of 5% higher than for CFC-12, and taking this to- gether we applied an adjustment of 1.05 to the CFC-11 data. This will make the CFC-11 and CFC-12 more consistent, but will keep the clear diﬀerence between these data, collected a decade earlier during late winter, and the rest of the cruise data sets. This cruise sampled the Greenland-Scotland Ridge, and dis- played a wider spread in the data vs. pressure (Figs. 2–5) due to the complex hydrography in the area, but still a tight relationship between CFC-11 and CFC-12 (Fig. 6). Never- theless, due to the limited overlap with other cruises in the Nordic Seas we did not consider this cruise for adjustments. However, the saturation of CFC-11 is very high and almost 10% higher than for CFC-12 and the North Atlantic group recommended a correction factor of 0.95 of the CFC-11 data (Steinfeldt et al., 2010). Since this will decrease the satura- tion diﬀerence the CFC-11 data have been adjusted accord- ing to this. 2.3.6 58AA20010527 (125) There was clearly an issue with the more shallow CFC-12 data for this cruise. The surface saturation was ∼120%, and showed large uncertainty, while CFC-11 was only slightly oversaturated. The depth proﬁles and the CFC relationship indicate that it is the data from the upper parts of the water column that are a problem. Due to this a careful re-check of the CFC-12 data was performed, resulting in recommenda- tions to ﬂag a rather large amount of the surface samples as questionable. The inversion suggested that the CFC-12 data was 7% too high and we applied an adjustment factor of 0.95. 2.3.2 316N20020530 (67) While near-surface CFC-11 and CFC-12 samples were fully saturated, the surface saturation of CFC-113 was the highest of all cruises. In qualitative accordance with this, the inver- sion suggested a correction of 0.80, but we applied a more modest adjustment of 0.9. Possibly the correction could have The inversion gave a strong oﬀset for CFC-12 in these data, with a suggested correction factor of 0.3. However, this oﬀset cannot be adjusted by a single correction factor; especially since the surface saturations implies that the CFC-12 values might be only 5% too high. Nevertheless, since the oﬀset Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 89 Figure 8. CFC-11 vs. CCl4. See Fig. 2 for speciﬁcations. Figure 8. CFC-11 vs. CCl4. See Fig. 2 for speciﬁcations. Figure 8. CFC-11 vs. CCl4. See Fig. 2 for speciﬁcations. implied from the inversion is too large to account for and inconsistent with the surface saturation, we ﬂagged the CFC- 12 data as questionable, which excluded these data in the ﬁnal CARINA data product. not alone motivate an adjustment. The inversion suggested a correction factor of 1.1 for the CFC-11 data, which would put the surface saturation in level with most of the Nordic Seas cruises. The adjustment also makes the deep-water values consistent with the values from the time-adjacent cruises (see Fig. 10), which justiﬁes the correction. The results of the WLSQ inversions of the cnaX crossovers also implied that the CCl4 is too high, and should be lowered by 10%. This is supported by the mean surface saturation that is 115%, and we thus adjusted the data with a factor of 0.9. www.earth-syst-sci-data.net/2/79/2010/ 2.3.5 58AA19950217 (119) There was a very large oﬀset suggested for CFC-113 from the inversion, and at the same time the surface saturation was the lowest seen for any of the cruises, for all measured CFCs, with rather large uncertainties (Fig. 9). The spread in the CFC-12 data was larger than for any of the other Nordic Seas cruises (not shown) and based on these results we ﬂagged the CFC-12 and CFC-113 data as questionable and thus not used in the ﬁnal data product. As mentioned above also CFC- 11 showed a low saturation with quite large scatter in the surface values. However, this can be expected for a winter cruise in the Greenland Sea, with a deep mixed layer, and can Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 90 0.4 0.6 0.8 1.0 1.2 1.4 1.6 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19940723 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 34AR19970805 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 Surface saturation CFC−11 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19940723 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 34AR19970805 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 CFC−12 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Surface saturation 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19940723 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 34AR19970805 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 CFC−113 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19940723 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 34AR19970805 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 CCl4 Figure 9. Surface saturations (upper 20 dbars) of the CFCs in the Nordic Seas cruises of CARINA. The error bars show the standard deviations. The cruises are sorted chronologically in order to see any trends in the data. 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19940723 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 34AR19970805 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 CFC−12 20228 11105 21105 31106 40224 40723 41028 50217 51108 60720 61030 61121 70414 70805 80801 90615 00527 10527 20420 20530 30922 CCl4 Figure 9. Surface saturations (upper 20 dbars) of the CFCs in the Nordic Seas cruises of CARINA. The error bars show the standard deviations. The cruises are sorted chronologically in order to see any trends in the data. 2.3.5 58AA19950217 (119) 0.6 0.8 1.0 1.2 1.4 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 Correction CFC−11 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 CFC−12 0.6 0.8 1.0 1.2 1.4 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 Correction CFC−113 18HU19820228 58JH19911105 58JH19921105 58JH19931106 58AA19940224 58JH19941028 58AA19950217 58JH19951108 74JC19960720 58JH19961030 58AA19961121 58JH19970414 58JH19980801 58JH19990615 58JH20000527 58AA20010527 77DN20020420 316N20020530 58GS20030922 CCl4 Figure 10. Suggested correction factors (and their standard deviations) of the CFCs from the cnaX inversions of the crossover analyses of the Nordic Seas data. The blue and red dots show the results before and after the adjustments were applied, respectively. 58JH19940723 and 34AR19970805 were not included in the ﬁnal inversions for any of the CFCs. The cruises are sorted chronologically in order to see any trends in the data. Correction Figure 10. Suggested correction factors (and their standard deviations) of the CFCs from the cnaX inversions of the crossover analyses of the Nordic Seas data. The blue and red dots show the results before and after the adjustments were applied, respectively. 58JH19940723 and 34AR19970805 were not included in the ﬁnal inversions for any of the CFCs. The cruises are sorted chronologically in order to see any trends in the data. Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 91 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 91 Figure 11. CFC-11 vs. pressure (dbar) for all Arctic Ocean cruises in CARINA. The red dots mark the data from each speciﬁc cruise, and the grey dots shows the CFC-11 data from all Arctic Ocean CARINA cruises. The inserted map shows the speciﬁc cruise track. Figure 11. CFC-11 vs. pressure (dbar) for all Arctic Ocean cruises in CARINA. The red dots mark the data from each speciﬁc cruise, and the grey dots shows the CFC-11 data from all Arctic Ocean CARINA cruises. The inserted map shows the speciﬁc cruise track. Figure 12. CFC-12. See Fig. 11 for speciﬁcations. 2.3.5 58AA19950217 (119) 2.3.7 58GS20030922 (128) The surface saturation was between 5 and 10% higher for CFC-12 than for CFC-11, and this is somewhat supported by the inversions, suggesting a correction factor of 0.85 for CFC-12, and a smaller one of 0.94 for CFC-11. Obviously, both CFCs cannot be adjusted since that would keep the dif- ference in surface saturations constant. An adjustment factor of 0.95 is applied to the CFC-12 data. 2.3.8 58JH19911105 (129) The WLSQ inversion implied that the CFC-11 values from this cruise were too high and suggested a downward correc- tion of almost 20%. The CCl4 values were, on the other hand, suggested to have an oﬀset of only 5%. The surface saturations were close to 100% for CFC-11 and ∼85% for CCl4. Unfortunately no CFC-12 data are available for this cruise, making any judgment of the CFC-11 values more dif- ﬁcult. However, the oﬀset in the deep water, seen from the Figure 12. CFC-12. See Fig. 11 for speciﬁcations. Figure 12. CFC-12. See Fig. 11 for speciﬁcations. 2.3.8 58JH19911105 (129) 2.3.7 58GS20030922 (128) The surface saturation was between 5 and 10% higher for CFC-12 than for CFC-11, and this is somewhat supported by the inversions, suggesting a correction factor of 0.85 for CFC-12, and a smaller one of 0.94 for CFC-11. Obviously, both CFCs cannot be adjusted since that would keep the dif- ference in surface saturations constant. An adjustment factor of 0.95 is applied to the CFC-12 data. The WLSQ inversion implied that the CFC-11 values from this cruise were too high and suggested a downward correc- tion of almost 20%. The CCl4 values were, on the other hand, suggested to have an oﬀset of only 5%. The surface saturations were close to 100% for CFC-11 and ∼85% for CCl4. Unfortunately no CFC-12 data are available for this cruise, making any judgment of the CFC-11 values more dif- ﬁcult. However, the oﬀset in the deep water, seen from the www.earth-syst-sci-data.net/2/79/2010/ Earth Syst. Sci. Data, 2, 79–97, 2010 92 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARI Figure 13. CFC-113. See Fig. 11 for speciﬁcations. Figure 14. CCl4. See Fig. 11 for speciﬁcations. inversion, is not supported by the surface saturation, where the latter agrees with the saturation of the cruises the few years after 1991. Thus, no adjustment is recommended for CFC-11. We also left CCl4 without any adjustment, but this and the suggested correction from the inversion support increase of the CCl4 values we applied an adjustment of 1 2.3.10 58JH19931106 (133) E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 92 Figure 13. CFC-113. See Fig. 11 for speciﬁcations. Figure 14. CCl4. See Fig. 11 for speciﬁcations. and the suggested correction from the inversion support an increase of the CCl4 values we applied an adjustment of 1.05. inversion, is not supported by the surface saturation, where the latter agrees with the saturation of the cruises the few years after 1991. Thus, no adjustment is recommended for CFC-11. We also left CCl4 without any adjustment, but this is a less certain case since the inversion and the surface satu- ration actually support each other. Nevertheless, when com- paring the surface saturation in the 1992 cruise (see Sect. 5.9) with this 1991 cruise the former should be adjusted in order to reach a higher consistency. 2.3.7 58GS20030922 (128) Due to this we decided to be somewhat cautious and leave the 1991 as they are. 2.3.10 58JH19931106 (133) The result from the cnaX WLSQ inversion suggests that the CFC-113 data are almost 20% too high. The surface satu- ration for CFC-113 was 85% during the cruise, while both CFC-11 and CFC-12 showed full saturation. The CFC-113 saturation is approximately 5% higher than the general mean for CFC-113, and we adjusted the CFC-113 data with a correction factor of 0.95. www.earth-syst-sci-data.net/2/79/2010/ 2.3.12 58JH19951108 (138) The surface saturation was 5% higher for CFC-12 than for CFC-11, with low uncertainty in both values. The inversion suggested a rather small upward correction of CFC-11 and an even smaller downward correction of CFC-12. Due to the fact that the inversion and the saturation diﬀerence point in the same direction we apply a 0.95 adjustment of the CFC-12 data. There were some diﬀerences in surface saturations in these data (Fig. 9), with CFC-11 and CCl4 being supersaturated by 10% and CFC-12 with 5%. CFC-113 on the other hand displayed one of the lowest surface saturations of all Nordic Seas data (<70%). The inversions suggested small correc- tions for CFC-11 and CFC-12, downward for the former and upward for the latter. Since the CCl4 saturation is as high as for CFC-11, and received no suggested oﬀset from the WLSQ inversion, we adjusted the CFC-12 data with 1.05, which will make these three CFCs consistent. The situation for CFC-113 is diﬀerent though. The inversion suggested that the data are too low, in agreement with the low satura- tion values, and that the correction factor should be 1.25. We follow the direction of this correction and applied an adjust- ment factor of 1.1, which is our upper limit of adjustment to tracer data. 2.3.11 58JH19940723 (135) There was a 5% saturation diﬀerence between CFC-11 and CFC-12, with the former being higher, but the inversions did not suggest any clear correction, but indicated that the CFC- 11 data are slightly too high. Since this is consistent with the diﬀerence in surface saturation we applied a correction factor of 0.95 of the CFC-11 data. The WLSQ inversions suggested a correction factor for CCl4 of 1.1. The surface saturation of CCl4 in these data was the lowest of all cruises in the Nordic Seas (<80%). Since both the low saturation The inversions identiﬁed large oﬀsets for CFC-11 and CFC- 12 for this cruise (not shown), and the saturations were gen- erally high with large uncertainties (Fig. 9). However, most of the stations were located in, or in the vicinity of, the over- ﬂow regions along the Greenland-Scotland Ridge, so a larger spread of the data could be expected. Nevertheless, the qual- ity of the data is questionable and the data will be ﬂagged as poor and not be included in the merged data ﬁle; this was also found by the North Atlantic group (Steinfeldt, et al., 2010). Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 93 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA Figure 15. CFC-11 vs. CFC-12 for the Arctic Ocean data. See Fig. 11 for speciﬁcations. Figure 15. CFC-11 vs. CFC-12 for the Arctic Ocean data. See Fig. 11 for speciﬁcations. Figure 16. CFC-11 vs. CFC-113. See Fig. 11 for speciﬁcations. 2.3.12 58JH19951108 (138) The surface saturation was 5% higher for CFC 12 than for 2.3.14 58JH19980801 (142) Figure 15. CFC-11 vs. CFC-12 for the Arctic Ocean data. See Fig. 11 for speciﬁcations. Figure 16. CFC-11 vs. CFC-113. See Fig. 11 for speciﬁcations. Figure 16. CFC-11 vs. CFC-113. See Fig. 11 for speciﬁcations. Figure 16. CFC-11 vs. CFC-113. See Fig. 11 for speciﬁcations. 2.3.14 58JH19980801 (142) 2.3.13 58JH19961030 (140) The surface saturation of CFC-113 was in the order of 70%, lower than for the other CFCs (Fig. 9). The inversion sug- gested a correction factor of 1.2, which is too high as com- pared to the saturation value, but we adjusted the data with 1.05. Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ 94 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CAR Figure 17. CFC-11 vs. CCl4. See Fig. 11 for speciﬁcations. 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 77DN19910726 06AQ19930806 18SN19940726 06AQ19960712 18SN19970803 18SN19970831 18SN19970924 Surface saturation CFC−11 77DN19910726 06AQ19930806 18SN19940726 06AQ19960712 18SN19970803 18SN19970831 18SN19970924 CFC−12 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Surface saturation 77DN19910726 06AQ19930806 18SN19940726 06AQ19960712 18SN19970803 18SN19970831 18SN19970924 CFC−113 77DN19910726 06AQ19930806 18SN19940726 06AQ19960712 18SN19970803 18SN19970831 18SN19970924 CCl4 Figure 18. Surface saturations (upper 20 dbars) of the CFCs in the Arctic Ocean cruises of CARINA. The error bars show the stan deviations. The cruises are sorted chronologically in order to see any trends in the data. 2.3.15 58JH19990615 (143) The inversion indicated a very large oﬀset for CFC-113 and 2.3.16 58JH20000527 (144) No consistency was seen in the surface saturations, w 94 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CAR Figure 17 CFC-11 vs CCl4 See Fig 11 for speciﬁcations E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 94 Figure 17. CFC-11 vs. CCl4. See Fig. 11 for speciﬁcations. s (upper 20 dbars) of the CFCs in the Arctic Ocean cruises of CARINA. The error bars show the standard ed chronologically in order to see any trends in the data. Figure 18. Surface saturations (upper 20 dbars) of the CFCs in the Arctic Ocean cruises of CARINA. The error bars show the standard deviations. The cruises are sorted chronologically in order to see any trends in the data. Figure 18. Surface saturations (upper 20 dbars) of the CFCs in the Arctic Ocean cruises of CARINA. The error bars show the standard deviations. The cruises are sorted chronologically in order to see any trends in the data. 2.3.18 77DN20020420 (179) The analytical method is the same as for the Nordic Seas data; see Sect. 2. All CFCs were sampled during this cruise, but there were some analytical problems with the CFC-113 and the CCl4 data, giving data of too low quality. As a result, these data were ﬂagged as questionable (by R. Key) in an earlier stage of the quality assessment and are thus not included in the CARINA data set. 2.3.16 58JH20000527 (144) The inversion indicated a very large oﬀset for CFC-113 and suggested a correction factor of 2.3. This is too high to give reliable data, especially since the surface saturation equalled the mean saturation of all CFC-113 cruise data, even if the uncertainty was relatively high. Based on this the CFC-113 data are ﬂagged as questionable and not included in the ﬁnal data product. No consistency was seen in the surface saturations, where CFC-11 was equilibrated, and CFC-12 and CCl4 was under- saturated and oversaturated, respectively. These diﬀerences are supported by the inversions that suggest correction fac- tors of 1.05 and 0.95, for CFC-12 and CCl4, respectively. Since these two controls are consistent we followed the rec- ommendations from the inversions for CFC-12 and CCl4 Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ 95 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA (Fig. 10). CFC-113 was clearly undersaturated (∼70%), with quite high uncertainty, but the inversion suggested a large oﬀ- set and correction of >1.5 and we ﬂagged the CFC-113 data as questionable. characteristics, and hide actual oﬀsets in the data. Moreover, due to the many diﬃculties connected to surveys in the Arctic Ocean the sampling cover of this area is much sparser than in the Nordic Seas, and thus crossover analysis are less suitable for these data. An overview of the Arctic Ocean CFC data included in CARINA can be found in Table 2, and the geographical lo- cation of the stations in Fig. 1. The description of the other Arctic Ocean cruises, 26 in total, and all other sampled pa- rameters can be found in Jutterstr¨om et al. (2010). The col- lection of CFC data contains ﬁve cruises, where the most re- cent one, in 1997, has been divided in three parts. Due to the sparse number of cruises, often covering diﬀerent parts of the Arctic Ocean, only one crossover was found, and this suggested only a small oﬀset. The second QC did instead follow most of the initial steps of the procedure used for the Nordic Seas data, including a careful check of the depth pro- ﬁles, CFC ratios and surface saturations. The CFC proﬁles and relationships are shown in Figs. 11–17. 2.3.17 74JC19960720 (176) The surface saturation was consistent for all CFCs and did not deviate from the overall mean for the Nordic Seas cruises; slightly below full saturation for all except CFC-113 that showed a saturation of 80%. The inversion only found a clear oﬀset for CCl4, and suggested that these data should be in- creased by 15%. However, the fact that this deviation was not observed in the surface saturation, led us to ﬂag the CCl4 data as questionable, and hence to exclude them from the ﬁ- nal data product. The other CFCs are included without any recommended adjustments. 2.4 Consistency Test of Final Nordic Seas CFC Data An evaluation of the consistency of the ﬁnal Nordic Seas CFC data was performed with a cnaX crossover analysis (Tanhua et al., 2010) of the data, after the recommended ad- justments had been applied. The corrections suggested by the WLSQ are shown in Fig. 10. As can be seen the adjusted data did not show oﬀsets larger than ±10% (depicted by the dashed lines), except a few exceptions (mentioned above). No trend could be seen in the CFC-11 or CFC-12 data. CFC- 113 and CCl4, on the other hand, show some indications of this; CFC-113 seem to be decreasing up to the end of the 1990s (even if this might not be statistically signiﬁcant due to the large variability) and then clearly higher values in 2003 (58GS20030922), while CCl4 shows an increasing trend dur- ing the 1990s, which might be somewhat surprising. As for the Nordic Seas data, the surface saturations were used as a quality control tool. The calculated values and their uncertainties are shown in Fig. 18. The overall means, and their standard deviations were, for CFC-11 0.88 ±0.06; CFC- 12 0.87±0.04; CFC-113 0.68±0.35; and CCl4 0.92±0.14. The saturations of CFC-11 and CFC-12 were stable, seen from the low uncertainty among all data. They were also con- sistent with each other; throughout the collection their rela- tionship diﬀered less than 2%, except for 1991 and the 1994 cruises. The large spread in the 1991 data, and the amount of reﬂagging now recommended for this cruise, makes it al- most impossible to suggest any reasonable adjustment. The 1994 data is better, though, but the diﬀerence in saturation between the diﬀerent CFCs is larger. 2.5 Arctic Ocean CFC data 2.6 Individual cruise assessments of the Arctic Ocean data From the proﬁles and the relationships, most of the CFC data from these cruises seem to be of good quality, with a few exceptions. The earliest cruise, with the Swedish icebreaker Oden in 1991 (77DN19910726; #177) showed a large spread (Figs. 11–12, 14–15, 17), and the metadata also stated that there were some problems with the general precision. There- fore these data were carefully re-checked, resulting in the recommendation to ﬂag a substantial amount of values as questionable, especially for CFC-11. 18SN19940724 (47) had some problem with the CFC-113 data, showing a large scatter, and these are now ﬂagged questionable. 2.7.1 18SN19940724 (47) Fogelqvist, E.: Determination of volatile halocarbons in seawa- ter, in: Methods of Seawater Analysis, 3rd edition, edited by: Grasshoﬀ, K., Kremling, K., and Ehrhardt, M., 501–519, Wiley- VCH, 1999. This was the only cruise that received any adjustments. Those were based on the diﬀerence in surface saturation be- tween CFC-11 and CFC-12, where the former was 4% lower, and both showed low uncertainty. For all other cruises the saturations are consistent, or with a somewhat higher satu- ration for CFC-11. Moreover, when comparing the CFC- 11/CFC-12 relationship among all cruises (see Fig. 15) the 1994 data does seem a bit too high in CFC-12 relative to CFC-11. In addition, the deep-water mean for this cruise is actually the lowest of all the Arctic cruises, which would then support an upward adjustment of the data. Therefore, we applied an adjustment of CFC-11 with 1.05. Huhn, O., Roether, W., Beining, P., and Rose, H.: Validity limits of carbon tetrachloride as an ocean tracer, Deep-Sea Res., 48, 2025–2049, 2001. Johnson, G. C., Robbins, P. E., and Huﬀord, G. E.: Systematic adjustments of hydrographic sections for internal consistency, J. Atmos. Ocean. Tech., 18, 1234–1244, 2001. Jutterstr¨om, S., Anderson, L. G., Bates, N. R., Bellerby, R., Johan- nessen, T., Jones, E. P., Key, R. M., Lin, X., Olsen, A., and Omar, Jutterstr¨om, S., Anderson, L. G., Bates, N. R., Bellerby, R., Johan- nessen, T., Jones, E. P., Key, R. M., Lin, X., Olsen, A., and Omar, A. M.: Arctic Ocean data in CARINA, Earth Syst. Sci. Data, 2, 71 78 2010 Jutterstr¨om, S., Anderson, L. G., Bates, N. R., Bellerby, R., Johan- nessen, T., Jones, E. P., Key, R. M., Lin, X., Olsen, A., and Omar, A. M.: Arctic Ocean data in CARINA, Earth Syst. Sci. Data, 2, 71–78, 2010, http://www.earth-syst-sci-data.net/2/71/2010/. A. M.: Arctic Ocean data in CARINA, Earth Syst. Sci. Data, 2, 71–78, 2010, http://www.earth-syst-sci-data.net/2/71/2010/. CFC-113 was supersaturated with ∼30%, with a very large uncertainty. In addition the data were very noisy (not shown). Together this suggests that the precision of the CFC-113 data is rather poor and we ﬂagged them as questionable. Karstensen, J., Schlosser, P., Wallace, D. W. R., Bullister, J. L., and Blindheim, J.: Water mass formation in the Green- land Sea during the 1990s, J. Geophys. Res., 110, C07022, doi:10.1029/2004JC002510, 2005. Acknowledgements. 2.5 Arctic Ocean CFC data The secondary QC of the Arctic Ocean cruises in CARINA was treated separated from the Nordic Seas cruises. The main reason is that the Arctic Ocean and the Nordic Seas are two hydrographically diﬀerent regions; the ice conditions af- fect the surface saturations of the CFCs, and the deep waters of the Arctic Ocean are less homogeneous than those in the Nordic Seas. Thus, diﬀerences revealed by comparing data from these two areas may both be confused with regional The saturation of CFC-113 is not as constant, however, seen from the very large uncertainty. When looking at the diﬀerent cruises (Fig. 18), 18SN19940724 stands out with a very high degree of supersaturation, but as mentioned pre- viously this cruise has a very large spread. When omitting www.earth-syst-sci-data.net/2/79/2010/ Earth Syst. Sci. Data, 2, 79–97, 2010 E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 96 these data the mean of the CFC-113 saturation is 0.54±0.13. This is very low, obviously, but it is hard to evaluate how reasonable this number is. Carbon Cycle Program. Additional support from the International Ocean Carbon Coordination Project IOCCP (Dr. Maria Hood) and the Hanse Institute for Advanced Study (HWK Delmenhorst, Germany) are gratefully acknowledged. The saturation of CCl4 has been very variable, as seen in Fig. 18; from a slight supersaturation with a large spread (77DN19910726 and 18SN19970831), to a clear undersat- uration with a small scatter (06AQ19960712). Edited by: V. Gouretski 2.7 Recommendations for the Arctic Ocean data 2.7 Recommendations for the Arctic Ocean data With only one crossover available it is clearly more diﬃcult to assess any adjustments to the data. Nevertheless, the sec- ondary QC identiﬁed some issues and some diﬀerences in the data that can support some adjustments. Most of the rec- ommendations for the Arctic Ocean data were on reﬂagging samples as questionable, and hence exclude some data from the ﬁnal data product. The motivation for this was to ob- tain as many reliable cruise data sets as possible. This is es- pecially true for the 77DN19910726 cruise, which, as men- tioned above, showed a generally larger spread in all CFCs compared to the other Arctic Ocean cruises (see Figs. 11, 14, 15, 17). However, except for the rather large amount of rec- ommended reﬂagging no other adjustments are suggested for this cruise. Anderson, L. G., Chierici, M., Fogelqvist, E., and Johannessen, T.: Flux of anthropogenic carbon into the deep Greenland Sea, J. Geophys. Res., 105(C6), 14339–14345, 2000. Blindheim, J. and Rey, F.: Water-mass formation and distribution in the Nordic Seas during the 1990s, ICES J. Mar. Sci., 61(5), 846–863, 2004. B¨onisch, G., Blindheim, J., Bullister, J. L., Schlosser, P., and Wal- lace, D. W. R.: Long-term trends of temperature, salinity, density, and transient tracers in the central Greenland Sea, J. Geophys. Res., 102(C8), 18553–18571, 1997. Bu, X. and Warner, M. J.: Solubilities of chloroﬂuorocarbon 113 in water and seawater, Deep-Sea Res., 42(7), 1151–1161, 1995. Bullister, J. L. and Weiss, R. F.: Anthropogenic chloroﬂuo- romethanes in the Greenland and Norwegian seas, Science, 221(4607), 265–268, 1983. Bullister, J. L. and Wisegarver, D. P.: The solubility of carbon tetra- chloride in water and seawater, Deep-Sea Res., 45, 1285–1302, 1998. The very low surface saturation of CCl4 in the 06AQ19960712 cruise (Fig. 18) might indicate that the data should be corrected, and this is somewhat supported by the relatively low mean in the deep water. However, the mag- nitude of such an adjustment is almost impossible to assess, and we will therefore leave these data without changes. Doney, S. C. and Bullister, J. L.: A chloroﬂuorocarbon section in the Eastern North Atlantic, Deep-Sea Res. Part I, 39(11–12), 1857– 1883, 1992. Falck, E. and Olsen, A.: Nordic Seas dissolved oxygen data in CA- RINA, Earth Syst. Sci. Data Discuss., 2, 537–553, 2009, http://www.earth-syst-sci-data-discuss.net/2/537/2009/. 2.7.1 18SN19940724 (47) 2.7.1 18SN19940724 (47) We would like to acknowledge all the tremendous work by diﬀerent people collecting the data described in this paper, the P.I.s for sharing the data and, not the least, the captains and crews on all vessels used during the diﬀerent surveys. This work has been possible due to support from the EU through IP CARBOOCEAN (511176) and through NOAA’s Global Key, R. M., Tanhua, T., Olsen, A., Hoppema, M., Jutterstr¨om, S., Schirnick, C., van Heuven, S., Kozyr, A., Lin, X., Velo, A., Wal- lace, D. W. R., and Mintrop, L.: The CARINA data synthesis project: introduction and overview, Earth Syst. Sci. Data Dis- cuss., 2, 579–624, 2009, http://www.earth-syst-sci-data-discuss.net/2/579/2009/. Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/ www.earth-syst-sci-data.net/2/79/2010/ E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA E. Jeansson et al.: Nordic Seas and Arctic Ocean CFC data in CARINA 97 Ronski, S. and Bud´eus, G.: Time series of winter convec- tion in the Greenland Sea, J. Geophys. Res., 110, C04015, doi:10.1029/2004JC002318, 2005. Olafsson, J. and Olsen, A.: Nordic Seas nutrients data in CARINA, Earth Syst. Sci. Data Discuss., in preparation, 2010. Olsen, A.: Nordic Seas total dissolved inorganic carbon data in CA- RINA, Earth Syst. Sci. Data, 1, 35–43, 2009a, http://www.earth-syst-sci-data.net/1/35/2009/. Steinfeldt, R., Tanhua, T., Bullister, J. L., Key, R. M., Rhein, M., Steinfeldt, R., Tanhua, T., Bullister, J. L., Key, R. M., Rhein, M., and Khler, J.: Atlantic CFC data in CARINA, Earth Syst. Sci. Data, 2, 1–15, 2010, http://www.earth-syst-sci-data.net/2/1/2010/. Olsen, A.: Nordic Seas total alkalinity data in CARINA, Earth Syst. Sci. Data, 1, 77–86, 2009b, http://www.earth-syst-sci-data.net/1/77/2009/. http://www.earth-syst-sci-data.net/2/1/2010/. Tanhua, T., van Heuven, S., Key, R. M., Velo, A., Olsen, A., and Schirnick, C.: Quality control procedures and methods of the CARINA database, Earth Syst. Sci. Data, 2, 35–49, 2010, http://www.earth-syst-sci-data.net/2/35/2010/. Olsen, A., Key, R. M., Jeansson, E., Falck, E., Olafsson, J., van Heuven, S., Skjelvan, I., Omar, A. M., Olsson, K. A., Anderson, L. G., Jutterstr¨om, S., Rey, F., Johannessen, T., Bellerby, R. G. J., Blindheim, J., Bullister, J. L., Pfeil, B., Lin, X., Kozyr, A., Schirnick, C., Tanhua, T., and Wallace, D. W. R.: Overview of the Nordic Seas CARINA data and salinity measurements, Earth Syst. Sci. Data, 1, 25–34, 2009, http://www.earth-syst-sci-data.net/1/25/2009/. Tanhua, T., Waugh, D. W. and Wallace, D. W. R.: Use of SF6 to es- timate anthropogenic CO2 in the upper ocean, J. Geophys. Res., 113, C04037, doi:10.1029/2007JC004416, 2008. Walker, S. www.earth-syst-sci-data.net/2/79/2010/ 2.7.1 18SN19940724 (47) J., Weiss, R. F., and Salameh, P. K.: Reconstructed histo- ries of the annual mean atmospheric mole fractions for the halo- carbons CFC-11, CFC-12, CFC-113 and carbon tetrachloride, J. Geophys. Res., 105(C6), 14285–14296, 2000. Rhein, M.: Ventilation rates of the Greenland and Norwegian Seas derived from distributions of the chloroﬂuoromethanes F11 and F12, Deep-Sea Res. A, 38(4), 485–503, 1991. Warner, M. J. and Weiss, R. F.: Solubilities of chloroﬂuorocarbons 11 and 12 in water and seawater, Deep-Sea Res., 32(12), 1485– 1497, 1985. Roether, W., Klein, B., and Bulsiewicz, K.: Apparent loss of CFC- 113 in the upper ocean, J. Geophys. Res., 106(C2), 2679–2688, 2001. Earth Syst. Sci. Data, 2, 79–97, 2010 www.earth-syst-sci-data.net/2/79/2010/
https://openalex.org/W3192188663	https://www.researchsquare.com/article/rs-721941/latest.pdf	English	null	Rhizobium inoculation enhanced the resistance of alfalfa and soil enzymatic activity in Cu-polluted soils	Research Square (Research Square)	2,021	cc-by	9,947	Rhizobium inoculation enhanced the resistance of alfalfa and soil enzymatic activity in Cu-polluted soils Chengjiao Duan Institute of Soil and Water Conservation CAS & MWR: Chinese Academy of Sciences and Ministry of Water Resources Institute of Soil and Water Conservation Yuxia Mei Huazhong Agriculture University Qiang Wang Northwest Agriculture and Forestry University Yuhan Wang Northwest Agriculture and Forestry University Qi Li Central China Normal University Maojun Hong Northwest Agriculture and Forestry University Sheng Hu Northwest Agriculture and Forestry University Shiqing Li Institute of Soil and Water Conservation CAS & MWR: Chinese Academy of Sciences and Ministry of Water Resources Institute of Soil and Water Conservation Linchuan Fang (  flinc629@hotmail.com ) Northwest Agriculture and Forestry University https://orcid.org/0000-0003-1923-7908 Research Article Keywords: Antioxidant enzymes, Copper stress, Legume-rhizobium, Soil enzymes, Plant-soil systems Rhizobium inoculation enhanced the resistance of alfalfa and soil enzymatic activity in Cu-polluted soils Chengjiao Duan Institute of Soil and Water Conservation CAS & MWR: Chinese Academy of Sciences and Ministry of Water Resources Institute of Soil and Water Conservation Yuxia Mei Huazhong Agriculture University Qiang Wang Northwest Agriculture and Forestry University Yuhan Wang Northwest Agriculture and Forestry University Qi Li Central China Normal University Maojun Hong Northwest Agriculture and Forestry University Sheng Hu Northwest Agriculture and Forestry University Shiqing Li Institute of Soil and Water Conservation CAS & MWR: Chinese Academy of Sciences and Ministry of Water Resources Institute of Soil and Water Conservation Linchuan Fang (  flinc629@hotmail.com ) Northwest Agriculture and Forestry University https://orcid.org/0000-0003-1923-7908 Research Article Keywords: Antioxidant enzymes, Copper stress, Legume-rhizobium, Soil enzymes, Plant-soil systems Posted Date: August 6th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-721941/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Article Keywords: Antioxidant enzymes, Copper stress, Legume-rhizobium, Soil enzymes, Plant-soil systems Posted Date: August 6th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-721941/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. Page 1/23 Abstract Although some studies have reported an important role of rhizobia in mitigating heavy metal toxicity, the regulatory mechanism of the alfalfa-rhizobium symbiosis system to resist copper (Cu) stress through biochemical reactions in the plant-soil system is still unclear. Hence, this study assessed the effects of rhizobium inoculation (i.e., Sinorhizobium meliloti CCNWSX0020) on the growth of alfalfa and soil enzyme activities under Cu stress. Our results showed that rhizobium inoculation markedly alleviated Cu- induced growth inhibition by increasing chlorophyll content, height and biomass and the contents of nitrogen and phosphorus in alfalfa. The content of malondialdehyde (MDA) was increased in both shoot and root of alfalfa under Cu stress. The application of rhizobium alleviated Cu-induced phytotoxicity by increasing the activity of antioxidant enzymes and soluble protein content of tissues and inhibiting the level of lipid peroxidation (i.e., MDA level). In addition, rhizobium inoculation improved soil nutrient cycling, increased soil enzyme activities (i.e., β-glucosidase activity and alkaline phosphatase) and microbial biomass nitrogen. Both Pearson correlation coefficient analysis and partial least squares path modeling (PLS-PM) identified that the interactions between soil nutrient content, enzyme activity, microbial biomass and plant antioxidant enzymes and oxidative damage could jointly regulate plant growth. This study provides comprehensive insights into the mechanism of action of the legume- rhizobium symbiosis system to mitigate Cu stress and provide an efficient strategy for phytoremediation of Cu-polluted soils. 1. Introduction In recent decades, copper (Cu) has led to severe soil and water pollution because of industrial production, sewage irrigation and heavy use of fertilizers and pesticides (Adrees et al. 2015; Huang et al. 2018; Saxena et al. 2020). Cu contamination of both water and soil not only directly threatens plants with severe toxicity (e.g., plant growth retardation, leaf shrinkage, and interference with a variety of metabolic processes) (Chen et al. 2015), but also endangers the living environment of soil organisms and damages soil quality, which ultimately has harmful effects on human (Huang et al. 2020; Chen et al. 2021). Cu ions are highly susceptible to absorbed by plant roots, competing with each other and transferred to other organs (Sun et al. 2015; Duan et al. 2018). Plant growth restriction, leaf shrinkage, nutrient scarcity, antioxidant enzyme activity reduction, and lipid peroxidation of cells are common phenomena of plant damage caused by Cu stress (Liu et al. 2016; Abbas et al. 2017). In response to metal-induced oxidative stress, plants have evolved integrated metal detoxification mechanisms that often regulate antioxidative enzymes and non-enzymatic antioxidants autonomously to alleviate excessive contents of reactive oxygen species (ROS) and malondialdehyde (MDA) (Chen et al. 2018; Fang et al. 2020; Zhou et al. 2020). For example, to effectively increase detoxification capacity, antioxidant enzyme activity of plants increases upon exposure to heavy metals (Mostofa et al. 2014), which favors plant growth as well as causes an enhancement in biomass and heavy metal uptake. Page 2/23 Soil enzymes are both mediators of most soil transformation processes and catalysts for organic matte decomposition and nutrient cycling (Ju et al. 2019; Aponte et al. 2020). Up to now, soil enzyme activities have been commonly used as an indicator of ecological health of terrestrial ecosystems under heavy metal contamination (Yang et al. 2016; Duan et al. 2018). Cu-contamination has deleterious effects on the abundance and activity of soil microorganisms, primarily in terms of soil basal respiration, microbial biomass and enzymatic activity, and even soil microbial activity (Molina-Santiago et al. 2019; Zheng et a 2019). Nutrient deficiency and high toxicity of heavy metal contaminated soils are two dominant factors limiting plant growth. The interactions between contaminated stressed plants and soils are extremely complex and the transfer of Cu is associated with plant-soil biochemical systems. 1. Introduction Therefore, to promote plant growth and optimize potential health risks, there is an urgent need to explore effective strategies that can mitigate metal phytotoxicity and improve soil quality. Recently, legumes have attracted interest due to their functions in the remediation of phytotoxicity in metal-polluted soils (Naveed et al. 2015; Duan et al. 2019; Ju et al. 2019). The symbiotic relationship between legumes and soil microorganisms, such as rhizobia, can promote plant growth directly or indirectly by increasing nitrogen (N) and phosphorus (P) uptake and enhancing plant defenses in heavy metal-contaminated soils. Therefore, legume-rhizobia symbiosis is widely used in phytoremediation of heavy metals (Dary et al. 2010; Kong et al. 2015). In addition, it has been shown that rhizobia can enhance the soil quality and benefit plant growth by enhancing the activity and diversity of soil microorganisms and reducing soil pathogens (Wang et al. 2016; Ju et al. 2019). Furthermore, inoculation with rhizobia was found to effective in reducing ROS levels and accelerating the ascorbate-glutathione cycle to protect plant seedlings from excessive Cu stress (Pajuelo et al. 2011; Chou et al. 2019). However, a detailed mitigation mechanism of legume-rhizobium mediated mitigation of Cu stress in the biochemical response of the plant-soil system are still limited. Alfalfa is a perennial forage grass that can survive in extreme environments and is widely distributed in the mining areas of northwest China (Cui et al. 2018a). Therefore, it is important to further improve the resistance of plant pastures to heavy metals, reduce their accumulation in tissues, and increase the biomass of plant for safe livestock production. In this study, a copper-resistant strain (Sinorhizobium meliloti) was selected as an exogenous additive. The main objectives of this study were: (1) to analyze the effects of rhizobium inoculation on the growth and physiological resistance of alfalfa; (2) to determine the regulatory mechanisms of rhizobium inoculation to alleviate Cu stress in plants through biochemical responses of the plant-soil system. These results will help us better understand the role of alfalfa-rhizobium symbiosis and the regulatory mechanisms against metallic Cu toxicity. 2.1. Experimental design The soil sample utilized in this study was collected from the soil (0-20 cm) of the uncontaminated soil within the Yangling District, Shaanxi, China. The properties of the soil were: pH, 8.49, total Cu, 13.2 mg kg- Page 3/23 1, soil organic carbon (SOC), 7.65 g kg-1, total nitrogen (TN), 0.2 g kg-1, total phosphorus (TP), 0.36 g kg- 1, soil organic carbon (SOC), 7.65 g kg-1, total nitrogen (TN), 0.2 g kg-1, total phosphorus (TP), 0.36 g kg- 1, soil organic carbon (SOC), 7.65 g kg-1, total nitrogen (TN), 0.2 g kg-1, total phosphorus (TP), 0.36 g kg- 1. The collected soil was air-dried, removed from impurities such as stones and plant debris and sieved. After that, CuSO4 solution was added, mixed thoroughly and aged in the dark at room temperature for three months. Ultimately, the experiment included five Cu-addition level: 0, 200, 400, 600 and 800 mg kg-1, respectively (denoted as Cu 0, Cu 200, Cu 400, Cu 600 and Cu 800, respectively). 1, soil organic carbon (SOC), 7.65 g kg-1, total nitrogen (TN), 0.2 g kg-1, total phosphorus (TP), 0.36 g kg- 1. The collected soil was air-dried, removed from impurities such as stones and plant debris and sieved. After that, CuSO4 solution was added, mixed thoroughly and aged in the dark at room temperature for three months. Ultimately, the experiment included five Cu-addition level: 0, 200, 400, 600 and 800 mg kg-1, respectively (denoted as Cu 0, Cu 200, Cu 400, Cu 600 and Cu 800, respectively). 1. The collected soil was air-dried, removed from impurities such as stones and plant debris and sieved. After that, CuSO4 solution was added, mixed thoroughly and aged in the dark at room temperature for three months. Ultimately, the experiment included five Cu-addition level: 0, 200, 400, 600 and 800 mg kg-1, respectively (denoted as Cu 0, Cu 200, Cu 400, Cu 600 and Cu 800, respectively). The Cu contaminated soil (600 g) was evenly packed into a plastic pot (10 cm diameter). Alfalfa seeds were sterilized in hydrogen peroxide solution (30%, v:v), then washed several times with distilled water, placed in seedling trays containing germination paper, which was placed in an incubator to await seed germination. Twenty seedlings showing the same growth characteristics were selected and transplanted into each pot. After the alfalfa has grown its first leaves, the plant roots are injected with a rhizobia bacterial solution. 2.1. Experimental design The strain we used was a metal-resistant rhizobia (Sinorhizobium meliloti CCNWSX0020) (Fan et al. 2011; Duan et al. 2019). The source and growth of S. meliloti can be seen in our previous study (Duan et al. 2019). The S. meliloti suspensions (20 mL) were added to the plant roots once a week (three times in total). The uninoculated controls received the same amount of sterile distilled water. There were three replicates of each treatment. Plants and soils were harvested on day 60 after the first inoculation, and indicators such as plant and soil enzyme activity were measured. 2.3. Assays of soil properties Soil pH was determined by conventional methods, and the detailed steps were referred to the previous study (Duan et al. 2019). The SOC and TN were determined using the K2CrO7–H2SO4 oxidation method and Kjeldahl method, respectively. Soil air-dried soils were wet digested with H2SO4 and HClO4, and TP was determined at 880 nm using an ultraviolet spectrophotometer (UV3200, Shimador, Japan). Soil NH4 +- N and NO3 −-N were determined using a continuous flow autoanalyzer. Dissolved organic carbon (DOC) in fresh soil samples was extracted with 50 mL of distilled water and then measured using a Liqui TOCII analyzer (Elementar, Germany). The available P (AP) was determined via sodium bicarbonate extraction. Total soil Cu content was determined using a modified USEPA Method 3051A. The procedure was as follows: the soil samples (sieved to 0.149 mm) were digested by fifteen mL of acid mixture (i.e., 8 mL HNO3, 2 mL HClO4 and 5mL HCl), diluted and fixed with distilled water, and finally the Cu concentration was determined by atomic absorption spectrophotometer. 2.2. Determination of plant indexes The chlorophyll content of plant leaves was extracted with 90% acetone, and then quantified using a spectrophotometer (Sobrino-Plata et al. 2014), the details of the method can be found in our previous study (Fang et al. 2020). Use a straightedge to measure alfalfa height and root length. The shoot and root were washed with sterilized deionized water and dried at 60 °C to constant weight. About 0.3 g of alfalfa samples were digested with 8 mL of HNO3 and 2 mL of HClO4 mixture, and then diluted to a certain volume with distilled water, and the Cu concentration was determined using an atomic absorption spectrophotometer (Hitachi, FAAS Z-2000, Japan). Total Cu uptake = shoot/root biomass Í shoot/root Cu concentration. The shoot and root samples were digested with H2SO4 and H2O2, then diluted and fixed with distilled water, and finally N and P content of tissue was determined by flow analyzer. Fresh shoots and roots were used to determine alfalfa MDA, soluble protein content and antioxidant enzyme activities. The MDA content of alfalfa tissues was determined using the kit provided by Suzhou Comin Biotechnology Co., Ltd (Suzhou, China). The procedure was as follows: 1 mL of the extraction was added to grind the shoot and root parts of the alfalfa. Then centrifuge at 8000 rpm for 15 min. The supernatant was aspirated and added to the color development solution in a boiling water bath for 30 min. After cooling and centrifugation, the MDA content in the supernatant was then determined. The soluble protein content was also determined according to the kit procedure. Plant antioxidant enzyme activities (i.e., total superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)) were also determined using the purchased kits. Fresh alfalfa tissue samples were first ground with 1 mL of the extraction and then centrifuged at 8000 rpm for 15 min. Page 4/23 Page 4/23 The supernatant was determined spectrophotometrically after the addition of the appropriate chromogenic agent to the supernatant. For detailed steps, please refer to the previous studies of our group (Chen et al. 2018; Duan et al. 2018). The supernatant was determined spectrophotometrically after the addition of the appropriate chromogenic agent to the supernatant. For detailed steps, please refer to the previous studies of our group (Chen et al. 2018; Duan et al. 2018). 2.4. Assays of soil enzyme activities and microbial biomass The methods for measuring soil enzyme activities including urease (UR), β-glucosidase (BG), alkaline phosphatase (ALP) and catalase were as previously described (Guan 1986; Duan et al. 2018). The UR, BG and ALP activities were determined using a spectrophotometer at 587, 400 and 578 nm, respectively. Among them, UR was used by the sodium phenolate colorimetric method, BG by the p-nitrophenol colorimetric method, and ALP by the sodium benzyl phosphate colorimetric method. Moreover, soil catalase activity was determined using the method of potassium permanganate titration. Microbial biomass carbon (MBC) and nitrogen (MBN) were analyzed using an improved method (Vance et al. 1987; Cui et al. 2018b). Weigh 10 g of fresh soil samples were fumigated with ethanol-free chloroform in the dark for 24 hours, and then extracted with 40 mL of 0.5 M potassium sulfate solution for 30 min. The supernatant was filtered and the extractable organic carbon (EOC) and nitrogen (EON) contents were determined simultaneously by LiquiTOC II analyzer (Elementar, Germany). Similarly, 10 g of unfumigated soil samples were taken to determine the EOC and EON. MBC and MBN were calculated from the difference of EOC and EON between fumigated and non-fumigated samples, respectively. 2.5. Statistical analyses Page 5/23 All the data used in this study were average values of replicated experiments. Analysis of variance (ANOVA) with the Duncan’s tests was conducted at a significant level of P < 0.05 by the SPSS 20 software. All bar graphs were plotted using Origin Pro 2021. Pearson correlation coefficient analysis and partial least squares path modeling (PLS-PM) analysis were performed in R software (version 3.6.2). The software packages "ggcorrplot" and "plspm" were used, respectively. The correlation heat map was used Page 5/23 Page 5/23 to compare and determine the relationships between different indicators, and PLS-PM was used to determine the direct and indirect factors affecting plant growth. to compare and determine the relationships between different indicators, and PLS-PM was used to determine the direct and indirect factors affecting plant growth. 3.1. Plant growth phenotype and the N and P content of alfalfa Based on Fig. 1, the shoot height, root length and biomass of alfalfa were increased as the increasing of Cu concentration in treatments, but in a way, rhizobium inoculation alleviated the growth inhibition of the alfalfa. For example, compared to the non-inoculated treatment, alfalfa height, root length and shoot and root biomass in the inoculated treatment were increased by 19.15%, 6.94%, 35.29% and 33.33%, respectively, under the Cu 800 treatment (Fig. 1). The chlorophyll content increased significantly after inoculation with rhizobium, and the increase of chlorophyll by inoculation with rhizobium was more distinct at the Cu concentration of 800 mg kg-1, which was 1.44 times higher than that of the non- inoculated treatment (Table 1). At Cu 800 mg kg-1, the chlorophyll a and b were significantly lower than that of other treatments. The N level of alfalfa shoot and root was superlative in the Cu 0 treatment, but did not differ significantly with increasing Cu stress (Table 1). Inoculation with rhizobium increased N content of shoot at the Cu concentrations of 0 mg kg-1 and 800 mg kg-1. Interestingly, the root N content was significantly increased by rhizobium inoculation. In addition, the P level in alfalfa tissues consistently decreased with the increasing Cu concentration (Table 1). Compared to the uninoculated treatment, inoculation with rhizobia significantly increased the P levels in the alfalfa tissues (Table 1), particularly for the Cu 600 and Cu 800 treatments (P < 0.05). 3.2. Differences Cu and Cu uptake by alfalfa It is noteworthy that the effect of rhizobia on copper concentration in difference parts of alfalfa was not uniform (Table 2). Specifically, exogenous addition of rhizobia significantly reduced the Cu concentration in the shoot but noticeably increased the Cu concentration in the root (P < 0.05). In the shoots, the Cu uptake by the inoculated plants was noticeably lower than that of the non-inoculated plants in the Cu 0, Cu 400 and Cu 600 treatments (P < 0.05). Interestingly, rhizobium inoculation decreased the Cu uptake of root in treatments with low Cu concentrations (0 or 200 mg kg-1). However, under high Cu concentrations, the Cu uptake of root was increased with by rhizobium inoculation. Except for Cu 0 treatment, the transfer coefficients of Cu were considerably less than 1.0 (Table 2). And the rhizobium inoculation increased Cu transfer coefficients in treatments with low Cu concentrations (0 or 200 mg kg-1). However, under high Cu concentrations, the Cu transfer coefficients were decreased with rhizobium inoculation. 3.3. Plant oxidative damage, soluble protein and antioxidant enzyme activity Compared with the uninoculated alfalfa, application of rhizobium reduced the CAT activity in shoot by 31.44%, 62.40%, 61.87% and 19.92% for the Cu 0, Cu 200, Cu 400 and Cu 800 treatments, respectively (P < 0.05); while application of rhizobia markedly enhanced the CAT activity in the Cu 600 treatment (P < 0.05) (Fig. 3c). The inoculation of rhizobium had no obvious effect on CAT activity in the alfalfa root. Differences in alfalfa tissue APX activity between uninoculated and inoculated plants were not consistent and were influenced by specific Cu treatments (Fig. 3d). And the application of rhizobium increased the alfalfa APX activity in the low Cu concentrations (0 or 200 mg kg-1). SOD activity of shoot was significantly and negatively correlated to the content of N and P (P < 0.01). However, the APX activity of alfalfa (shoot and root) was significantly and positively correlated with the content of N and P, respectively (P < 0.05) (Fig. 5). 3.3. Plant oxidative damage, soluble protein and antioxidant enzyme activity Exploring the effect of heavy metals on lipid peroxidation damage by measuring the MDA content of lipid peroxidation reaction (Fig. 2a). Based on Fig. 2a, the MDA content was increased in both shoots and Page 6/23 roots as the increasing of Cu concentration in treatments, but in a way, rhizobium inoculation reduced the accumulation of MDA in plant tissues. The content of MDA in the shoot and root parts of Cu 800 mg kg-1 increased by 29.2% and 41.4%, respectively, compared with that of Cu 0 mg kg-1 (Fig. 2a). In the Cu 400, Cu 600 and Cu 800 treatment, the reduction of shoot after inoculation of rhizobium was 13.8%, 17.7% and 17.8%, respectively; and reduction in the root was 17.6%, 18.4% and 24.6%, respectively. This indicating that inoculation of rhizobium had a greater relieving effect on roots than aboveground parts. Additionally, MDA content in shoots and roots was noticeably negatively correlated to N and P content (P < 0.001) content (Fig. 5). In non-inoculated plants, the highest shoot and root soluble protein contents were detected in the Cu 800 treatment, which was 1.83 and 1.21 times higher compared to the Cu 0 treatments, respectively (Fig. 2b). Compared with the non-inoculated treatment, the rhizobium-inoculated plants exhibited higher soluble protein content in the alfalfa tissue (Fig. 2b). In the Cu 400, Cu 600 and Cu 800 treatment, the reduction of shoot after inoculation of rhizobium was 47.9%, 29.8% and 20.8%, respectively; and reduction in the root was 19.5%, 8.8% and 16.1%, respectively. In addition, soluble protein content in roots was significantly positively correlated to N content (P < 0.05) (Fig. 5b). The SOD activity in the shoots of non-inoculated plants showed no obvious change, while the SOD activity in the roots of alfalfa showed a downward trend under copper stress (Fig. 3a). Rhizobium inoculation significantly increased SOD activity of the shoot under Cu 600 mg kg-1 (P < 0.05). Plants inoculated with rhizobium exhibited higher SOD activity in the roots compared to uninoculated plants (Fig. 3a). For example, in the Cu 600 treatment, the increases of shoot and root after inoculation of rhizobium was 22.1% and 65.3%, respectively (Fig. 3b). The highest root POD activity was exhibited in the Cu 600 treatment. Rhizobium inoculation significantly increased the POD activity in the shoots and roots (P < 0.05). 3.5. Driving factors affecting plant growth The N content of shoot and root showed the strongest and positive correlations with DOC, β-glucosidase, alkaline phosphatase, urease and SOC, but the strongest and negative correlations with MBN, NH4 --N, AP and Cu concentrations (P < 0.05) (Fig. S2). In addition, the plant P showed the strongest positive correlation with MBC, soil enzyme activities, and SOC; and strongest negative correlation with MBN, NH4 -- N, TN, AP and Cu concentrations (P < 0.05) (Fig. S2). We used PLS-PM to further reveal the effects of plant physiological indicators, soil physicochemical properties and soil biochemical indicators on alfalfa growth (Fig. 6). The PLS-PM method showed that plant antioxidant enzyme activities, soil nutrient content and soil enzyme activities had a significant positive direct effect (0.35, 0.28 and 0.42, respectively; P < 0.01) on plant growth (Fig. 6a). The application of S. meliloti (0.34), soil microbial biomass (0.61), soil enzyme activities (0.82), soil nutrient (0.28), and plant antioxidant enzyme activities (0.35) had positive total effects on the plant growth, whereas the plant oxidative damage (-0.13) induced negative total effects (Fig. 6b). 3.4. Soil nutrient, enzyme activities and microbial biomass The soil Cu concentration decreased significantly under rhizobium inoculation (P <0.05) (Table 3). Rhizobium inoculation significantly increased NO3 --N and NH4 +-N content in Cu 400 and Cu 800 treatments (P < 0.05). In the inoculated treatment, the highest AP content was detected in the Cu 600 mg kg-1, which was higher of 42.19% and 27.93% compared to the Cu 0 and the Cu 800 treatments, respectively (Table 3). However, the effect of rhizobium inoculation on AP was not obvious. And the Page 7/23 Page 7/23 rhizobium inoculation increased the DOC content in the low Cu concentrations (200 or 400 mg kg-1), on the contrary, application of rhizobia significantly reduced the DOC at the Cu concentration of 600 mg kg-1 (P < 0.05). rhizobium inoculation increased the DOC content in the low Cu concentrations (200 or 400 mg kg-1), on the contrary, application of rhizobia significantly reduced the DOC at the Cu concentration of 600 mg kg-1 (P < 0.05). The changes of soil enzymes (i.e., catalase, UR, BG and ALP) were shown in Fig. 4. In the non-inoculated treatment, the catalase activity in Cu 0 was significantly higher than that in other treatments, the increasing Cu concentration strongly inhibited catalase activity. Rhizobium inoculation remarkably increased catalase activity in Cu 400 and Cu 600 treatments (P < 0.05). The inoculation of rhizobia had a notable effect on the BG and ALP activity. However, the inoculation of rhizobium only enhanced the UR activity in the low Cu concentrations (0 or 200 mg kg-1). Rhizobium inoculation significantly increased MBN in Cu 200, Cu 400 and Cu 600 treatments (P < 0.05) (Fig. S1). 4. Discussion Plants grown in Cu-contaminated soils could suffer from physiological dysfunction, growth inhibition, reduced biomass, and metal accumulation, among other phenomena (Duan et al. 2019; Saeid and Abooalfazl 2019; Fang et al. 2020). In this study, inoculation of rhizobium (i.e., S. meliloti) not only increased the plant height and biomass of alfalfa, but also increased its chlorophyll content (Table 1; Fig. 1), indicating that alfalfa-rhizobium symbiosis plays an important role in soil Cu bioremediation. Plants grown in Cu-contaminated soils could suffer from physiological dysfunction, growth inhibition, reduced biomass, and metal accumulation, among other phenomena (Duan et al. 2019; Saeid and Abooalfazl 2019; Fang et al. 2020). In this study, inoculation of rhizobium (i.e., S. meliloti) not only increased the plant height and biomass of alfalfa, but also increased its chlorophyll content (Table 1; Fig. 1), indicating that alfalfa-rhizobium symbiosis plays an important role in soil Cu bioremediation. Moreover, our results showed that the inoculation of rhizobia significantly promoted Cu accumulation in alfalfa roots (Table 2). Stambulska and Bayliak (2019) found that the pathway that reduces the toxic effects of heavy metals on legumes may be due to their preferential accumulation in the root nodules of legume-rhizobium symbiosis. Chlorophyll is an important substance for photosynthesis in plants, and chlorophyll content has a direct impact on photosynthetic efficiency (Tang et al. 2021). Inoculation with rhizobium significantly increased chlorophyll content (Chou et al. 2019), which helped to promote photosynthetic reaction rate and alleviate the toxic effects of Cu stress. Copper stress reduces the total N Moreover, our results showed that the inoculation of rhizobia significantly promoted Cu accumulation in alfalfa roots (Table 2). Stambulska and Bayliak (2019) found that the pathway that reduces the toxic effects of heavy metals on legumes may be due to their preferential accumulation in the root nodules of legume-rhizobium symbiosis. Chlorophyll is an important substance for photosynthesis in plants, and chlorophyll content has a direct impact on photosynthetic efficiency (Tang et al. 2021). Inoculation with rhizobium significantly increased chlorophyll content (Chou et al. 2019), which helped to promote photosynthetic reaction rate and alleviate the toxic effects of Cu stress. Copper stress reduces the total N Page 8/23 Page 8/23 content in plant tissues and affects the N fixation of alfalfa, but inoculation with rhizobium significantly increases the N content of alfalfa roots (Table 1), providing sufficient source of N for plant growth and alleviating alfalfa toxicity. 4. Discussion In addition, the effect of rhizobium inoculation on N content of the shoots was remarkably lower than that of the roots (Table 1). The nodules probably function in the root system and are the main nitrogen-fixing organ. Moreover, this indicates that the Cu-resistant strain (i.e., S. meliloti) is able to survive at 0–800 mg kg-1 Cu concentrations and promote alfalfa nitrogen fixation. Heavy metal- tolerant rhizobia affect the biological effectiveness of metals in soil, mainly because rhizobia promote plant growth by regulating biological nitrogen fixation (Duan et al. 2019; Shen et al. 2019; Shen et al. 2020). The P content in alfalfa increased significantly after application with rhizobia at 600 and 800 mg kg-1 Cu concentration (Table 1). These results demonstrate that alfalfa-rhizobium symbiosis alleviates Cu stress in soils by increasing biomass, N and P content, and chlorophyll content of alfalfa. Oxidative damage and antioxidant enzyme activities can effectively reflect the intensity of toxicity suffered by plant cells, and are important for understanding the metal-specific toxicity mechanisms in plants (Chen et al. 2018; Romero-Puertas et al. 2018). Excessive accumulation of Cu led to the production of large amounts of MDA in alfalfa seedlings, which intensified oxidative stress in cells (Fig. 2). Moreover, the MDA contents were significantly and negatively correlated to both plant N and P content (Fig. 5). The high level of MDA caused severe damage to plant cell membrane and inhibited plant uptake of N, P and other nutrients. Under Cu stress, plants inoculated with rhizobia significantly reduced the MDA content in the aboveground and roots (Fig. 2). The results of PLS-PM further supported that the rhizobium inoculation had a strong negative effect on MDA (Fig. 6). This indicates that plants inoculated with rhizobium suffered less oxidative damage and had a higher antioxidant capacity under Cu stress. These results suggest that inoculation with rhizobium can effectively reduce the damage to alfalfa by reactive oxygen species under Cu stress. The soluble protein content of plants can indicate the physiological and biochemical reactions in plants, and the higher the content, the more vigorous the metabolic activity, which is conducive to better resistance of plants to external stresses (Yang et al. 2019). Plants inoculated with rhizobium exhibited higher soluble protein content in alfalfa tissue compared to uninoculated plants (Fig. 2b). 4. Discussion This finding indicates that the addition of rhizobia improved the symbiotic performance of alfalfa and promoted an increase in soluble protein content, especially under non-optimal conditions. Meanwhile, alfalfa has high antioxidant enzyme activities that protect their cells from oxidative damage caused by external environmental stresses (i.e., higher MDA levels) (Filis et al. 2016; Hojati et al. 2017). Cu stress reduced the SOD activity in alfalfa tissue (Fig. 3a). Cu-contamination induced destruction of membranes and enzyme systems results in the reduce of SOD activity, especially at high Cu concentration (600 and 800 mg kg-1). Interestingly, inoculation with rhizobia had a more pronounced effect on the increase of SOD activity in alfalfa roots (Fig. 3a). This result indicates that rhizobium application enhanced superoxide dismutase activity in tissues and improved their resistance to adversity stresses. Furthermore, in comparison with uninoculated alfalfa tissue, the activities of POD and APX in shoot and root of inoculated alfalfa were increased at different levels of symbiosis development (Fig. 3). Moreover, our results showed that a Page 9/23 Page 9/23 significant positive correlation between APX activities and the N and P content of alfalfa (Fig. 5). Heavy metal resistant rhizobacteria in soil can directly affect soil Cu concentration through chelation, precipitation and biosorption. In addition, the alfalfa-rhizobium symbiotic system can promote plant nitrogen fixation, stimulate the release of phytohormones and antioxidant enzyme activities, and ultimately reduce the toxicity in alfalfa and promote alfalfa growth (Hao et al. 2014; Fang et al. 2020). Nonetheless, addition of rhizobia reduced the Cu-induced CAT activities in the shoots, except for the Cu 600 treatment (Fig. 3c). It suggests that rhizobium inoculated in the shoots inhibit the damage caused by excess hydrogen peroxide and therefore did not need to induce more CAT activities to eliminate the production of hydrogen peroxide caused by Cu concentration in the alfalfa tissues (Cao et al. 2017; Beiyuan et al. 2021). Hence, it is hypothesized that rhizobia addition mitigates Cu-toxicity in alfalfa by increasing antioxidant enzyme activities and soluble protein content. Soil enzyme activity was an important indicator to characterize the fertility and health of soils contaminated with toxic metals. Rhizobium inoculation significantly increased catalase activity in Cu 400 and Cu 600 treatments (Fig. 4a). Some studies showed that the soil catalase could achieve detoxification by changing the valence reference of heavy metal ions (Yang et al. 2016; Fang et al. 2017). 4. Discussion Meanwhile, inoculation with rhizobium significantly promoted alkaline phosphatase activity (Fig. 4d). Phosphatase in soils was mainly produced by microbial activity and was critical for mobilizing the organic forms of P (Duan et al. 2019; Wei et al. 2019). This suggests that soil catalase and phosphatase activities were positively influenced directly and indirectly by the legume-rhizobia symbiosis. There was a significant negative correlation between urease, phosphatase and catalase activities and Cu content (Fig. S2), it was shown that the legume-rhizobia symbiosis regulated the rate of enzyme synthesis by Cu or altered the interference of enzyme-producing microbial community toxicity, thus reducing metal toxicity in soil (Yu et al. 2019; Fang et al. 2020). Moreover, N and P contents of alfalfa were significantly positively correlated with activities of four soil enzymes (Fig. S2). Further, PLS-PM results showed a significant positive total effect of soil enzymes and nutrient content on plant growth following S. meliloti application (Fig. 6). Based on the above information, we found that the legume-rhizobium symbiosis system promoted alfalfa growth in Cu-contaminated soil by regulating plant physiological and biochemical properties, soil nutrients, and soil enzyme activities. Funding This work was financially supported by the National Natural Science Foundation of China (41977031, 41671406), National Funds for Distinguished Young Scientists of Shaanxi Province (2020JC-31). Consent for publication: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests. Competing interests: The authors declare no competing interests. 5. Conclusions Our results showed that excessive accumulation of Cu in alfalfa exhibited varied toxicity symptoms, including decreased biomass, chlorotic leaves, and inhibition of antioxidant enzyme activities. Rhizobium inoculation significantly improved the growth of alfalfa under Cu stress due to effectively improving detoxification ability. Legume-rhizobium symbiosis system resists Cu-induced oxidative stress in alfalfa tissues by enhancing antioxidant enzyme activities to scavenge superfluous MDA. Moreover, soil catalase, phosphatase, and β-glucosidase, as well as MBN, were higher in the rhizobium inoculated treatment compared to the non-inoculated control. These results can improve our understanding of the mechanism of microbial inoculation to mitigate Cu toxicity and provide guidance for pasture cultivation in heavy metal contaminated areas. Page 10/23 Author contribution Chengjiao Duan: writing - original draft, writing - review & editing, visualization; Yuxia Mei: Methodology, Writing - review & editing; Qiang Wang: Methodology, Writing - review & editing; Yuhan Wang: Methodology, Writing - review & editing; Qi Li: Investigation, Methodology; Maojun Hong: Investigation; Sheng Hu: Investigation; Shiqing Li: Writing - review & editing; Linchuan Fang: Funding acquisition, Methodology, Resources, Supervision, Writing - review & editing. Data availability All data generated or analyzed during this study are included in this article References 1. 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Table 1 The chlorophyll, nitrogen and phosphorus content in alfalfa tissue. Treatments Chlorophyll content (mg g-1) Nitrogen (g kg-1) Phosphorus (g kg-1) a b Total Shoot Root Shoot Root Cu 0 Control 0.62 ± 0.01 Ab 0.42 ± 0.02 Aa 1.04 ± 0.02 ABb 21.1 ± 0.37 Ab 17.5 ± 0.17 Aa 1.25 ± 0.01 Ab 0.81 ± 0.01 Ab S.meliloti 0.66 ± 0.02 BCa 0.47 ± 0.03 Aa 1.13 ± 0.03 Ba 23.1 ± 0.41 Aa 17.2 ± 0.18 Aa 1.51 ± 0.03 Aa 1.33 ± 0.02 Aa Cu 200 Control 0.63 ± 0.01 Ab 0.43 ± 0.01 Aa 1.06 ± 0.01 Ab 20.0 ± 0.39 Ba 15.9 ± 0.29 Bb 1.2 ± 0.01 Ba 0.76 ± 0.01 Ba S.meliloti 0.74 ± 0.02 Aa 0.48 ± 0.02 Aa 1.22 ± 0.03 Aa 21.4 ± 0.93 Ba 17.2 ± 0.23 Aa 1.24 ± 0.01 Ba 0.74 ± 0.02 Ba Cu 400 Control 0.56 ± 0.02 Aa 0.41 ± 0.01 Aa 0.97 ± 0.01 Bb 17.1 ± 0.11 Ca 14.3 ± 0.07 Cb 1.09 ± 0.03 Ca 0.60 ± 0.01 Ca S.meliloti 0.62 ± 0.01 Ca 0.42 ± 0.01 ABa 1.04 ± 0.01 Ca 17.8 ± 0.30 Ca 15.2 ± 0.09 Ca 0.97 ± 0.02Db 0.63 ± 0.01 Ca Cu 600 Control 0.61 ± 0.03 Aa 0.40 ± 0.02 Aa 1.01 ± 0.04 ABb 17.7 ± 0.32 Ca 14.4 ± 0.12 Cb 0.90 ± 0.01 Db 0.59 ± 0.01 Cb S.meliloti 0.71 ± 0.01 ABa 0.45 ± 0.01 Aa 1.16 ± 0.01 ABa 18.4 ± 0.05 Ca 15.6 ± 0.02 Ca 0.97 ± 0.02 Da 0.64 ± 0.02 Ca Cu 800 Control 0.38 ± 0.04 Bb 0.22 ± 0.01 Bb 0.61 ± 0.02 Cb 17.1 ± 0.18 Cb 14.7 ± 0.15 Cb 0.74 ± 0.01 Eb 0.51 ± 0.01 Db S.meliloti 0.52 ± 0.03 Da 0.37 ± 0.01 Ba 0.88 ± 0.02 Da 18.9 ± 0.38 Ca 16.2 ± 0.07 Ba 1.05 ± 0.02 Ca 0.55 ± 0.02 Da Note: The capitalized letters indicate significant differences between different Cu concentrations, whereas the lower-case letters indicate significant differences between non-inoculated and inoculated alfalfa under the same Cu concentration condition (P < 0.05). Each value represents the mean ± SE (n = 3). Table 3 Effect of rhizobium inoculation on soil physicochemical properties. the lower-case letters indicate significant differences between non-inoculated and inoculated alfalfa under the same Cu concentration condition (P < 0.05). TF (translocation factor: shoot concentration/root concentration). Each value represents the mean ± SE (n = 3). Tables bl d h l k f lf lf Table 2 Cu concentrations and the total uptake of Cu in alfalfa tissue. Page 15/23 Note: The capitalized letters indicate significant differences between different Cu concentrations, whereas Treatments Cu concentrations (mg kg-1) Total uptake (μg plant-1) TF Shoot Root Shoot Root Cu 0 Control 8.54 ± 0.07 Da 24.9 ± 0.56 Ea 3.25 ± 0.03 Ba 9.30 ± 0.21 Da 0.34 ± 0.01 Ab S.meliloti 6.45 ± 0.12 Db 3.06 ± 0.22 Eb 2.68 ± 0.05 Bb 1.18 ± 0.08 Eb 2.13 ± 0.19 Aa Cu 200 Control 12.3 ± 0.90 Ca 42.8 ± 0.92 Da 2.96 ± 0.22 Ba 14.3 ± 0.31 Ca 0.29 ± 0.03 Bb S.meliloti 10.4 ± 0.15 Ba 26.1 ± 0.04 Db 3.41 ± 0.05 Aa 10.3 ± 0.02 Db 0.40 ± 0.01 Ba Cu 400 Control 15.9 ± 0.27 Ba 50.4 ± 0.33 Cb 2.45 ± 0.04 Ca 15.6 ± 0.10 Bb 0.32 ± 0.01 Ba S.meliloti 8.54 ± 0.08 Cb 64.6 ± 0.32 Ca 1.89 ± 0.02 Cb 22.4 ± 0.11 Ba 0.13 ± 0.01 Ca Cu 600 Control 28.7 ± 0.56 Aa 93.3 ± 1.20 Ab 4.13 ± 0.08 Aa 18.8 ± 0.24 Ab 0.31 ± 0.01 Ba S.meliloti 15.7 ± 0.70 Ab 119 ± 0.75 Aa 3.16 ± 0.14 Ab 31.5 ± 0.20 Aa 0.13 ± 0.01 Cb Cu 800 Control 13.2 ± 0.70 Ca 55.2 ± 2.89 Bb 1.49 ± 0.08 Da 9.06 ± 0.47 Db 0.24 ± 0.02 Ca S.meliloti 7.50 ± 0.70 CDb 83.1 ± 0.68 Ba 1.31 ± 0.12 Da 19.9 ± 0.16 Ca 0.09 ± 0.01 Cb the lower-case letters indicate significant differences between non-inoculated and inoculated alfalfa under the same Cu concentration condition (P < 0.05). TF (translocation factor: shoot concentration/root concentration). Each value represents the mean ± SE (n = 3). the lower-case letters indicate significant differences between non-inoculated and inoculated alfalfa under the same Cu concentration condition (P < 0.05). TF (translocation factor: shoot concentration/root concentration). Each value represents the mean ± SE (n = 3). the lower-case letters indicate significant differences between non-inoculated and inoculated alfalfa under the same Cu concentration condition (P < 0.05). TF (translocation factor: shoot concentration/root concentration). Each value represents the mean ± SE (n = 3). Tables The capitalized letters indicate significant differences between different Cu concentrations, whereas the lower-case letters indicate significant differences between non-inoculated and inoculated alfalfa under the same Cu concentration condition (P < 0.05). Each value represents the mean ± SE (n = 3). Tables Page 16/23 Page 16/23 Page 16/23 Treatments Cu concentration (mg kg-1) NO3 --N (mg kg- 1) NH4 +-N (mg kg- 1) TN (g kg-1) AP (mg g- 1) SOC (g kg-1) DOC (mg kg- 1) Cu 0 Control 8.23 ± 0.36 Ea 2.67 ± 0.04 ABb 0.44 ± 0.01 Da 0.77 ± 0.01 Aa 18.3 ± 0.59 Eb 6.66 ± 0.11 Aa 133 ± 3.93 ABa S.meliloti 5.34 ± 0.17 Eb 2.94 ± 0.07 Ba 0.43 ± 0.01 Ca 0.76 ± 0.01 Ca 22.8 ± 0.67 Da 6.65 ± 0.20 Aa 138 ± 2.80 ABa Cu 200 Control 194 ± 0.75 Da 2.83 ± 0.03 Aa 0.41 ± 0.01 Da 0.77 ± 0.01 Aa 21.7 ± 0.09 Da 6.67 ± 0.14 Aa 136 ± 0.71 Ab S.meliloti 185 ± 0.48 Db 2.73 ± 0.03 Ca 0.42 ± 0.01 Ca 0.78 ± 0.01 ABa 21.8 ± 0.19 Da 6.63 ± 0.18 Aa 144 ± 2.50 Aa Cu 400 Control 336 ± 1.09 Ca 2.71 ± 0.05 ABb 1.84 ± 0.01 Ab 0.77 ± 0.01 Aa 35.8 ± 0.86 Ba 6.54 ± 0.13 Aa 97.8 ± 2.24 Cb S.meliloti 310 ± 3.74 Cb 3.22 ± 0.05 Aa 2.31 ± 0.01 Aa 0.79 ± 0.01 Aa 35.5 ± 1.42 Ba 6.57 ± 0.09 Aa 113 ± 1.47 Da Cu 600 Control 536 ± 7.06 Ba 2.61 ± 0.08 Ba 1.57 ± 0.01 Ba 0.78 ± 0.01 Aa 38.7 ± 1.08 Aa 6.75 ± 0.02 Aa 135 ± 0.86 Aa S.meliloti 505 ± 7.29 Bb 2.71 ± 0.04 Ca 1.63 ± 0.01 Ba 0.78 ± 0.01 ABa 39.4 ± 0.34 Aa 6.45 ± 0.05 Ab 124 ± 1.92 Cb Cu 800 Control 757 ± 8.76 Aa 2.65 ± 0.02 Bb 1.21 ± 0.01 Cb 0.77 ± 0.01 Aa 27.3 ± 1.23 Ca 6.40 ± 0.07 Aa 124 ± 4.52 Ba S.meliloti 708 ± 7.16 Ab 3.12 ± 0.07 Aa 1.63 ± 0.01 Ba 0.77 ± 0.01 BCa 28.4 ± 0.35 Ca 6.62 ± 0.04 Aa 133 ± 5.74 BCa Note: TN: total nitrogen; AP: available phosphorus; SOC: soil organic carbon; DOC: dissolved organic Note: TN: total nitrogen; AP: available phosphorus; SOC: soil organic carbon; DOC: dissolved organic Note: TN: total nitrogen; AP: available phosphorus; SOC: soil organic carbon; DOC: dissolved organic Note: TN: total nitrogen; AP: available phosphorus; SOC: soil organic carbon; DOC: dissolved organic carbon. Figures Page 17/23 Figure 1 The effect of inoculation with S. meliloti on length and biomass in alfalfa tissue with different Cu concentration treatments. Figure 1 The effect of inoculation with S. meliloti on length and biomass in alfalfa tissue with different Cu concentration treatments. Figure 1 Figure 1 The effect of inoculation with S. meliloti on length and biomass in alfalfa tissue with different Cu concentration treatments. The effect of inoculation with S. meliloti on length and biomass in alfalfa tissue with different Cu concentration treatments. Page 18/23 Figure 2 The effect of inoculation with S. meliloti on MDA and soluble protein in alfalfa tissue with different Cu concentration treatments. Figure 2 The effect of inoculation with S. meliloti on MDA and soluble protein in alfalfa tissue with different Cu concentration treatments. The effect of inoculation with S. meliloti on MDA and soluble protein in alfalfa tissue with different Cu concentration treatments. The effect of inoculation with S. meliloti on MDA and soluble protein in alfalfa tissue with different Cu concentration treatments. Page 19/23 Figure 3 The effect of inoculation with S. meliloti on plant antioxidant enzyme activities in alfalfa tissue. Figure 3 The effect of inoculation with S. meliloti on plant antioxidant enzyme activities in alfalfa tissue. The effect of inoculation with S. meliloti on plant antioxidant enzyme activities in alfalfa tissue. The effect of inoculation with S. meliloti on plant antioxidant enzyme activities in alfalfa tissue. The effect of inoculation with S. meliloti on plant antioxidant enzyme activities in alfalfa tissue. The effect of inoculation with S. meliloti on plant antioxidant enzyme activities in alfalfa tissue. Page 20/23 Figure 4 The effect of inoculation with S. meliloti on soil enzyme activities under different Cu concentration treatments. Figure 4 Figure 4 The effect of inoculation with S. meliloti on soil enzyme activities under different Cu concentration treatments. The effect of inoculation with S. meliloti on soil enzyme activities under different Cu concentration treatments. The effect of inoculation with S. meliloti on soil enzyme activities under different Cu concentration treatments. Page 21/23 Figure 5 A correlation heat map illustrating pairwise relationships between plant nutrient elements and plant properties in alfalfa shoot (a) and root (b) based on Pearson correlationcoefficient analysis. Figure 5 Figure 6 Partial least squares path modeling (PLS-PM) disentangling major pathways of the effects of S. meliloti, soil microbial biomass, enzyme activity, soil nutrient, plant oxidative damage and plant antioxidant enzyme activities on plant growth. Partial least squares path modeling (PLS-PM) disentangling major pathways of the effects of S. meliloti, soil microbial biomass, enzyme activity, soil nutrient, plant oxidative damage and plant antioxidant enzyme activities on plant growth. Figure 5 A correlation heat map illustrating pairwise relationships between plant nutrient elements and plant properties in alfalfa shoot (a) and root (b) based on Pearson correlationcoefficient analysis. Page 22/23 Page 22/23 Page 22/23 Page 22/23 SupplementaryMaterial.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryMaterial.docx Page 23/23
https://openalex.org/W2256418771	https://hal.archives-ouvertes.fr/hal-01274518/document	English	null	IL-17RA in Non-Hematopoietic Cells Controls CXCL-1 and 5 Critical to Recruit Neutrophils to the Lung of Mycobacteria-Infected Mice during the Adaptive Immune Response	PloS one	2,016	cc-by	10,907	IL-17RA in Non-Hematopoietic Cells Controls CXCL-1 and 5 Critical to Recruit Neutrophils to the Lung of Mycobacteria-Infected Mice during the Adaptive Immune Response Robin Lombard, Emilie Doz-Deblauwe, Florence Carreras, Mathieu Epardaud, Yves Le Vern, Dominique Buzoni, Nathalie Winter Immune Response ard, Emilie Doz-Deblauwe, Florence Carreras, Mathieu Epardaud, Yves Le Vern, Dominique Buzoni, Nathalie Winter To cite this version: Robin Lombard, Emilie Doz-Deblauwe, Florence Carreras, Mathieu Epardaud, Yves Le Vern, et al.. IL-17RA in Non-Hematopoietic Cells Controls CXCL-1 and 5 Critical to Recruit Neutrophils to the Lung of Mycobacteria-Infected Mice during the Adaptive Immune Response. PLoS ONE, 2016, 11 (2), 18 p. 10.1371/journal.pone.0149455. hal-01274518 Distributed under a Creative Commons Attribution 4.0 International License Robin Lombard, Emilie Doz☯, Florence Carreras☯, Mathieu Epardaud, Yves Le Vern, Dominique Buzoni-Gatel, Nathalie Winter* INRA, Université de Tours, UMR 1282, Infectiologie et Santé Publique, Nouzilly, France INRA, Université de Tours, UMR 1282, Infectiologie et Santé Publique, Nouzilly, France ☯These authors contributed equally to this work. * nathalie.winter@tours.inra.fr ☯These authors contributed equally to this work. * nathalie.winter@tours.inra.fr HAL Id: hal-01274518 https://hal.science/hal-01274518v1 Submitted on 15 Feb 2016 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 RESEARCH ARTICLE Abstract During chronic infection with Mycobacterium tuberculosis (Mtb), bacilli multiplication is con- strained within lung granulomas until excessive inflammation destroys the lung. Neutrophils are recruited early and participate in granuloma formation, but excessive neutrophilia exac- erbates the tuberculosis disease. Neutrophils thus appear as potential targets for therapeu- tic interventions, especially in patients for whom no antibiotic treatment is possible. Signals that regulate neutrophil recruitment to the lung during mycobacterial infection need to be better understood. We demonstrated here, in the mouse model, that neutrophils were recruited to the lung in two waves after intranasal infection with virulent Mtb or the live atten- uated vaccine strain Bacillus Calmette Guérin (BCG). A first wave of neutrophils was swiftly recruited, followed by a subsequent adaptive wave that reached the lung together with IFN- γ- and IL-17A-producing T cells. Interestingly, the second neutrophil wave did not partici- pate to mycobacteria control in the lung and established contacts with T cells. The adaptive wave was critically dependent on the expression of IL-17RA, the receptor for IL-17A, expressed in non-hematopoietic cells. In absence of this receptor, curtailed CXCL-1 and 5 production in the lung restrained neutrophil recruitment. CXCL-1 and 5 instillation reconsti- tuted lung neutrophil recruitment in BCG-infected IL17RA-/- mice. OPEN ACCESS Citation: Lombard R, Doz E, Carreras F, Epardaud M, Le Vern Y, Buzoni-Gatel D, et al. (2016) IL-17RA in Non-Hematopoietic Cells Controls CXCL-1 and 5 Critical to Recruit Neutrophils to the Lung of Mycobacteria-Infected Mice during the Adaptive Immune Response. PLoS ONE 11(2): e0149455. doi:10.1371/journal.pone.0149455 Editor: Selvakumar Subbian, Public Health Research Institute at RBHS, UNITED STATES Received: December 16, 2015 Accepted: January 31, 2016 Published: February 12, 2016 Editor: Selvakumar Subbian, Public Health Research Institute at RBHS, UNITED STATES Editor: Selvakumar Subbian, Public Health Research Institute at RBHS, UNITED STATES Editor: Selvakumar Subbian, Public Health Research Institute at RBHS, UNITED STATES Received: December 16, 2015 Accepted: January 31, 2016 Published: February 12, 2016 y p p p expressed in non-hematopoietic cells. In absence of this receptor, curtailed CXCL-1 and 5 production in the lung restrained neutrophil recruitment. CXCL-1 and 5 instillation reconsti- tuted lung neutrophil recruitment in BCG-infected IL17RA-/- mice. Copyright: © 2016 Lombard 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. IL-17RA in Non-Hematopoietic Cells Controls CXCL-1 and 5 Critical to Recruit Neutrophils to the Lung of Mycobacteria-Infected Mice during the Adaptive Immune Response Robin Lombard, Emilie Doz☯, Florence Carreras☯, Mathieu Epardaud, Yves Le Vern, Dominique Buzoni-Gatel, Nathalie Winter* IL-17RA and Lung Neutrophils in Mycobacterial Infection currently corresponds to about one third of the world population [3]. Vaccination with M. bovis Bacillus Calmette Guérin (BCG), a live attenuated strain, induces a strong and long-last- ing immune response. However, BCG provides high levels of protection only against the most severe forms of TB and, despite broad vaccination coverage, BCG is unable to control global pandemics of TB [4]. The WHO has declared the fight against TB to be a global priority. In latently infected individuals, CD4 and CD8 T and B cells that are recruited to the lung together with innate cells, form a specific multicellular structure, the granuloma [5]. Excessive inflam- mation within the granuloma leads to caseification and lung tissue destruction. The roles of macrophages in mycobacterial killing and evasion, and of dendritic cells in linking innate and adaptive responses to mycobacteria are well established [6]. The role played by neutrophils is more debated. They are among the first cells to respond to mycobacterial infection and partici- pate in the onset of adaptive immunity [7, 8] and granuloma formation [9]. However, chronic neutrophilia is involved in TB physiopathology, although the mechanisms underlying neutro- phil accumulation long after primary infection are not entirely clear [10–12]. currently corresponds to about one third of the world population [3]. Vaccination with M. bovis Bacillus Calmette Guérin (BCG), a live attenuated strain, induces a strong and long-last- ing immune response. However, BCG provides high levels of protection only against the most severe forms of TB and, despite broad vaccination coverage, BCG is unable to control global pandemics of TB [4]. The WHO has declared the fight against TB to be a global priority. In latently infected individuals, CD4 and CD8 T and B cells that are recruited to the lung together with innate cells, form a specific multicellular structure, the granuloma [5]. Excessive inflam- mation within the granuloma leads to caseification and lung tissue destruction. The roles of macrophages in mycobacterial killing and evasion, and of dendritic cells in linking innate and adaptive responses to mycobacteria are well established [6]. The role played by neutrophils is more debated. They are among the first cells to respond to mycobacterial infection and partici- pate in the onset of adaptive immunity [7, 8] and granuloma formation [9]. However, chronic neutrophilia is involved in TB physiopathology, although the mechanisms underlying neutro- phil accumulation long after primary infection are not entirely clear [10–12]. Competing Interests: The authors have declared that no competing interests exist. IL-17 cytokines play an important role in inflammation. The best characterized member of this large family is IL-17A. IL-17F is closely related to IL-17A and these two molecules can form heterodimers with different effects on the fine-tuning of the inflammatory response depending on the pathological context [13]. IL-17 cytokines signal through receptors of the IL- 17R family consisting of five subunits, which can assemble in different combinations to form diverse functional receptors. The IL-17RA subunit is common to several receptors used by at least four ligands containing IL-17A or F proteins [14]. IL-17 receptors mediate signaling through pathways generally associated with innate immunity and they connect the innate and adaptive arms of the immune response [14]. IL-17RA is expressed ubiquitously, and particu- larly in non hematopoietic epithelial cells, endothelial cells and fibroblasts [14]. In response to mycobacterial infection, IL-17A is produced principally by CD4+ CD8- αβ+ T cells, which are also known as Th17 cells [15], and Uδ+ T cells [16, 17]. Vaccine-induced Th17 cells favor the recruitment of protective Th1 cells in response to Mtb infection [15], IL-17A contributes to the formation of a mature granuloma [17, 18] and is required to constrain multiplication of Mtb clinical isolates [19] demonstrating beneficial effects. However, IL-17A is also detrimental because its unrestricted production leads to lung tissue destruction [10]. We investigated how neutrophils were recruited to the lung in mice inoculated by the intra- nasal (i.n.) route with high dose of live attenuated BCG or low dose of virulent Mtb. While BCG multiplication in the lung was controlled by the adaptive response, Mtb was not. In both situations we observed that, in addition to neutrophils recruited early in infection, a second adaptive wave of neutrophils was recruited to the lung, together with T cells. IL-17RA expressed by non-hematopoietic cells, was critically involved in the adaptive wave of neutrophil recruitment. In absence of this receptor, even though CXCL-2 was produced, CXCL-1 and 5 production in the lung was curtailed and neutrophils were not recruited. CXCL-1 and 5 instil- lation restored lung recruitment to the lung in IL-17RA-/- BCG-infected mice. Introduction Following exposure to virulent Mycobacterium tuberculosis (Mtb), one of the three leading infectious cause of human mortality [1], a large number of individuals do not show evidence of T-cell sensitization suggesting that innate mechanisms in the lung may clear infection [2]. In others, the adaptive immune response, characterized by a delayed hypersensitivity reaction to tuberculin, is initiated. However, this is generally not enough to eradicate all bacilli and most people remain latently infected with Mtb. The estimated latent tuberculosis (TB) reservoir Funding: This work was supported by the Agence Nationale pour la Recherche (FR) Grant N° ANR- 008-MIEN-001 17MycImPact to NW, the Région Centre (FR) 2013-00085470 to NW and the Région Centre (FR) fellowship to RL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 1 / 18 Ethics statement and mouse treatments Experimental protocols complied with French law (Décret: 2001–464 29/05/01) and EEC regu- lations (86/609/CEE) for the care and use of laboratory animals and were carried out under Authorization for Experimentation on Laboratory Animals Number B-37-201. Our animal protocol (number 2012-06-14) was approved by the “Val de Loire” Ethics Committee for Ani- mal Experimentation (CEEA VdL) and was registered with the French National Committee for Animal Experimentation. 2 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 IL-17RA and Lung Neutrophils in Mycobacterial Infection Six- to eight-week-old C57BL/6 male mice were obtained from SAS Janvier (Le Genest Saint Isle—France); IL-17RA-/- mice originally produced by Ye and coworkers [20] were reared in the INRA specific pathogen-free resident animal facility. Mice, anesthetized by i.p. injection of keta- mine/xylasine cocktail, received 5x106 CFU of BCG or 103 CFU of Mtb under 20 μl in each nos- tril. To deplete the neutrophil adaptive wave, mice were i.p. injected on day 17, 19 and 21 following BCG or Mtb inoculation with 200 μg of NIMPR14 mAb [21] which specifically depletes Ly-6G+ neutrophils, control mice were injected with the same quantity of IgG2b Ab (both produced in the laboratory).We produced hematopoietic chimeras, by injecting 7x106 BM cells from either WT or IL-17RA-/- donor mice intravenously into WT or IL-17RA-/- mice. Six weeks later, we checked the genotype of the donor BM, by performing PCR on blood cells. CXCL-1 and 5 chemokines (R&D systems, Minneapolis, MN, USA) were mixed in PBS and instilled i.n. to mice at day 20 μl after BCG infection. Each mouse received 100 μg of each che- mokine under 20 μl in each nostril. Control mice received equivalent volume of PBS. Bacterial strains and CFU determination Wild-type BCG strain 1173P2 Pasteur [22] and recombinant Myc 409 strains expressing EGFP [7] were grown in Beck-Proskauer medium, supplemented with hygromycin (50 μg/ml) for Myc 409. The Mtb strain laboratory adapted strain from human origin H37Rv was cultured in 7H9 medium supplemented with ADC (5% BSA fraction V, 2% dextrose, 0.003% beef catalase and 0.85% NaCl; BD Microbiology Systems, San Jose, CA, USA). The fast growing non patho- genic species M. smegmatis was grown in LB medium (Becton Dickinson, USA) Bacteria were harvested at mid-exponential growth phase and frozen at -70°C. CFU were counted after plat- ing dilutions on Middlebrook 7H11 agar supplemented with OADC (ADC supplemented with 0.005% oleic acid, BD Microbiology Systems). For the preparation of heat-killed (HK) BCG, bacilli suspended in PBS were heated for 30 min at 80°C. For lung CFU determination, tissues were disrupted with ceramic beads (Lysing Matrix D, MP Biomedicals Europe, Illkirch, France) in PBS, in a Ribolyser apparatus (Fastprep-24, MP Biomedicals), in accordance with the manu- facturer’s instructions. Appropriate dilutions were plated on 7H11 plates supplemented with OADC and CFU were counted after 21 days. For determination of neutrophil bacterial killing activity cells were infected with M. smegmatis or BCG at a MOI of 10. After 20 h of incubation, neutrophils were lyzed and bacterial CFU were determined after plating. PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 Lung and bone marrow cell preparation, antibodies, flow cytometry and cell sorting Lungs were perfused with 10 ml PBS. Single-cell suspensions were obtained from lung tissue after 1 h of treatment at 37°C with 1.5 mg/ml collagenase D and 40 units/ml DNAse A (Roche, Mannheim, Germany) in RPMI 1640 complete medium supplemented with 10% FBS (Gibco BRL, Life Technologies, Paisley, UK), 2 mM L-glutamine (Gibco), 100 U penicillin and 100 μg/ ml streptomycin (Gibco). Cells were filtered through a nylon cell strainer with 100 μm pores (BD Falcon) and suspended in complete medium for stimulation with HK BCG. For the collec- tion of cells after Broncho Alveolar Lavage (BAL), mice were killed and their lungs were washed four times with 0.5 ml PBS each time. The washing fluid from all four washes was pooled. BAL cells were washed once in PBS and were then suspended in complete medium. Bone marrow neutrophils were purified from bone-marrow by positive magnetic sorting (Mil- tenyi Biotec, Bergish Gladbach, Germany) after labeling with anti-Ly-6G PE-conjugated Ab (clone 1A8) as previously described [23]. For flow cytometry, cells were incubated for 20 min with 2% total mouse serum, and then with PBS supplemented with 5% FCS and 0.1% total mouse serum, for staining of the surface markers CD11b (clone M1/70), Ly6G (clone 1A8), Ly- 3 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 IL-17RA and Lung Neutrophils in Mycobacterial Infection 6C (clone AL-21), CD3ε (clone 145-2C11), CD4 (clone L3T4), γδ (clone GL3) all from BD Bio- sciences (San Rose, CA, USA) Before intracellular staining for IFN-γ and IL-17A, T cells were incubated with 1 μg/ml ionomycin and 25 ng/ml M phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Saint Louis, MO, USA) and HK BCG and treated for 4 h at 37°C with 5 μg/ml brefeldin A (Sigma-Aldrich). For the purification of lung neutrophils on days 1 and 23, CD11b+ Ly-6C+ Ly-6G+ cells were sorted on a MoFlo1 high-speed cell sorter (Beckman Coul- ter, Fort Collins, CO, USA) with Summit Software (Beckman Coulter). Neutrophils were recovered in complete medium and immediately stimulated with HK-BCG. Detection of chemokines and cytokines by ELISA, measure of Reactive Oxygen Species and Myeloperoxidase activity For the measurement of antigen-specific cytokine production by lung cells, we dispensed 105 lung tissue cells in individual wells of 96-well plates. The cells were incubated at 37°C, under an atmosphere containing 5% CO2, and were stimulated with the equivalent of 106 CFU of HK BCG. Supernatants were harvested 72 h later and assayed for IL-17A and IFN-γ by ELISA. CXCL-1, 2 and 5 production was measured in BAL fluids and lung tissue homogenates by ELISA. Commercial kits were from R&D Systems (Minneapolis, MN, USA) Myeloperoxidase (MPO) activity was measured immediately after sorting and Reactive Oygen Species (ROS) produced by neutrophils were assessed after overnight stimulation with HK BCG (equivalent MOI of 10). 3x105 neutrophils resuspended in 450 μl of PBS were lyzed by adding 450 μl of 0.05% Triton X100. After sonication and clearance by centrifugation for 10 min at 4°C and 12,000 x g. MPO was determined by mixing 30 μl of lysate with 70 μl of 3, 3’, 5, 5’-tetramethylbenzidine (Sigma Aldrich). After stopping the reaction with hydrochloric acid (Interchim, Montluçon, France), MPO activity was determined by measuring absorbance at 450 nm. ROS were measured in neutrophils after the addition of 10 μM ROS fluorescent detec- tion reagent (Invitrogen) and incubation for 60 min at 37°C. Green fluorescence was read in a flow cytometer (FACS Calibur, BD Biosciences). Immunohistochemistry on mycobacteria-infected lungs In situ lung neutrophil infection was analyzed in mice on day 1 or 23 after infection with 5x106 CFU of the green fluorescent BCG strain Myc409 [7]. Freshly collected lungs were incubated overnight at 4°C in 4% paraformaldehyde, 10% sucrose (w/vol in PBS) and then immersed in 30% sucrose (w/vol in PBS) for 4 h. Lung tissue was snap frozen in OCT compound (CellPath, Powys, UK). 8 μm-thick lung sections collected on Superfrost Plus slides (Thermo Fischer Sci- entific) were air-dried and stored at -80°C. Cryosections were blocked for 30 min with Fc- blocking antibody 2.4 G2 (BD Biosciences), washed in PBS with 2% FCS and incubated over- night at 4°C with anti-Ly-6G (clone1A8), (BD Biosciences) followed by incubation with Alexa 594 conjugated anti-rat (Invitrogen) and anti CD3ε (145-2C11, APC conjugated, Miltenyi Bio- tec) for the localization of T cells and neutrophils. Slides were washed in PBS with 2% and mounted with Fluoromount-G (eBioscience) including DAPI. Images were captured with a confocal Leica TCS SP8 microscope. Neutrophils reach the lung in two waves following intranasal infection with attenuated BCG or virulent Mtb We decided to investigate the kinetics of neutrophil recruitment to the lung in response to live attenuated BCG that is controlled by the host adaptive response, or virulent Mtb that is not. 5x106 CFU of live attenuated BCG or 103 CFU of virulent Mtb were administered i.n. to target the lung. Cells from the airspace, obtained after bronchoalveolar lavage (BAL), and from enzyme-digested lung tissue were analyzed by FACS. CD11b, Ly-6C and Ly-6G triple positive neutrophils, with characteristic polylobed nucleus (Fig 1A), were distinguished from inflam- matory monocytes and myeloid-derived suppressor cells [24]. The day after BCG instillation, neutrophils were abundant in both lung tissue and airspace where they accounted for a high proportion of CD11b+ cells. They rapidly decreased after day 3. However, a second, larger peak of neutrophils was observed at day 23 (Fig 1B). In the airspace, neutrophils also appeared in two waves, at day 1 and 23 (S1 Fig). A kinetic study with additional time points showed that the second wave of neutrophil appeared in the lung between days 15 and 17 (S1 Fig). By day 56 after BCG instillation, the number of neutrophils declined (Fig 1B). Following administration of 103 CFU of virulent Mtb, we detected neutrophils in the lung tissue at day one, albeit in smaller numbers than in mice receiving high dose of BCG (Fig 1C). After a slight decrease at day 7, neutrophil numbers again increased by day 23. Neutrophils continued to accumulate in Mtb-infected lung tissue at day 56 (Fig 1C). Similar kinetics was observed in the airspace (S1 Fig). CD11b, Ly-6C, Ly-6G triple positive lung tissue neutrophils sorted by flow cytometry at day 1 and day 23 after BCG infection were then compared (Fig 2). y g They displayed similar polymorphonuclear morphology (Fig 2A). In order to analyze their infection status in vivo, we used the green fluorescent BCG strain Myc409 [7]. Smaller numbers of BCG-infected neutrophils were recovered from lung tissue on day 23 than on day 1 (Fig 2B) despite a higher peak of recruitment (Fig 1B). Among all lung tissue neutrophils on day 1, 6% were infected with BCG whereas on day 23, less than 1% of neutrophils carried fluorescent bacilli (Fig 2C). Even though BCG phagocytosis by neutrophils seemed less frequent on day 23 than day 1, it remained detectable. Neutrophils were purified from the lung on day 1 or 23 after BCG instillation. Statistical analysis For analysis of in vivo data, results from two independent experiments were pooled. PRISM Graphpad 5.0 software (San Diego, CA, USA) was used for statistical analyses. We used the Kruskal-Wallis followed by Dunn’s post hoc test to analyze the kinetics of neutrophil recruit- ment to the lung and the nonparametric unpaired Mann-Whitney test for all other compari- sons. ( P < 0.05, P < 0.01 and P < 0. 001). 4 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 IL-17RA and Lung Neutrophils in Mycobacterial Infection Neutrophils reach the lung in two waves following intranasal infection with attenuated BCG or virulent Mtb Immature neutrophils were also purified from the bone-marrow for compari- son. Cells were infected in vitro with BCG or the fast growing species M. smegmatis. After over- night incubation, intracellular bacterial CFU were determined and compared to the initial inoculum. Lung neutrophils were more effective than bone-marrow to eliminate bacilli. How- ever lung neutrophils purified on day 1 or 23 displayed similar killing efficacy (Fig 2D). Myelo- peroxidase (MPO) activity and reactive oxygen species (ROS) production by the different neutrophils was measured after in vitro BCG infection. All subsets displayed similar MPO activity (Fig 2E). Bone marrow neutrophils produced less ROS than lung tissue neutrophils. However lung neutrophils purified on day 1 or 23 post BCG infection did not display signifi- cant difference in ROS production (Fig 2E). Therefore, activated neutrophils recruited to the lung on day 1 or 23 after BCG infection were highly similar in phenotype, antimicrobial mole- cules production and killing ability. IL-17RA and Lung Neutrophils in Mycobacterial Infection equivalent load of heat-killed (HK) bacilli similar numbers of neutrophils were recruited to the Fig 1. Neutrophils reach the lung in two waves following mycobacterial infection. Wild-type C57BL/6 mice were infected with 5x106 CFU of BCG or 103 CFU of Mtb and euthanized at the time points indicated. Cells were obtained from the lung tissue or airspace from individual animals. (a) Combined labeling with anti-CD11b, anti-Ly-6C and anti-Ly-6G antibodies was used to identify neutrophils among the cells present in the lung. The characteristic polylobed nucleus of neutrophils was observed in CD11b Ly-6C Ly-6G triple-positive cells from the R2 gate after flow cytometry sorting and May Grünwald Giemsa labeling. Slides were observed under an Eclipse 80 microscope (Nikon instrument) equipped with a 40 x objective. (b, c) We determined the numbers of neutrophils present in the lung tissue by flow cytometry for C57BL/6 mice killed on days 1, 3, 7, 23 and 56 post infection. Lung neutrophil numbers were determined after infection with BCG (b) or Mtb (c) Data from two independent experiments (n = 5–10). Kruskal-Wallis with Dunn’s post hoc test P<0.05, * P<0.01, *** P< 0.001. doi:10.1371/journal.pone.0149455.g001 Fig 1. Neutrophils reach the lung in two waves following mycobacterial infection. Wild-type C57BL/6 mice were infected with 5x106 CFU of BCG or 103 CFU of Mtb and euthanized at the time points indicated. Cells were obtained from the lung tissue or airspace from individual animals. (a) Combined labeling with anti-CD11b, anti-Ly-6C and anti-Ly-6G antibodies was used to identify neutrophils among the cells present in the lung. The characteristic polylobed nucleus of neutrophils was observed in CD11b Ly-6C Ly-6G triple-positive cells from the R2 gate after flow cytometry sorting and May Grünwald Giemsa labeling. Slides were observed under an Eclipse 80 microscope (Nikon instrument) equipped with a 40 x objective. (b, c) We determined the numbers of neutrophils present in the lung tissue by flow cytometry for C57BL/6 mice killed on days 1, 3, 7, 23 and 56 post infection. Lung neutrophil numbers were determined after infection with BCG (b) or Mtb (c) Data from two independent experiments (n = 5–10). Kruskal-Wallis with Dunn’s post hoc test P<0.05, * P<0.01, * P< 0.001. doi:10.1371/journal.pone.0149455.g001 equivalent load of heat-killed (HK) bacilli, similar numbers of neutrophils were recruited to the lung. Neutrophils reaching the lung on day 23 do not control mycobacteria in i o We then investigated the correlation between mycobacteria multiplication in vivo and the lung influx of neutrophils. One day after i.n. administration of 5x106 CFU of live BCG or an 5 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 IL-17RA and Lung Neutrophils in Mycobacterial Infection Fig 2. Day 1 or 23 lung neutrophils present similar killing abilities ex vivo. C57BL/6 mice were infected with 5x106 CFU of BCG (a, d, e) or the green fluorescent recombinant BCG strain Myc 409 (b, c) (a) CD11b, Ly-6C, Ly-6G triple positive neutrophils recruited to the lung on day 1 or 23 were sorted by flow cytometry and stained with May Grünwald Giemsa. (b) Neutrophils carrying the green fluorescent BCG were analyzed by flow cytometry in five individua animals. Their numbers on day 1 and 23 are represented and mean +/-SD per lung. (c) In the same animals, the percentage of rBCG-gfp-infected, among all neutrophils are represented on day 1 or 23. (d) Lung neutrophils, sorted by flow cytometry at day 1 and 23 from BCG-infected mice or bone marrow neutrophils from naïve mice were infected with M. smegmatis or BCG at a MOI of 10. Bacterial killing was determined as the ratio of the initial inoculum to CFU obtained after overnight incubation with neutrophils, with 100% killing corresponding to no cell-associated bacilli. (e) The amounts of MPO and ROS bactericidal compounds produced by lung neutrophils purified at day 1 or 23 after BCG infection were compared with those produced by BM neutrophils. The results are shown as means ± SD for triplicate wells from two independent experiments (n = 6) Fig 2. Day 1 or 23 lung neutrophils present similar killing abilities ex vivo. C57BL/6 mice were infected with 5x106 CFU of BCG (a, d, e) or the green fluorescent recombinant BCG strain Myc 409 (b, c) (a) CD11b, Ly-6C, Ly-6G triple positive neutrophils recruited to the lung on day 1 or 23 were sorted by flow cytometry and stained with May Grünwald Giemsa. (b) Neutrophils carrying the green fluorescent BCG were analyzed by flow cytometry in five individual animals. Their numbers on day 1 and 23 are represented and mean +/-SD per lung. (c) In the same animals, the percentage of rBCG-gfp-infected, among all neutrophils are represented on day 1 or 23. (d) Lung neutrophils, sorted by flow cytometry at day 1 and 23 from BCG-infected mice or bone marrow neutrophils from naïve mice were infected with M. smegmatis or BCG at a MOI of 10. By contrast, on day 23 the neutrophil peak was detected only in mice inoculated with live BCG (Fig 3A). We then assessed the multiplication of attenuated BCG and virulent Mtb multiplication in the lung. As expected, by day 23 BCG CFU had decreased 100 times as compared to initial inoculum whereas virulent Mtb CFU had increased 100 times (Fig 3B). Therefore, the two 6 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 The number of BCG or Mtb CFU present in the lung was determined on day 1 and 23 post infection. (b-d) The data shown are the mean ± SD of (log10) CFU counts from individual animals (n = 5–6), P<0.01 Fig 3. Neutrophils from the second wave do not kill mycobacteria in vivo. (a) Lung tissue neutrophil numbers from individual C57BL/6 mice were determined after flow cytometry analysis on day 1 and 23 after instillation with 5x106 CFU of BCG or an equivalent load of heat-killed (HK) BCG. Data from two independent experiments (n = 8–9), *P<0.001. (b) C57BL/6 mice (n = 5) were infected with 5x106 CFU of BCG or 103 CFU of Mtb. From day 1 to 42, the numbers of CFU in lungs were determined after bacterial growth on plates. (c-d) The impact of neutrophil depletion on bacterial control was measured by injecting mAb NIMPR14 or similar amounts of IgG2b control mAb into C57BL/6 mice 5, 3 and 1 day before infection with 5x106 CFU of BCG (c) or 103 CFU of Mtb (d) for the first neutrophil wave, or on day 17, 19 and 22 post infection for the second. The number of BCG or Mtb CFU present in the lung was determined on day 1 and 23 post infection. (b-d) The data shown are the mean ± SD of (log10) CFU counts from individual animals (n = 5–6), P<0.01 doi:10.1371/journal.pone.0149455.g003 doi:10.1371/journal.pone.0149455.g003 IL-17RA and Lung Neutrophils in Mycobacterial Infection Fig 3. Neutrophils from the second wave do not kill mycobacteria in vivo. (a) Lung tissue neutrophil numbers from individual C57BL/6 mice were determined after flow cytometry analysis on day 1 and 23 after instillation with 5x106 CFU of BCG or an equivalent load of heat-killed (HK) BCG. Data from two independent experiments (n = 8–9), *P<0.001. (b) C57BL/6 mice (n = 5) were infected with 5x106 CFU of BCG or 103 CFU of Mtb. From day 1 to 42, the numbers of CFU in lungs were determined after bacterial growth on plates. (c-d) The impact of neutrophil depletion on bacterial control was measured by injecting mAb NIMPR14 or similar amounts of IgG2b control mAb into C57BL/6 mice 5, 3 and 1 day before infection with 5x106 CFU of BCG (c) or 103 CFU of Mtb (d) for the first neutrophil wave, or on day 17, 19 and 22 post infection for the second. The number of BCG or Mtb CFU present in the lung was determined on day 1 and 23 post infection. (b-d) The data shown are the mean ± SD of (log10) CFU counts from individual animals (n = 5–6), P<0.01 doi:10 1371/journal pone 0149455 g003 trophils from the second wave do not kill mycobacteria in vivo. (a) Lung tissue neutrophil numbers from individu 6 ond wave do not kill mycobacteria in vivo. (a) Lung tissue neutrophil numbers from individual C57BL/6 mice were 6 Fig 3. Neutrophils from the second wave do not kill mycobacteria in vivo. (a) Lung tissue neutrophil numbers from individual C57BL/6 mice were determined after flow cytometry analysis on day 1 and 23 after instillation with 5x106 CFU of BCG or an equivalent load of heat-killed (HK) BCG. Data from two independent experiments (n = 8–9), **P<0.001. (b) C57BL/6 mice (n = 5) were infected with 5x106 CFU of BCG or 103 CFU of Mtb. From day 1 to 42, the numbers of CFU in lungs were determined after bacterial growth on plates. (c-d) The impact of neutrophil depletion on bacterial control was measured by injecting mAb NIMPR14 or similar amounts of IgG2b control mAb into C57BL/6 mice 5, 3 and 1 day before infection with 5x106 CFU of BCG (c) or 103 CFU of Mtb (d) for the first neutrophil wave, or on day 17, 19 and 22 post infection for the second. Bacterial killing was determined as the ratio of the initial inoculum to CFU obtained after overnight incubation with neutrophils, with 100% killing corresponding to no cell-associated bacilli. (e) The amounts of MPO and ROS bactericidal compounds produced by lung neutrophils purified at day 1 or 23 after BCG infection were compared with those produced by BM neutrophils. The results are shown as means ± SD for triplicate wells from two independent experiments (n = 6) doi:10.1371/journal.pone.0149455.g002 waves of lung neutrophils were not directly correlated to the mycobacterial load. We then selectively depleted neutrophils on days 1 and 23 by injecting anti-Ly-6G NIMPR14 Ab (S1 Fig and [24]) either before mycobacterial infection or at days 17, 19 and 21 after infection. Neither of the neutrophil depletion had a measurable impact on BCG CFU in the lungs (Fig 3C). In Mtb infected mice, a moderate although significant decrease in CFU was observed when neu- trophils were depleted in comparison to control mice (Fig 3D) indicating that the neutrophils had a negative impact on Mtb control. Therefore neutrophils reaching the lung on day 23 had no (BCG) or negative (Mtb) impact on mycobacterial multiplication in situ. PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 7 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 Neutrophils from the second wave reach the lung at the same time as T cells with which they colocalize in the lung parenchyma We then analyzed T cell recruitment to the lung. Interestingly, coincident with the second peak of neutrophils, both lung CD3+ CD4+ αβ and CD3+ CD4- γδ T cells peaked at day 23 in the BCG- and Mtb-infected mice (Fig 4A). In a more detailed kinetic study, we observed that the second wave of neutrophils reached the lung at day 15 or 17 together with T cells (S1 Fig). CD3+ CD4- γδ T cells were more numer- ous in Mtb than in BCG-infected mouse lungs (Fig 4A). As T-cell derived IFN-γ [12] and IL- 17A [10] could be involved in the lung influx of neutrophils, we analyzed the production of these two cytokines in the lung. Both cytokines peaked at day 23 in total lung cells isolated from BCG or Mtb-infected mice (Fig 4B). IFN-γ production in response to Mtb infection was slightly higher than to BCG. By contrast, Il-17A production was 20 times more elevated in Mtb as compared to BCG infected mice indicating a more sustained inflammation in the first model. Expression of the il-17a gene but not the other genes from the il-17 family [14] was up regulated in the lung at day 23 (S2 Fig). Consistent with previous reports [16–18], CD3+ CD4+ αβ T cells were the chief producers of IFN-γ, whereas CD3+ CD4- γδ T cells produced most of the IL-17A (S2 Fig). 8 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 IL-17RA and Lung Neutrophils in Mycobacterial Infection Fig 4. Neutrophils from the second wave reach the lung at the same time as effector T cells with which they establish intimate contacts. tissue cells from wild-type C57BL/6 mice, infected with 5x106 CFU of BCG or 103 CFU of Mtb, were labeled on day 1, 3, 7 and 23 and analyzed by cytometry. The numbers of CD3+ CD4+ conventional T cells or CD3+ γδ + T cells per mouse were determined (n = 4–5), P<0.05. (b) On day 1 and Fig 4. Neutrophils from the second wave reach the lung at the same time as effector T cells with which they establish intimate contacts. (a) Lung tissue cells from wild-type C57BL/6 mice, infected with 5x106 CFU of BCG or 103 CFU of Mtb, were labeled on day 1, 3, 7 and 23 and analyzed by flow cytometry. IL-17RA and Lung Neutrophils in Mycobacterial Infection infection with BCG or Mtb, 105 total lung cells were cultured for three days in the presence of HK-BCG (equivalent to 106 CFU). IL-17A and IFN-γ concentrations present in the supernatant were determined by ELISA (n = 8–10). Mann Whitney P<0.05, *P<0.001. (c-f) Representative confocal images of day 1 (c, d) or 23 (e, f) lung 8 μm cryosections from mice infected with green fluorescent BCG Myc 409 (green) and labeled with anti-Ly-6G for neutrophils (red), and anti-CD3 for T cells (cyan) and counterstained with DAPI for nuclei (blue). Merge triple or quadruple fluorescence of lung sections observed under low (c, e) or high magnification (d, f) showed neutrophil and BCG co localisation and absence of T cells on day 1 whereas neutrophils and T cell clusters were observed on day 23 (e). Higher-magnification (f) showed that on day 23, neutrophils barely co localized with mycobacteria and established close interactions with T cells (white arrowheads). Images were acquired with a confocal Leica TCS SP8 microscope equipped with x63 objective and were analyzed with the Leica LAS AF software. doi:10.1371/journal.pone.0149455.g004 We then analyzed colocalization of neutrophils with BCG or T cells in lungs on day 1 or 23 following administration of the green fluorescent Myc409 strain. On day 1, the neutrophils mostly colocalized with BCG (Fig 4C and 4D), even though many bacilli were also observed inside Ly-6G negative cells, most probably alveolar macrophages (data not shown) and CD3+ T cells were not observed. By contrast, on day 23 the second wave of Ly-6G+ neutrophils mostly clustered with CD3+ T cells (Fig 4E) and the two cell types established close contacts (Fig 4F). At that time point BCG inside Ly-6G+ neutrophils were barely observed. Thus neutro- phils that appeared in the lung on day 23 correlated with arrival of IFN-γ—and IL-17A-pro- ducing T cells in the lung but were not involved in direct mycobacterial control. IL-17RA on non-hematopoietic cells is necessary to control recruitment of neutrophils to the lung during the adaptive response Since IL-17A that peaked on day 23 is a key regulator of the mycobacterium granuloma [25], we decided to investigate neutrophil lung influx in IL-17RA-/- mice [20], in which signaling by most members of the IL-17 family is blocked [26]. The day after BCG infection, neutrophils reached the lung tissue slightly less efficiently in IL-17RA-/- than WT mice while in Mtb- infected mice, neutrophils reached the lungs in similar numbers whatever the genetic back- ground (Fig 5A). By contrast, on day 23 the influx of neutrophils to the lung tissue (Fig 5A) or airspace (data not shown) was severely impaired in Mtb or BCG-infected IL mice. The mycobacterial load was similar in WT and IL-17RA-/- mice 23 days after BCG or Mtb infection (Fig 5B) showing again that neutrophils on day 23 did not play a major role in mycobacterial control (Fig 5B). Since IL-17RA is ubiquitously expressed, we decided to investigate hematopoietic and non- hematopoietic IL-17RA contribution to neutrophil recruitment. To this end, lethally irradiated recipient WT or IL-17RA-/- mice were reconstituted with donor WT or IL-17RA-/- BM cells. All cells from control WT!WT mice expressed IL-17RA whereas IL-17RA-/-! IL- Since IL-17RA is ubiquitously expressed, we decided to investigate hematopoietic and non- hematopoietic IL-17RA contribution to neutrophil recruitment. To this end, lethally irradiated recipient WT or IL-17RA-/- mice were reconstituted with donor WT or IL-17RA-/- BM cells. All cells from control WT!WT mice expressed IL-17RA whereas IL-17RA-/-! IL- 17RA-/-mice were totally IL-17RA-deficient. Reconstitution was complete after six weeks (data not shown) and the mice were inoculated i.n. with 5x106 CFU of BCG. Twenty-three days later, we analyzed the adaptive neutrophil recruitment in lung tissue and airspace. WT mice reconstituted with IL-17RA-/- BM cells (IL-17RA-/-!WT) displayed lung tissue (Fig 5C) or air- space (data not shown) neutrophil numbers similar to control WT!WT mice. By contrast, adaptive lung neutrophil recruitment was severely impaired in WT! IL-17RA-/- as in IL- 17RA-/-! IL-17RA-/- mice where IL-17RA-/- expression by non hematopoietic cells was abro- gated (Fig 5C). Neutrophils from the second wave reach the lung at the same time as T cells with which they colocalize in the lung parenchyma The numbers of CD3+ CD4+ conventional T cells or CD3+ γδ + T cells per mouse were determined (n = 4–5), P<0.05. (b) On day 1 and 23 after PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 9 / 18 doi:10.1371/journal.pone.0149455.g005 IL-17RA regulates CXL-1 and 5 production in the lungs during the adaptive response CXCL-1 (KC), CXCL-2 (MIP2) and CXCL-5 (LIX) are three main CXCR2 ligands involved in neutrophil recruitment [27]. We next investigated whether IL-17RA controlled the production of these chemokines in the lungs BCG or Mtb-infected WT and IL-17RA-/- mice. On day 23 10 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 IL-17RA and Lung Neutrophils in Mycobacterial Infection Fig 6. IL-17RA controls the lung production of CXCL-1 and 5 after BCG or Mtb infection WT or IL-17RA-/- mice were infected with 5x106 CFU of BCG (a, b) or 103 CFU of Mtb (c, d) and production of CXCL-1 and 5 was analyzed by ELISA in BAL fluid (A, C) or lung tissue homogenates (b, d) on and 23 infection. Data from n = 4–5 animals are represented. P<0.05. Fig 6. IL-17RA controls the lung production of CXCL-1 and 5 after BCG or Mtb infection WT or IL-17RA-/- mice were infected with 5x106 CFU of BCG (a, b) or 103 CFU of Mtb (c d) and production of CXCL 1 and 5 was analyzed by ELISA in BAL fluid (A C) or lung tissue homogenates (b d) on and 23 infection Fig 6. IL-17RA controls the lung production of CXCL-1 and 5 after BCG or Mtb infection WT or IL-17RA-/- mice were infected with 5x106 CFU of BCG (a, b) or 103 CFU of Mtb (c, d) and production of CXCL-1 and 5 was analyzed by ELISA in BAL fluid (A, C) or lung tissue homogenates (b, d) on and 23 infection. Data from n = 4–5 animals are represented. P<0.05. doi:10.1371/journal.pone.0149455.g006 after infection, ELISA was performed on the BAL fluids and cleared lung tissue homogenates to measure the three chemokines. CXCL-2 was detected in lung homogenates only and did not differ between WT and IL-17RA-/- mice (data not shown). In BCG infected mice, we observed an impaired production of both CXCL-1 and 5 in IL-17RA-/- mice as compared to WT (Fig 6A and 6B). The same trend was observed in Mtb infected animals although it reached the statistical level only for CXCL-1 in lung tissue homogenates (Fig 6C and 6D). Therefore IL-17RA expres- sion in the lungs was required for optimal production of two of the three main CXCR2 ligands i.e. CXCL-1 and 5 after lung infection with BCG. The same trend was observed for Mtb. IL-17RA and Lung Neutrophils in Mycobacterial Infection Fig 5. IL-17RA in non hematopoietic cells regulates recruitment of neutrophils to the lung during the adaptive phase of the immune response. Neutrophils from C57BL/6 WT or IL-17RA-/- mice infected with 5x106 CFU of BCG or 103 CFU of Mtb or mock-treated with PBS, were analyzed by flow cytometry the day following infection or 23 days later. Numbers of neutrophils per mouse (n = 7–9) were determined in lung tissues. P<0.05, P<0.01, *P<0.001. (b) Number of CFU recovered from lungs from WT and IL-17RA-/- mice (n = 5–6) 23 days after instillation of 5x106 CFU of BCG or 103 CFU Mtb (c) Recipient WT and IL-17RA-/- mice were lethally irradiated and reconstituted with 7x106 bone marrow cells from WT or IL donor mice. On day 23 a infection with 5x106 CFU of BCG, numbers of neutrophils recruited to the lung tissue cells was determined after flow cytometry analysis (n = 5–6) P<0.0 P<0.01. doi:10.1371/journal.pone.0149455.g005 IL-17RA and Lung Neutrophils in Mycobacterial Infe Fig 5. IL-17RA in non hematopoietic cells regulates recruitment of neutrophils to the lung during the adaptive phase of the immune response. (a) Neutrophils from C57BL/6 WT or IL-17RA-/- mice infected with 5x106 CFU of BCG or 103 CFU of Mtb or mock-treated with PBS, were analyzed by flow cytometry the day following infection or 23 days later. Numbers of neutrophils per mouse (n = 7–9) were determined in lung tissues. P<0.05, P<0.01, P<0.001. (b) Number of CFU recovered from lungs from WT and IL-17RA-/- mice (n = 5–6) 23 days after instillation of 5x106 CFU of BCG or 103 CFU of Mtb (c) Recipient WT and IL-17RA-/- mice were lethally irradiated and reconstituted with 7x106 bone marrow cells from WT or IL donor mice. On day 23 after infection with 5x106 CFU of BCG, numbers of neutrophils recruited to the lung tissue cells was determined after flow cytometry analysis (n = 5–6) P<0.05, P<0.01. PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 11 / 18 Discussion In response to mycobacterial infection, neutrophils may exert protective or deleterious func- tions [28, 29]. In order to gain more insight into pathways that control neutrophil recruitment to the lung during mycobacterial infection, we decided to use two different models. We instilled into the lung either a high dose of the live attenuated strain BCG, that induced a high early inflammatory response and was later controlled by the host immune response, or a low dose of fully virulent Mtb that induced less initial inflammation but displayed unrestricted multiplica- tion. In these two opposite situations we observed that neutrophil recruitment to the lung was biphasic. In addition to the swift recruitment of neutrophils during the first 24 hours after bac- terial entry into the lung, a second wave of neutrophils reached the lung at the same time as primed T cells. These data, obtained after Mtb or BCG administration targeting the natural site of infection, i.e. the lung are in agreement with pioneering work carried out by Appelberg and coworkers who showed T cell-dependent chronic neutrophilia in the peritoneum of M. avium or Mtb infected mice [30–32]. However our data differ regarding the role played by these T-cell dependent neutrophils. While Pedrosa et al [33] reported that neutrophils played a protective role, in our case T-cell dependent neutrophils had no measurable—for BCG—or negative—for Mtb—impact (our data and [34]) on bacilli growth in vivo, despite in vitro killing ability. We think that this discrepancy is mostly due to differences in inoculation routes and to the antibody RB6-8C5 that was used by Pedrosa et al that is now established to deplete other cells in addition to neutrophils [35]. Instillation of HK BCG resulted in a barely detectable second wave of neutro- phil recruitment, indicating that the persistence of mycobacteria, inducing a sustained protective Fig 7. CXCL-1 and 5 intranasal inoculation restores neutrophil recruitment to the lungs of BCG-infected IL-17RA-/- mice WT or IL-17RA-/- mice were infected with 5x106 CFU of BCG. On day 20 after infection, a mixture of recombinant CXCL-1 and 5 in PBS, 100 μg each, was instilled into 5 animals while controls received PBS only. On day 23, neutrophils recruited into the airspace (a) or lung tissue (b) were analyzed by flow cytometry. CXCL-1 and 5 instillation recapitulates the neutrophil recruitment default in lungs of IL-17RA-/- mice during the adaptive response We next decided to assess if the default of CXCL-1 and 5 production in IL-17RA-/- mice during the adaptive phase of the immune response to mycobacterial infection was responsible for the impaired recruitment of neutrophils to the lung. WT and IL-17RA-/- mice were infected with BCG and on day 20 they received an instillation of a mixture of 100 μg of each of the two che- mokines CXCL-1 and 5. Control animals received a similar volume of PBS. Three days later CD11b, Ly-6C, Ly-6G triple positive neutrophils recruited to the airspace and lung tissue were analyzed by flow cytometry and counted. In WT mice similar numbers of neutrophils were 12 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 IL-17RA and Lung Neutrophils in Mycobacterial Infection observed into the air space (Fig 7A) and lung tissue (Fig 7B) after instillation of PBS or the two chemokines. By marked contrast, in IL-17RA-/- mice, instillation of CXCL-1 and 5, and not PBS, allowed recruitment of neutrophils to the air space (Fig 7A) and the lung tissue (Fig 7B) with levels comparable to WT animals. Therefore, the defective neutrophil recruitment to the lung observed in IL-17RA-/- mice during the onset of the adaptive response after BCG infection, could be restored by local introduction of CXCL-1 and 5. Discussion In BCG infected mice we observed that, at the time of adaptive neutrophil recruitment to the lung, production of CXCL-1 and 5 was impaired in IL-17RA deficient mice as compared to WT. Interestingly, production of CXCL-2, the third main ligand for CXCR2, was observed in presence or absence of IL-17RA. A similar trend was also observed in Mtb- infected mice although it did not reach statistical significance. Despite similar CXCL-2 produc- tion in IL-17RA-/- and WT mice, recruitment of adaptive neutrophils was highly impaired in IL-17RA-/- mouse lungs indicating that CXCL-1 and 5 were the most important. This was con- firmed after local instillation of CXCL-1 and 5 that restored adaptive neutrophils recruitment in IL-17RA-/- BCG-infected mice. The three ligands may have different affinities for CXCR2 expressed by adaptive neutrophils and CXCL-2 may not be well recognized. Alternatively, CXCL-1 or 5, but not 2, may signal via a neutrophil chemokine receptor different from CXCR2 specifically expressed by adaptive neutrophils. Although we cannot exclude that other chemo- kines such as CXCL-3, 6, 7 and 8, signaling via CXCR1 or 2 in humans [27], or CXCL15/lung- kine [41] are involved in neutrophil recruitment during the adaptive phase of the immune response, our data indicate that CXCL-1 and 5 are the chief drivers. Several reports have highlighted the importance of IL-17A production in the control of mycobacterial infection. Memory Th17 cells induced by vaccination mediate an optimal Th1 protective response against Mtb in the mouse lung [15] and IL-17A is critically involved in mature granuloma formation, which helps to constrain mycobacterial multiplication [17, 18] and is required to restrict multiplication of the hypervirulent Mtb strain HN878 [19]. We observed no difference in the control of BCG or lab adapted Mtb H37Rv multiplication between WT and IL-17RA-/- mice, from day 1 to 56, in agreement with previous data obtained after short term low-dose infection with Mtb [42]. However, IL-17RA might play an important role in longer terms [43] i.e. when the Mtb bacilli load becomes more important. Nevertheless, the IL-17A-driven exacerbation of inflammation observed in several circumstances may lead to tissue destruction. IL-17A is overproduced in response to high mycobacterial loads in the mouse [10] or in humans infected with multidrug-resistant Mtb strains resulting in high anti- gen loads [44], leading to an unfavorable clinical outcome. The IL-17A/IL-17RA pathway thus appears to be something of a double-edged sword in the control of Mtb-induced disease. Discussion Numbers of neutrophils present in 5 BAL pooled from 2 to 3 mice (a) or in lung tissue from 10 to 12 individual animals from two independent experiments are depicted (b). P<0.05 doi:10.1371/journal.pone.0149455.g007 Fig 7. CXCL-1 and 5 intranasal inoculation restores neutrophil recruitment to the lungs of BCG-infected IL-17RA-/- mice WT or IL-17RA-/- mice were infected with 5x106 CFU of BCG. On day 20 after infection, a mixture of recombinant CXCL-1 and 5 in PBS, 100 μg each, was instilled into 5 animals while controls received PBS only. On day 23, neutrophils recruited into the airspace (a) or lung tissue (b) were analyzed by flow cytometry. Numbers of neutrophils present in 5 BAL pooled from 2 to 3 mice (a) or in lung tissue from 10 to 12 individual animals from two independent experiments are depicted (b). * P<0.05 doi:10.1371/journal.pone.0149455.g007 doi:10.1371/journal.pone.0149455.g007 PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 13 / 18 IL-17RA and Lung Neutrophils in Mycobacterial Infection immune response [36, 37], was required for this second wave. Furthermore, they barely co local- ized with bacteria in the lung, and built up intimate contacts with T cells. These observations emphasize the ability of this second wave of neutrophils to take part to the adaptive immune response and we propose to define them as “adaptive neutrophils”. We also established that the IL-17A/F-IL-17RA pathway was a key regulator of the adaptive wave of neutrophil recruitment to lungs infected with Mtb or BCG. This pathway was less important for the influx of early neutrophils. The signals involved in early neutrophil recruit- ment to the lung remain to be established, but it is possible that several compounds from the mycobacterial cell wall, recognized by neutrophil-expressed pathogen recognition receptors, are directly involved [38]. The IL-17RA-driven neutrophil recruitment pathway was rather connected to the onset of the adaptive T cell response, whether it was induced by an attenuated or a virulent strain. IL-17RA is ubiquitously expressed [14] and most cells are therefore potentially able to respond to IL-17A. However, we established, in studies on BM-chimeric mice, that IL-17RA expression by non-hematopoietic cells controlled the recruitment of adaptive neutrophils. In response to IL-17A or F, the lung epithelium produces CXCR2 ligands, such as CXCL-1 (KC), CXCL-2 (MIP2) and CXCL-5 (LIX), which act as powerful attractants of neutrophils [39]. In the lung, CXCL5 is the principal ligand responsible for sustained neutrophil attraction after Mtb infection [40]. PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 Discussion We observed that during the adaptive response, neutrophils did not play an important role in mycobacterial control. However, neutrophils were recruited to the lung at the same time as T PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 14 / 18 IL-17RA and Lung Neutrophils in Mycobacterial Infection cells with which they established intimate contacts, suggesting their potential role in T cell reg- ulation. Indeed we have previously shown in vitro that neutrophils, when in contact with BCG- infected dendritic cells and T cells, were able to produce IL-10 that specifically shut down Th17 CD4 T cells through their IL-10 receptor [23]. Such immunosuppressive IL-10 producing neu- trophils were also described in vivo, in the lung of Mtb-infected mice by Zhang et al [34] where they had a negative impact on Mtb control as we observed in our experiments. Whether neutrophils have positive or negative impact in TB in humans remains a matter of debate. Whatever their role, these cells represent an important target for the development of new host-directed therapies to combat TB that are urgently needed to combat multidrug resis- tant strains [45]. In that respect our findings in the mouse model indicate that the IL-17RA- driven production of CXCL-1 and 5 in the lung is a new research axis to be considered. Supporting Information S1 Fig. Kinetics of cell recruitment to the lung after BCG or Mtb infection. C57BL/6 WT mice were inoculated by the intranasal route with 5x106 CFUs of BCG (all panels except b) or 103 CFUs of Mtb (b) and euthanized on indicated days. Cells from the airspace recovered after bronchoalveloar lavage (a, b) or enzyme lung-tissue digestion (c, e) were analyzed by flow cytometry with antibodies, as indicated in materials and methods. CD11b+ Ly-6C+ Ly-6G+ neutrophils (a-d) and CD3+ CD4+ γδ- and CD3+ CD4- γδ+ T cells (e) from four independent mice were counted P<0.05. (d) Three injections of NIMP-R14 Ab or isotype control were administered i.p to mice on days 17, 20 and 22 after BCG-infection. Neutrophils present in lung tissue were numerated on day 23 (n = 6) P< 0,005. (PDF) S2 Fig. IL-17 family genes expression in lungs from BCG or Mtb infected mice and IL-17A production by lung T cell subtypes. C57BL/6 mice were infected i.n. with 5x106 CFUs of BCG or 103 CFUs of Mtb or treated with PBS. (a) Lungs were harvested on day 1 or 23 and processed for mRNA isolation and cDNA preparation. Quantitative real-time PCR was performed on the cDNA as described in supporting materials and methods. The expression of the il-17 a, c, e and f genes was normalized with respect to the mean expression levels of three housekeeping genes: hrprt-1, rel-4 and ppia. The data shown are means ± SD of four to five individual mice. The only gene displaying significant upregulation was il-17a, which was more strongly expressed on day 23 than on day 1, with similar levels of expression in BCG- and Mtb-infected mice. (b) Lung cells from day 23 BCG-infected mice were stimulated by incubation overnight with HK BCG and PMA ionomycin and then stained for intracellular IL-17A and IFN-γ. Among the CD3+ T cells, CD4+ γδ- T cells were the main producers of IFN-γ, whereas CD3+ γδ+ T cells mainly produced IL-17A. 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Khader SA, Bell GK, Pearl JE, Fountain JJ, Rangel-Moreno J, Cilley GE, et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacte- rium tuberculosis challenge. Nat Immunol. 2007; 8(4):369–77. Acknowledgments We thank Dr Bernhard Ryffel (CNRS, Orléans, France) for the gift of IL-17RA-/- breeding pairs and Dr Fabrice Laurent, from our laboratory, for IFN-γ R-/- mice. We warmly thank all mem- bers of the Plate Forme d’Infectiologie Experimentale mouse facility from the INRA Val de Loire Center for their expertise and kindness. We would also like to express our gratitude to Sylvie Darche and Dr James Di Santo from the Pasteur Institute in Paris for their help with the PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 15 / 18 IL-17RA and Lung Neutrophils in Mycobacterial Infection generation of chimeric mice, and to Mario Delgado and François Meurens from our laboratory for help with RT-qPCR experiments and data interpretation. Author Contributions Conceived and designed the experiments: NW RL. Performed the experiments: RL ED FC ME YLV. Analyzed the data: NW RL ED FC ME DBG. Contributed reagents/materials/analysis tools: YLV. Wrote the paper: NW RL ED. Conceived and designed the experiments: NW RL. Performed the experiments: RL ED FC ME YLV. Analyzed the data: NW RL ED FC ME DBG. Contributed reagents/materials/analysis tools: YLV. Wrote the paper: NW RL ED. PLOS ONE \| DOI:10.1371/journal.pone.0149455 February 12, 2016 References Role of chemokines in the pathogenesis of acute lung injury. 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https://openalex.org/W2899245523	https://europepmc.org/articles/pmc6215444?pdf=render	English	null	Do subterranean mammals use the Earth’s magnetic field as a heading indicator to dig straight tunnels?	PeerJ	2,018	cc-by	8,192	ABSTRACT Distributed under Submitted 27 March 2018 Accepted 23 September 2018 Published 31 October 2018 Corresponding author Hynek Burda, hynek.burda@uni-due.de Academic editor Darren Burke Additional Information and Declarations can be found on page 10 DOI 10.7717/peerj.5819 Copyright 2018 Malewski et al. Distributed under Creative Commons CC-BY 4.0 Subjects Animal Behavior, Biodiversity, Ecology, Zoology Keywords Burrow systems, Magnetoreception, Mole-rats, Orientation, Subterranean rodents Subjects Animal Behavior, Biodiversity, Ecology, Zoology Keywords Burrow systems, Magnetoreception, Mole-rats, Orientation, Subterranean rodents How to cite this article Malewski et al. (2018), Do subterranean mammals use the Earth’s magnetic ﬁeld as a heading indicator to dig straight tunnels?. PeerJ 6:e5819; DOI 10.7717/peerj.5819 Do subterranean mammals use the Earth’s magnetic ﬁeld as a heading indicator to dig straight tunnels? Sandra Malewski1, Sabine Begall1,2, Cristian E. Schleich3, C. Daniel Antenucci3 and Hynek Burda1,2 1 Department of General Zoology, University of Duisburg-Essen, Essen, Germany 2 Department of Game Management and Wildlife Biology, Czech University of Agriculture, Prague, Czech Republic 1 Department of General Zoology, University of Duisburg-Essen, Essen, Germany 2 Department of Game Management and Wildlife Biology, Czech University of Agriculture, Prague, Czech Republic 3 3 Laboratorio de Ecología Fisiológica y del Comportamiento, Instituto de Investigaciones Marinas y Costeras (IIMyC), Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas (CONICET), Universidad Nacional de Mar del Plata, Mar del Plata, Buenos Aires, Argentina Subjects Animal Behavior, Biodiversity, Ecology, Zoology Keywords Burrow systems, Magnetoreception, Mole-rats, Orientation, Subterranean rodents ABSTRACT Subterranean rodents are able to dig long straight tunnels. Keeping the course of such “runways” is important in the context of optimal foraging strategies and natal or mating dispersal. These tunnels are built in the course of a long time, and in social species, by several animals. Although the ability to keep the course of digging has already been described in the 1950s, its proximate mechanism could still not be satisfactorily explained. Here, we analyzed the directional orientation of 68 burrow systems in ﬁve subterranean rodent species (Fukomys anselli, F. mechowii, Heliophobius argenteocinereus, Spalax galili, and Ctenomys talarum) on the base of detailed maps of burrow systems charted within the framework of other studies and provided to us. The directional orientation of the vast majority of all evaluated burrow systems on the individual level (94%) showed a signiﬁcant deviation from a random distribution. The second order statistics (averaging mean vectors of all the studied burrow systems of a respective species) revealed signiﬁcant deviations from random distribution with a prevalence of north–south (H. argenteocinereus), NNW–SSE (C. talarum), and NE–SW (Fukomys mole-rats) oriented tunnels. Burrow systems of S. galili were randomly oriented. We suggest that the Earth’s magnetic ﬁeld acts as a common heading indicator, facilitating to keep the course of digging. This study provides a ﬁeld test and further evidence for magnetoreception and its biological meaning in subterranean mammals. Furthermore, it lays the foundation for future ﬁeld experiments. Subterranean rodents are able to dig long straight tunnels. Keeping the course of such “runways” is important in the context of optimal foraging strategies and natal or mating dispersal. These tunnels are built in the course of a long time, and in social species, by several animals. Although the ability to keep the course of digging has already been described in the 1950s, its proximate mechanism could still not be satisfactorily explained. Here, we analyzed the directional orientation of 68 burrow systems in ﬁve subterranean rodent species (Fukomys anselli, F. mechowii, Submitted 27 March 2018 Accepted 23 September 2018 Published 31 October 2018 Corresponding author Hynek Burda, hynek.burda@uni-due.de Academic editor Darren Burke Additional Information and Declarations can be found on page 10 DOI 10.7717/peerj.5819 Copyright 2018 Malewski et al. INTRODUCTION Across the globe, about 250 rodent species, belonging to different unrelated taxa (six families, 38 genera), have convergently adapted to permanent life in self-excavated extensive underground burrow systems (for review see Begall, Burda & Schleich, 2007; Lacey, 2000; Nevo, 1999). Burrow systems inhabited by single individuals (solitary species) or families (social species) can reach lengths of several hundred meters or, in social Distributed under Creative Commons CC-BY 4.0 species, even kilometers (Brett, 1991; Šklíba et al., 2012; Šumbera et al., 2003, 2008, 2012). Here, the animals live, communicate, orientate in space and time in a dark, monotonous sensory environment free of most orientation cues which are available aboveground. Especially light propagation is highly limited in subterranean burrow systems, so that vision is of little use for subterranean rodents there (Kott et al., 2014). Since environmental cues are mostly lacking in the subterranean ecotope, rodents are forced to rely on idiothetic cues to orientate which are, however, prone to accumulations of errors. Consequently, an external reference that could be used for orientation and navigation would be of high value (Moritz et al., 2007). While the question, how these animals can orientate in their complex underground maze, has been repeatedly addressed (Burda, 1987; Eloff, 1951; Kimchi, Etienne & Terkel, 2004; Kimchi & Terkel, 2001), one interesting sensory ecological aspect remained understudied. How do subterranean mammals manage to keep the course of digging? This question was ﬁrst raised approximately 70 years ago in 1951 when Eloff mentioned the “remarkable ability (of the South-African Cryptomys mole-rat) to follow a direction or ﬁnd a spot where it previously bored a tunnel” (Eloff, 1951, p. 145). The African mole-rat’s ability to maintain its course while digging long, straight tunnels (Eloff, 1951) gave rise to speculations on possible orientation cues: air currents (De Graaff, 1972; Eloff, 1958; Poduschka, 1978) and acoustic cues (Eloff, 1951; Müller & Burda, 1989; Rosevear, 1969) have been discussed, as well as internal mechanisms based on kinesthetic and/or vestibular cues (Teroni et al., 1988). However, none of these mechanisms provide a satisfactory explanation for the highly efﬁcient directional orientation observed in Cryptomys hottentotus (Eloff, 1951). Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 INTRODUCTION ( ff ) Inspired by the mentioned enigmatic ability, about 40 years after Eloff, Burda (1987) postulated that the mole-rats might use magnetic cues to orientate and navigate, an ability which was then explicitly proven in the laboratory repeatedly, speciﬁcally for the Zambian Ansell’s mole-rat (Fukomys anselli, formerly assumed to represent the same species as the South African C. hottentotus and in papers published before 2006 named so) (Burda, 1987; Burda et al., 1990; Malewski et al., 2018; Marhold, 1997; Thalau et al., 2006; Wegner, Begall & Burda, 2006). The phenomenon of magnetoreception, meaning an animal’s sensory ability to extract information from the Earth’s magnetic ﬁeld, has been investigated since the 1960s (for review see Begall, Burda & Malkemper, 2014; Wiltschko & Wiltschko, 1995). However, studies with the purpose to unravel the function of the mammalian magnetic sense are still rare (Holland et al., 2006; Kimchi, Etienne & Terkel, 2004). Following the ﬁnding that African mole-rats are magnetosensitive (Burda et al., 1990), Lovegrove, Körtner & Körtner (1992) investigated directional orientation of burrow systems of Damaraland mole-rats (F. damarensis) in the ﬁeld, inferring that the “orientation of the burrow system with respect to a speciﬁc compass orientation may represent an intrinsic requirement of successful geomagnetic orientation and direction ﬁnding” (Lovegrove, Körtner & Körtner, 1992, p. 631). However, no correlation between the Earth’s magnetic ﬁeld and the burrow system’s orientation was found. Years later, Schleich & Antinuchi (2004) performed a comparable study on burrow systems of Talas tuco-tucos (Ctenomys talarum), and achieved also negative results. However, in both Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 2/15 studies, the data were analyzed by means of a Chi2-test, which does not take the speciﬁc characteristics of circular data into account (e.g., 10 is close to 350), and which is therefore less powerful than circular statistics. Furthermore, the previously used “polygon method” expressed directional alignment of the burrow system through a line connecting the most distant points of a polygon surrounding the burrow system. Since this method ignores all the shorter side branches of a burrow system within the circumferential polygon, a new, more detailed and accurate method would be of avail. In our study, we revive the question raised by Eloff (1951), and test if subterranean rodents use the Earth’s magnetic ﬁeld as a heading indicator to keep a straight course of digging. INTRODUCTION Keeping a digging direction, for example, during foraging (Burda, 1987) or during natal or mating dispersal (Hickman, 1990; Nevo, 1999; Rado, Wollberg & Terkel, 1992) would be advantageous, as digging curvy tunnels would imply increased or even devastating energetic costs; note that costs for digging are between 360 and up to 3,400 times higher than if the animal moves the same distance above ground (Vleck, 1979). We may assume that straight tunnels longer than, for example, one m are not the product of a single digging bout, and in the case of social species, not even of a single individual (Jarvis et al., 1994). This fact points out the necessity of a heading indicator to keep the course of digging. While diverse heading indicators (visual or olfactory landmarks, sun position, wind direction) can be used to keep the course of locomotion aboveground, availability of such cues is restricted underground. To test the hypothesis of a magnetic heading indicator, we analyzed the directional orientation of complete burrow systems excavated in the ﬁeld of ﬁve subterranean rodent species, three of which are already known to be magnetosensitive (Oliveriusová et al., 2012): two Fukomys mole-rat species (F. anselli and F. mechowii), the silvery mole-rat (Heliophobius argenteocinereus), the blind mole rat (Spalax galili), and the Talas tuco-tuco (C. talarum) by means of two different methods. Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 MATERIAL AND METHODS ote: Number of mapped burrow systems, locality and year of excavation, as well as references to the studies describing burrow architecture of the respective species li t d Note: Number of mapped burrow systems, locality and year of excavation, as well as references to the studies describing burrow architecture of the respective species are listed. directionality, since the original purpose of the excavation was not to study burrow orientation, but to obtain data about other aspects of subterranean life (e.g., burrow architecture). In our study, the systems were digitally analyzed twice, by applying two different methods: the “polygon method,” which was applied already in previous studies (Lovegrove, Körtner & Körtner, 1992; Schleich & Antinuchi, 2004), and the “long tunnel method,” introduced by us here (Fig. S1). Since the animals’ original digging direction (unidirectional) cannot be deduced from the excavations or drawings, bidirectional analysis was the method of choice, that is, data are doubled (modulo 360) before being analyzed, and the resulting mean vector is then back-converted, thus ranging in the interval (0; 180). All axial values are reported as XX/XX (N/S). The “polygon method” (Figs. S1A and S1C) is based on drawing a convex polygon connecting the outermost points of a burrow system. A diagonal, marking the longest distance within the polygon, was drawn, representing the main axis of the burrow system. The direction of the longest distance within the polygon was measured blindly (i.e., The polygon method (Figs. S1A and S1C) is based on drawing a convex polygon connecting the outermost points of a burrow system. A diagonal, marking the longest distance within the polygon, was drawn, representing the main axis of the burrow system. The direction of the longest distance within the polygon was measured blindly (i.e., without knowing the direction of magnetic North) by means of a digital compass rose. Subsequently, all measured values were unblinded by normalizing the data relative to the direction of true magnetic North. without knowing the direction of magnetic North) by means of a digital compass rose. Subsequently, all measured values were unblinded by normalizing the data relative to the direction of true magnetic North. The “long tunnel method” (Figs. S1B and S1E) weights the magnetic axial direction of the branches within a burrow system depending on their length. Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 MATERIAL AND METHODS Our dataset consisted of 68 burrow systems of ﬁve subterranean rodent species (F. anselli, F. mechowii, H. argenteocinereus, C. talarum, S. galili), belonging to three nonrelated families with different geographic distribution, and representing both solitary or social lifestyles (Table 1). All burrow systems were mapped within the framework of previous ﬁeld ecological studies (Lövy et al., 2015; Schleich & Antinuchi, 2004; Šklíba, Šumbera & Chitaukali, 2010; Šklíba et al., 2009, 2012; Šumbera et al., 2003, 2008, 2012), and the maps were provided to us for this study. They were digitized true to scale by means of Quantum GIS (QGIS Geographic Information System, www.qgis.osgeo.org) for burrow systems of silvery mole-rats, Fukomys mole-rats, and Spalax, and by means of ImageJ (Schneider, Rasband & Eliceiri, 2012) for burrow systems of Talas tuco-tucos. We pooled the data of both species of Fukomys mole-rats in order to increase the sample size. It should be noted that both species are phylogenetically related, live in the same habitat in the same geographic region and have the same lifestyle. The direction of magnetic North was noted as accompanying information as usual in geographical mapping. The data has been collected blindly with respect to the analysis of compass Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 3/15 Table 1 Tested rodent species (sorted alphabetically by their common names). Species Family Social/ solitary Number of burrow systems Locality Year of excavation Reference, burrow systems were originally used for Ansell’s mole-rat (Fukomys anselli) Bathyergidae Social 7 Lusaka East Forest Reserve (Zambia) 2010 Šklíba et al. (2012) Giant mole-rat (Fukomys mechowii) Bathyergidae Social 2 Ndola Hill Forest Reserve (Zambia) 2009 Šumbera et al. (2012) Silvery mole-rat (Heliophobius argenteocinereus) Bathyergidae Solitary 31 Blantyre, Mulanje, Mpalaganga (Malawi) 2000, 2005 Šklíba et al. (2009), Šklíba, Šumbera & Chitaukali (2010), and Šumbera et al. (2003, 2008), and additional previously unpublished burrow systems (cf. raw data) Talas tuco-tuco (Ctenomys talarum) Ctenomyidae Solitary 19 Mar de Cobo, Buenos Aires (Argentina) 1988, 1989 Schleich & Antinuchi (2004) Upper Galilee Mountains blind mole rat (Spalax galili) Spalacidae Solitary 9 Upper Galilee Mountains (Israel) 2012 Lövy et al. (2015) Note: Number of mapped burrow systems, locality and year of excavation, as well as references to the studies describing burrow architecture of the respective species are listed. Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 MATERIAL AND METHODS The underlying hypothesis concerning this approach was the following: The longer the distance, which was dug in a speciﬁc magnetic direction, the stronger is the animal’s preference for that particular direction. To apply this method, ﬁrst, we determined the directions of all straight tunnels of a burrow system blindly (i.e., the evaluator did not know the direction of magnetic North). We deﬁned a “straight tunnel” as straight segment including small turns/curves which ended at a clear change in direction of more than 30 (see Fig. S1E). Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 Only straight tunnels that were at least as long as 5% of the polygon’s circumference were included in our data set. The tunnels’ axial directions were measured by means of a digital compass rose. After unblinding the data, vector analysis of each straight tunnel direction weighted by the tunnels’ lengths was performed to calculate the mean direction for each burrow system separately. Therefore, the vector’s direction was treated as value and its length as frequency (see frequency editor of Oriana 4.0, Kovach computing systems). All drawings of polygons and diagonals as well as their evaluations were performed with Fiji (Schindelin et al., 2012). Circular statistics (Batschelet, 1981) and vector analyses were calculated by using Oriana 4.0 (Kovach computing systems). Axial mean values were calculated for each species and each method (for the “long tunnel method,” the mean vector (grand mean) was calculated over the mean axial directions of the respective tunnels). The Rayleigh-test of uniformity was used to test for signiﬁcant deviations from a random distribution. Watson–Williams F-test (pairwise comparison) was applied to compare the burrow systems’ directions achieved by both methods for each species, respectively. RESULTS By applying the “long tunnel method,” the axial orientation of the vast majority of all evaluated burrow systems (94%) showed a signiﬁcant deviation from a random distribution, that is, in 64 out of the 68 analyzed tunnel systems the straight tunnel segments were oriented signiﬁcantly in a certain axial compass direction (Fukomys: 100%; H. argenteocinereus: 90%; C. talarum: 95%; S. galili: 100%). Considering the second order statistics (averaging the mean vectors of all analyzed burrow systems of a respective genus or species), a signiﬁcant deviation from random orientation was found for the Fukomys mole-rats, the silvery mole-rat, and the Talas tuco-tuco (Fig. 1). An axial directional preference for the North–South axis was observed for the silvery mole-rat (Fig. 1A), while it was slightly shifted to NNW/SSE in the Talas tuco-tuco (Fig. 1B). The Fukomys mole-rats showed a preference for the NE/SW (Fig. 1C). No prevailing magnetic direction of the studied burrows was found in the blind mole rat (S. galili) (Fig. 1D). Since most burrow systems (N = 31) were available for the silvery mole rats Since most burrow systems (N = 31) were available for the silvery mole-rats, we analyzed whether the tunnel systems’ lengths and locality of excavation (Blantyre, Mpalaganga, Mulanje; Table 1) had an effect on the orientation of the mean axial direction. A signiﬁcant deviation from random orientation was found for systems which were shorter than 100 m (N = 15, m = 1/181, SD = 31, P = 0.007; mean length = 57 m, SD = 26 m), in contrast to systems which were longer than 100 m (N = 16, m = 15/195, SD = 48, P = 0.406; mean length = 192 m, SD = 68 m). With respect to the locality of excavation, a signiﬁcant deviation from random orientation was only observable in one out of three localities (Blantyre: N = 17, m = 5/185, SD = 34, P = 0.015; Mpalaganga: N = 10, m = 169/349, SD = 52, P = 0.702; Mulanje: N = 4, m = 20/200, SD = 30, P = 0.272). Furthermore, we did not ﬁnd statistical sex-dependent differences in mean orientation for any of the solitary species (statistics not shown). The “polygon method” revealed that the alignment of the burrow systems of the Talas tuco-tuco deviated signiﬁcantly from random orientation (Fig. 1F). A similar strong tendency was Malewski et al. Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 DISCUSSION Figure 1 illustrates that the “polygon method” provides less consistent results than the “long tunnel method.” Since the “polygon method” has been employed in previous studies (Lovegrove, Körtner & Körtner, 1992; Schleich & Antinuchi, 2004), we decided to use this method and present its results for the sake of comparability, too. However, the “polygon method” is generally suitable only for rather linear instead of circular burrow systems, where the long diagonal within a polygon represents the directional alignment of the whole burrow system. In a more circular system, several diagonals of similar lengths can be drawn (Fig. S1D). Various ecological factors inﬂuence the geometry of a burrow system. Radiality (high branching) of a burrow system is expected to increase with uniformity and predictability of food resources, age of the burrow system, and with the number of inhabitants (Romañach et al., 2005; Sichilima et al., 2008; Thomas, Swanepoel & Bennett, 2016). Moreover, the architecture of burrow systems can vary seasonally and in dependence on the inhabitant’s sex (e.g., dispersing and mate-seeking individuals might build more linear tunnels) (Antinuchi & Busch, 1992; Nevo, 1999; Šklíba et al., 2012; Šumbera et al., 2003, 2008, 2012). Indeed, the index of circularity (formula: 4π (area)/perimeter2, 0 = linear, 1 = circular; Romañach et al., 2005) identiﬁes 60% of the studied burrow systems of the solitary silvery mole-rats (Fig. 2A) and 70% of the systems of the solitary tuco-tucos as being rather linear (with an index <0.7 as exemplarily deﬁned threshold separating the upper third as being circular), while all systems of the social Fukomys mole-rats exhibited values >0.7 (Fig. 2B). In contrast to the “polygon method,” the “long tunnel method” revealed constant results for different species independent from the respective indices of circularity. Having applied this method, we found a signiﬁcant deviation from random axial orientation for burrowing longer straight tunnels along a certain axis, which was speciﬁc for each burrow system. Moreover, the second order statistics (averaging mean vectors of all burrow systems of a respective genus or species) revealed signiﬁcant deviation from random orientation in all the tested species but S. galili (Fig. 1), including a prevalence of NNW/SSE in case of Talas tuco-tucos and NE–SW in case of Fukomys mole-rats. RESULTS (2018), PeerJ, DOI 10.7717/peerj.5819 5/15 Figure 1 Burrow system’s orientation. Directional orientation of the burrow systems (estimated b “long tunnel method,” A–D) and the prevailing direction of burrows (estimated by the “po method,” E–H) of the tested rodent species—the silvery mole-rat H. argenteocinereus (A, E), tuco-tuco C. talarum (B, F), Fukomys mole-rats Ansell’s mole-rat F. anselli and the giant mo F. mechowii (C, G), and the Upper Galilee Mountains blind mole rat S. galili (D, H) (sorted by sa size)—relative to magnetic North (0). Two mirrored dots represent the axial direction of one bu system, which is in case of the long tunnel method an axial mean vector calculated over all directio straight tunnel segments weight by the segments’ lengths. The double-headed arrow indicates the (g mean vector, and the inner circle marks the signiﬁcance level of 0.05 (Rayleigh test). Sample size mean axial direction (m), circular standard deviation (SD), and P-value are given for each tested sp Full-size  DOI: 10.7717/peerj.5819 Figure 1 Burrow system’s orientation. Directional orientation of the burrow systems (estimated by the “long tunnel method,” A–D) and the prevailing direction of burrows (estimated by the “polygon method,” E–H) of the tested rodent species—the silvery mole-rat H. argenteocinereus (A, E), Talas tuco-tuco C. talarum (B, F), Fukomys mole-rats Ansell’s mole-rat F. anselli and the giant mole-rat F. mechowii (C, G), and the Upper Galilee Mountains blind mole rat S. galili (D, H) (sorted by sample size)—relative to magnetic North (0). Two mirrored dots represent the axial direction of one burrow system, which is in case of the long tunnel method an axial mean vector calculated over all directions of straight tunnel segments weight by the segments’ lengths. The double-headed arrow indicates the (grand) mean vector, and the inner circle marks the signiﬁcance level of 0.05 (Rayleigh test). Sample size (N), mean axial direction (m), circular standard deviation (SD), and P-value are given for each tested species. Full-size  DOI: 10.7717/peerj.5819/ﬁg-1 Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 6/15 observable for the silvery mole-rat’s systems (Fig. 1E). The burrow systems of the Fukomys mole-rats (Fig. 1G) and the blind mole rat (Fig. 1H) showed random directional orientation using the “polygon method”. The axial directional orientation of the burrow systems as assessed by the “polygon method” (Figs. 1A–1D) and the prevailing direction of the burrows estimated by the “long tunnel method” (Figs. RESULTS 1E–1H) did not differ signiﬁcantly except for the Fukomys mole-rats (Watson–Williams F-test (pairwise comparisons for each species): Fukomys mole-rats: P = 0.042, silvery mole-rat: P = 0.423, tuco-tuco: P = 0.455, blind mole rat: P = 0.636), however, a greater scatter was observable by applying the “polygon method.” Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 DISCUSSION The fact that the studied burrow systems have been uncovered in different regions (even continents) and areas (e.g., grassland, woodland, mountainous areas), and thus accompanying different habitats, as well as in different years (1988–2012; Table 1), support the assumption that the Earth’s magnetic ﬁeld seems to be a globally common and Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 Figure 2 Examples of burrow systems of a (A) solitary (here Heliophobius argenteocinereus) and (B) social species (here Fukomys anselli). Index of circularity (0 = linear, 1 = circular; Romañach et al., 2005) for (A) = 0.42, and (B) = 0.83. Full-size  DOI: 10.7717/peerj.5819/ﬁg-2 Figure 2 Examples of burrow systems of a (A) solitary (here Heliophobius argenteocinereus) and (B) social species (here Fukomys anselli). Index of circularity (0 = linear, 1 = circular; Romañach et al., 2005) for (A) = 0.42, and (B) = 0.83. Full-size  DOI: 10.7717/peerj.5819/ﬁg-2 stable potential heading indicator. Accordingly, we suggest that the studied species use magnetic cues as heading indicator to keep the course of digging. This hypothesis does not exclude that the animals use also further, locally available heading indicators, for this purpose. Indeed, magnetoreception has been proved in the laboratory for F. anselli (Burda et al., 1990; Marhold, Wiltschko & Burda, 1997), F. mechowii (Oliveriusová et al., 2012), and H. argenteocinereus (Oliveriusová et al., 2012). Interestingly, magnetoreception was not proved in laboratory maze experiments in C. talarum (Schleich & Antinuchi, 2004), which, however, could have been due to an inappropriate experimental design as discussed by the authors themselves (Schleich & Antinuchi, 2004). On the other hand, magnetoreception has been proven in Spalax judaei (Kimchi & Terkel, 2001; Marhold et al., 2000), a close relative of S. galili for which no directional preference for digging was found here. The reasons, why no directional digging preference was found in S. galili, can be manifold. All analyzed burrow systems originate from a single small area in the Upper Galilee Mountains, and six of nine examined burrow systems were located in basaltic soils. Basaltic soils are generally characterized by rather strong magnetic signatures and the studied region is known for magnetic anomalies (Eppelbaum, Ben-Avraham & Katz, 2004) that might compromise magnetic orientation. Furthermore, the population density in basaltic soil is ﬁve times higher than in rendzina soil (Lövy et al., 2015). Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 DISCUSSION The avoidance of crossing a neighboring burrow system might inﬂuence the burrow systems orientation and thus possibly override orientation via magnetic cues. Therefore, it is of great interest to analyze comparative data on directionality of burrow systems of Spalax from other geographic regions and other habitats (cf., Nevo et al., 1995; Reyes, Nevo & Saccone, 2003; Savic, 1973; Yağci, Coşkun & Aşan, 2010). stable potential heading indicator. Accordingly, we suggest that the studied species use magnetic cues as heading indicator to keep the course of digging. This hypothesis does not exclude that the animals use also further, locally available heading indicators, for this purpose. Indeed, magnetoreception has been proved in the laboratory for F. anselli (Burda et al., 1990; Marhold, Wiltschko & Burda, 1997), F. mechowii (Oliveriusová et al., 2012), and H. argenteocinereus (Oliveriusová et al., 2012). Interestingly, magnetoreception was not proved in laboratory maze experiments in C. talarum (Schleich & Antinuchi, 2004), which, however, could have been due to an inappropriate experimental design as discussed by the authors themselves (Schleich & Antinuchi, 2004). On the other hand, magnetoreception has been proven in Spalax judaei (Kimchi & Terkel, 2001; Marhold et al., 2000), a close relative of S. galili for which no directional preference for digging was found here. The reasons, why no directional digging preference was found in S. galili, can be manifold. All analyzed burrow systems originate from a single small area in the Upper Galilee Mountains, and six of nine examined burrow systems were located in basaltic soils. Basaltic soils are generally characterized by rather strong magnetic signatures and the studied region is known for magnetic anomalies (Eppelbaum, Ben-Avraham & Katz, 2004) that might compromise magnetic orientation. Furthermore, the population density in basaltic soil is ﬁve times higher than in rendzina soil (Lövy et al., 2015). The avoidance of crossing a neighboring burrow system might inﬂuence the burrow systems orientation and thus possibly override orientation via magnetic cues. Therefore, it is of great interest to analyze comparative data on directionality of burrow systems of Spalax from other geographic regions and other habitats (cf., Nevo et al., 1995; Reyes, Nevo & Saccone, 2003; Savic, 1973; Yağci, Coşkun & Aşan, 2010). stable potential heading indicator. Accordingly, we suggest that the studied species use magnetic cues as heading indicator to keep the course of digging. DISCUSSION While using a magnetic compass as a heading indicator, in general, the animals could follow every direction, and indeed, species-speciﬁc differences in chosen nest-building directions were already reported repeatedly (for review see Oliveriusová et al., 2012). However, the North–South axis might be the direction of choice. A preference for this axis, as observed in our study for silvery mole-rats and Talas tuco-tucos, is common in studies on magnetic orientation (Malkemper et al., 2015; Oliveriusová et al., 2014; Phillips, 1986) and magnetic alignment (for review see Begall et al., 2013; Malkemper, Painter & Landler, 2016), and is observable in diverse contexts: landing in waterfowl (Hart et al., 2013), escape in roe deer (Obleser et al., 2016), activity in cattle and deer (Begall et al., 2008), ﬂamingos (Nováková et al., 2017), corvids (Pleskač et al., 2017), wild boars and warthogs (Červený et al., 2017), carps (Hart et al., 2012). Considering that species-speciﬁc burrowing behavior in North American Peromyscus rodents was reported to have a genetic basis (Dawson, Lake & Schumpert, 1988; Hu & Hoekstra, 2017; Metz et al., 2017; Weber, Peterson & Hoekstra, 2013), we would like to point to the possibility that “magnetic digging” might be genetically determined, too, representing a highly interesting research question. While using a magnetic compass as a heading indicator, in general, the animals could follow every direction, and indeed, species-speciﬁc differences in chosen nest-building directions were already reported repeatedly (for review see Oliveriusová et al., 2012). Since magnetic and further orientation cues are not exclusive, apart from the Earth’s magnetic ﬁeld, several other cues might inﬂuence the alignment of the burrow systems. The most crucial external cues might be food resources (Heth et al., 2002; Romañach et al., 2005), soil condition (implying the presence of stones and other obstacles) (Ebensperger & Bozinovic, 2000; Kimchi, Reshef & Terkel, 2005; Luna, Antinuchi & Busch, 2002; Zuri & Terkel, 1997), and landscape characteristics (e.g., dunes, vegetation, slopes, water currents), and even subsurface geological fractures might function as orientation cues (Olson & Pollard, 1989). Presence of food resources inﬂuences goal-directed burrowing at shorter distances (Eloff, 1951; Voigt, 2014). Since geophytes are usually clumped, and can be located at a limited distance by smelling plant kairomones diffused in the soil (Heth et al., 2002; Lange et al., 2005), an optimal foraging strategy is expected to involve digging straight scouting tunnels with perpendicular shorter branches in fertile areas (following kairomone cues). DISCUSSION This hypothesis does not exclude that the animals use also further, locally available heading indicators, for this purpose. Indeed, magnetoreception has been proved in the laboratory for F. anselli (Burda et al., 1990; Marhold, Wiltschko & Burda, 1997), F. mechowii (Oliveriusová et al., 2012), and H. argenteocinereus (Oliveriusová et al., 2012). Interestingly, magnetoreception was not proved in laboratory maze experiments in C. talarum (Schleich & Antinuchi, 2004), which, however, could have been due to an inappropriate experimental design as discussed by the authors themselves (Schleich & Antinuchi, 2004). On the other hand, The burrow systems of Heliophobius analyzed here (N = 31) were excavated at three different localities, characterized by different landscapes (Blantyre, N = 17: grassland; Mpalaganga, N = 10: woodland; Mulanje, N = 4: mountainous) and thus habitats. A signiﬁcant deviation from random axial orientation was only observable for the systems from Blantyre. The larger scatter of mean vectors at Mulanje might be partially explained A signiﬁcant deviation from random axial orientation was only observable for the systems from Blantyre. The larger scatter of mean vectors at Mulanje might be partially explained Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 8/15 by the lower sample size. Secondly, at Mpalaganga and Mulanje, several systems were excavated in cultivated areas, where higher density and uniformity of food resources might have had inﬂuenced foraging strategies and thus also the burrow architecture. Another factor, which seems to affect the burrow system’s orientation of silvery mole-rats is the system’s length—while shorter systems (<100 m) were oriented signiﬁcantly in North–South direction (every other direction would have been equally plausible), longer systems (>100 m) were not. This is, however, not surprising, considering that the larger a system becomes, the stronger an original alignment might be overridden or masked. Trying to explain these ﬁndings, we have to consider that a system’s size presumably grows with its age. Consequently, a system’s axial orientation may rather be observable when it is shorter (in statu nascendi) compared to when it is longer and older. Trying to explain these ﬁndings, we have to consider that a system’s size presumably grows with its age. Consequently, a system’s axial orientation may rather be observable when it is shorter (in statu nascendi) compared to when it is longer and older. Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 Competing Interests The authors declare that they have no competing interests. CONCLUSION AND OUTLOOK To conclude, the Fukomys mole-rats, the silvery mole-rat, as well as the Talas tuco-tuco were shown to dig their burrow systems with respect to a certain axis, and are assumed to use the Earth’s magnetic ﬁeld as a possible heading indicator. In future studies, animals might be released at a new site so that the genesis of the new burrow system might be monitored in situ as, for example, done by Voigt (2014). Furthermore, digging experiments should be performed under controlled laboratory conditions to unequivocally experimentally demonstrate that subterranean rodents use magnetic cues to keep a heading direction during digging. DISCUSSION Although season is expected to affect the digging activity (Sichilima et al., 2008) and general architecture of the burrow system (Šumbera et al., 2003), it is highly unlikely that its main orientation, if present, changes seasonally, wherefore we do not assume signiﬁcant seasonal inﬂuences. Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 9/15 ACKNOWLEDGEMENTS We thank R. Šumbera and M. Lövy for providing most of the data and for their helpful comments on the manuscript. Further thanks are due to J. Šklíba for offering digital maps of tunnel systems of the Ansell’s mole-rats, W. N. Chitaukali, V. Dvoráková, M. Elichová, E. Hrouzková, H. Konvičková, O. Kott, J. Kubová, V. Mazoch, J. Ritter as well as all local helpers who assisted in the ﬁeld, and N. Oberste for assistance with the analyses. Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: PhD fellowship of the German National Academic Foundation (Studienstiftung des deutschen Volkes). The following grant information was disclosed by the authors: PhD fellowship of the German National Academic Foundation (Studienstiftung des deutschen Volkes). PhD fellowship of the German National Academic Foundation (Studienstiftung des deutschen Volkes). PhD fellowship of the German National Academic Foundation (Studienstiftung des deutschen Volkes). “EVA4.0”: No. CZ.02.1.01/0.0/0.0/16_019/0000803. “EVA4.0”: No. CZ.02.1.01/0.0/0.0/16_019/0000803. Grant Agency of the Czech Republic (project. nr. 15-21840S) Grant Agency of the Czech Republic (project. nr. 15-21840S). Funding S. Malewski was funded by a PhD fellowship of the German National Academic Foundation (Studienstiftung des deutschen Volkes). H. Burda and S. Begall received support funded by grant “EVA4.0,” No. CZ.02.1.01/0.0/0.0/16_019/0000803 ﬁnanced by OP RDE and H. Burda the Grant Agency of the Czech Republic (project. nr. 15-21840S). We acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 Author Contributions Sandra Malewski conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, authored or reviewed drafts of the paper, approved the ﬁnal draft. Sandra Malewski conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, authored or reviewed drafts of the paper, approved the ﬁnal draft. Malewski et al. (2018), PeerJ, DOI 10.7717/peerj.5819 Sabine Begall conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the ﬁnal draft. Cristian E. Schleich performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, approved the ﬁnal draft. Carlos D. Antenucci performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, approved the ﬁnal draft. Hynek Burda conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the ﬁnal draft. Data Availability The following information was supplied regarding data availability: The raw data are provided at https://www.uni-due.de/imperia/md/images/fb10_bio/ allgemeine_zoologie/Malewski_et_al._raw_data.xlsx. The raw data are provided at https://www.uni-due allgemeine_zoologie/Malewski_et_al._raw_data.xlsx. Supplemental Information Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.5819#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.5819#supplemental-information. Malewski et al. 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https://openalex.org/W4213243932	https://www.mdpi.com/2306-5338/9/2/37/pdf?version=1645101886	English	null	Integrated Hydrological Modeling to Analyze the Effects of Precipitation on Surface Water and Groundwater Hydrologic Processes in a Small Watershed	Hydrology	2,022	cc-by	11,201	Citation: Paudel, S.; Benjankar, R. Integrated Hydrological Modeling to Analyze the Effects of Precipitation on Surface Water and Groundwater Hydrologic Processes in a Small Watershed. Hydrology 2022, 9, 37. https://doi.org/10.3390/ hydrology9020037 Academic Editor: Alain Dezetter Received: 8 January 2022 Accepted: 14 February 2022 Published: 17 February 2022 Citation: Paudel, S.; Benjankar, R. Integrated Hydrological Modeling to Analyze the Effects of Precipitation on Surface Water and Groundwater Hydrologic Processes in a Small Watershed. Hydrology 2022, 9, 37. https://doi.org/10.3390/ hydrology9020037 Keywords: integrated hydrological modeling; groundwater; surface water; MIKE SHE; precipitation sources; watershed Article Integrated Hydrological Modeling to Analyze the Effects of Precipitation on Surface Water and Groundwater Hydrologic Processes in a Small Watershed Sabin Paudel 1,2 and Rohan Benjankar 1,* Sabin Paudel 1,2 and Rohan Benjankar 1,* 1 Department of Civil Engineering, Southern Illinois University Edwardsville, Edwardsville, IL 62026, USA; spaudel@illinois.edu 1 Department of Civil Engineering, Southern Illinois University Edwardsville, Edwardsville, IL 62026, USA; spaudel@illinois.edu 2 Prairie Research Institute, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA * Correspondence: rbenjan@siue.edu; Tel.: +1-618-650-2814 2 Prairie Research Institute, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA * Correspondence: rbenjan@siue.edu; Tel.: +1-618-650-2814 2 Prairie Research Institute, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA * Correspondence: rbenjan@siue.edu; Tel.: +1-618-650-2814 Abstract: The main objective of this study is to evaluate the performance of the integrated hydrologi- cal model, MIKE SHE in a small watershed to analyze the effect of two different precipitation sources on model outputs (groundwater elevation and surface water ﬂows). The model was calibrated and validated with observed groundwater elevations and surface water ﬂows measured at the United States Geological Survey (USGS) gage stations in the basin. The model calibration performance for surface water ﬂows (R = 0.80, MAE= 0.20 m3/s, BIAS = −0.14 m3/s, NSE = 0.59) and groundwater elevations (R = 0.74, MAE = 0.45 m, BIAS = 0.08 m, NSE = 0.35) showed that the model was able to predict hydrological processes based on forcing variables in a small watershed. The analysis did not show the model with precipitation at the nearer (NOAA-Edwardsville) gauge station has better performance than the farther gauge station (NOAA-St. Louis). The quantitative analyses for the most sensitive model output variable suggested that precipitation uncertainties had noticeable impacts on surface water ﬂows (0.81% to 11.19%), than groundwater elevations (0.06% to 0.07%), with an average of 6.71% and 0.66%, respectively. Our results showed noticeable differences in simulated surface water ﬂows in spring (12.9%) and winter (36%) seasons compared to summer (11.4%) and fall (4.6%) as a result of difference (6% to 18%) in precipitation, which indicated that uncertainties in precipitation impact simulated surface water ﬂows in a small watershed vary with different seasons. Our analyses have shown that precipitation affects the simulated hydrological processes and care should be taken while selecting input datasets (i.e., precipitation) for better hydrological model performance, speciﬁcally for surface water ﬂows. hydrology hydrology 1 Department of Civil Engineering, Southern Illinois University Edwardsville, Edwardsville, IL 62026, USA; spaudel@illinois.edu 2 Prairie Research Institute, University of Illinois at Urbana-Champaign, Champaign, IL 61820, USA * Correspondence: rbenjan@siue.edu; Tel.: +1-618-650-2814 1. Introduction Rainfall data measured at gauge stations may not be able to capture accurate spatial variability [21,22]. To lower the effects of such variation on runoff volume and timing, spatially distributed rainfall data sources can be used [23]. Meanwhile, continuous monitoring of precipitation and other meteorological parameters such as wind speed, temperature, solar radiation, and stream runoff is also crucial to analyze the watershed water balance [24]. Therefore, hydrological monitoring is important to understand the real effect of precipitation on hydrologic responses, including groundwater [24–26]. g Past studies used different precipitation sources (point and spatially distributed), e.g., gauges, radar, satellite, etc. [27–31]. A gauge station is a major source of precipitation data for hydrological analysis [32] and yields better results using multiple rain gauge stations within the watershed [33]. A study showed that poorly distributed rain gauge sta- tions impact model results [34], forcing to recalibrate the model with different precipitation sources [28,35]. National Oceanic and Atmospheric Administration (NOAA) provide the best rainfall data measurement; however, they are not error-free [36]. Site-speciﬁc rainfall data distribution can cause an error in the hydrological model results [37]. Radar-based and Climate Forecast System Reanalysis (CFSR) rainfall data have received increasing attention for hydrologic analyses because of the large area coverage [31,35,38,39]. Over the last couple of decades, various studies have been designed to determine the effect of precipitation on hydrologic responses. The impacts vary signiﬁcantly de- pending on the type of precipitation, hydrologic models, and watershed characteris- tics [15,16,21,28,31,33,35,40–45]. For example, the sensitivity of spatial precipitation distri- bution to the surface-runoff response depends on the model scale [42,46]. Lopes [47] showed that spatial distribution of precipitation had signiﬁcant effects on the runoff mechanism, irrespective of scales in the Walnut Gulch watershed, Arizona. Moreover, Guo et al. [48] found better calibration results with a ﬁne spatial resolution of precipitation data. Previous studies showed that the hydrological model performance was better with CFSR data, compared to traditionally observed weather data [43]. Another study showed that PRISM-based (Parameter-elevation Relationships on Independent Slopes Model) pre- cipitation provides a better streamﬂow prediction than CFSR and gauge data within a watershed [35]. Furthermore, they found better results with NCDC (National Climatic Data Center) gauge data than for the CFSR, due to the close proximity of NCDC stations to the watershed boundary. Uncertainties in rainfall result in parametric uncertainty in a distributed hydrological model and simulated ﬂows [39]. 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Water-resources management has been a challenging task for water managers, hydrol- ogists, and ecologists to fulﬁll various demands such as energy, agricultural, industrial, municipal, ﬂood control, and ecological processes, etc. [1]. For example, water-resource management has a direct negative impact on the riverine ecosystem, and therefore, ecosys- tem restoration has focused on restoring ecological ﬂows [2–4]. g g Hydrological models (e.g., SWAT, DRAINMOD, HEC-HMS, etc.) have been used to simulate hydrological responses. However, some of these models do not consider direct interaction with groundwater [5,6]. Stand-alone groundwater models (e.g., MODFLOW) have been used in past studies to simulate groundwater-ﬂow processes [7]. The integrated hydrological (surface and groundwater) model MIKE SHE has been successfully tested in watersheds with different characteristics in the USA [8] and around the world [5,9]. One of the major advantages of MIKE SHE is that it considers a dynamic interaction between groundwater and surface water ﬂows in streams and rivers. The advantages of Copyright: © 2022 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/). https://www.mdpi.com/journal/hydrology Hydrology 2022, 9, 37. https://doi.org/10.3390/hydrology9020037 Hydrology 2022, 9, 37 2 of 18 integrated models are highlighted by various recent studies [9–13]. Since MIKE SHE is a spatially distributed model (grid-based), it can incorporate spatial variability of physical and meteorological parameters [8] compared to lumped models such as, for example, SWAT [14] and HEC-HMS [6]. integrated models are highlighted by various recent studies [9–13]. Since MIKE SHE is a spatially distributed model (grid-based), it can incorporate spatial variability of physical and meteorological parameters [8] compared to lumped models such as, for example, SWAT [14] and HEC-HMS [6]. Hydrological modeling requires meteorological data such as precipitation, wind speed, atmospheric temperature, and solar radiation to simulate surface water ﬂow, sediment transport, and water quality [15]. An accurate spatial and temporal representation of pre- cipitation is crucial to predict hydrological responses and water balance within a watershed since it is the most signiﬁcant model input parameter [16–18]. Therefore, both the spatial and temporal variation of precipitation is very important for better hydrological model performance and watershed management [19,20]. 2.1. Study Area A small watershed (298 km2) (hereafter Canteen-Cahokia Watershed) within the Canteen-Cahokia Watershed (HUC-10) is considered for this study, which is located about 16 km northeast of downtown city of St. Louis, Missouri, and drains into the Mississippi River (Figure 1). The watershed comprises 30% agricultural ﬁeld, mostly plain land with mild slopes. 1. Introduction A study showed that the er- rors due to model parameters are similar or even higher than the errors due to rainfall uncertainties [49]. The simulated surface water ﬂow is not only affected by the change in precipitation, but also by evapotranspiration and groundwater contribution [50–53]. These studies indicated that uncertainties in the hydrological model simulated ﬂows due to change in precipitation spatially and temporally (i.e., months and seasons). Besides surface water ﬂows, groundwater systems are inﬂuenced by change in precip- itation because they are typically recharged from the ground surface. For example, autumn and winter precipitation has a noticeable impact on groundwater level than in spring and summer seasons, where air temperature is driving groundwater ﬂuctuations [50]. Increased precipitation results in elevated groundwater elevations in some areas, whereas lowered in Hydrology 2022, 9, 37 3 of 18 3 of 18 other areas [52]. Another study concluded that groundwater storage changes do not reﬂect the long-term trend in precipitation, but the change is due to alteration of evapotranspira- tion, and reduction in snowmelt [51]. Studies have shown mixed results for correlations between precipitation and groundwater elevations [50–52]. It is apparent that researchers found the mixed performance of the hydrological model speciﬁcally for surface water, based on different precipitation sources (e.g., gauge stations, CFSR, NEXRAD, etc.) and their proximity to the watershed. However, the performance of an integrated hydrological model based on the precipitation measured at different gauge stations (e.g., NOAA), for predicting groundwater elevations and surface water ﬂows in a small watershed is lacking. It is unclear if groundwater elevations or surface water ﬂows are the most sensitive to precipitation. Furthermore, the knowledge about how uncertainties of precipitation input in the hydrological model impact simulated surface water ﬂows in different seasons lacking. This study is focused on changes in groundwater elevations and surface water flows due to differences in measured precipitation at NOAA-St. Louis and NOAA-Edwardsville gauges in a small watershed using integrated hydrological model MIKE SHE. Specifically, we used calibrated model with CFSR based meteorological and precipitation data to quantify, i. Most sensitive variable (i.e., groundwater elevations or surface water flows). ii. Seasonal variations of difference in surface water flows with respect to change in precipitation. 2.2. Integrated Hydrological Modeling—MIKE SHE Integrated hydrological models dynamically couple surface water and groundwater ﬂow processes at a wide range of spatial scales [28,54]. This study used an integrated hydrologic modeling system (200 m × 200 m grid size), MIKE SHE to simulate ground- water and surface water ﬂows (DHI) [55]. The diffusive wave approximation method was used to simulate the overland ﬂow in the horizontal direction, whereas Richard’s equation simulates the unsaturated ﬂow along the vertical direction. Water movement in SZ (saturated zone) was simulated using the Boussinesq equation [56]. Kristensen and Jensen’s method was applied to describe the evapotranspiration, considering interception from the canopy, evaporation from the soil, and plants transpiration [57]. MIKE Hydro River is a one-dimensional (1D) river modeling software (Version 2019) based on the dynamic wave approximation of the Saint Venant equation [58]. The cou- pling of MIKE SHE and MIKE Hydro River allows an interaction between surface water hydrodynamics and groundwater ﬂow regimes [59,60]. The Saint Venant diffusive wave approximation method was used to predict the overland ﬂow. Manning’s number (inverse of manning’s roughness), initial water depth, and detention storage parameters were con- sidered for the simulation of overland ﬂows. The channel network alignment for the model was digitized along the perineal streams in the watershed (Figure 1). The cross-section geometry along the channels was extracted from a one-meter resolution LiDAR DEM. Hydrology 2022, 9, 37 4 of 18 Figure 1. Location of Canteen-Cahokia watershed showing National Oceanic and Atmospheric Administration (NOAA) precipitation gauges, Climate Forecast System Reanalysis (CFSR) stations, and USGS groundwater (GW-GC: USGS Groundwater Station# 384656089582001 at Glen Carbon, Illinois) and surface water gauge stations (SW-GC: USGS surface water ﬂow station# 05588720 at Glen Carbon, Illinois). SW = surface water, GW = groundwater, GC = Glen Carbon, PB = Pontoon Beach. 2.3. Input Data Figure 1. Location of Canteen-Cahokia watershed showing National Oceanic and Atmospheric Administration (NOAA) precipitation gauges, Climate Forecast System Reanalysis (CFSR) stations, and USGS groundwater (GW-GC: USGS Groundwater Station# 384656089582001 at Glen Carbon, Illinois) and surface water gauge stations (SW-GC: USGS surface water ﬂow station# 05588720 at Glen Carbon, Illinois). SW = surface water, GW = groundwater, GC = Glen Carbon, PB = Pontoon Beach. 2.5. Model Calibration Parameters Optimization The calibration of MIKE SHE and MIKE Hydro River models are performed simulta- neously as the change of a parameter in one model can affect another model’s results [58]. Groundwater elevation and surface water ﬂow from April 2005 to March 2009 were used for the calibration process, whereas from April 2009 to September 2011 for the validation. However, additional 2 years were added as a model warm-up period before the calibration and validation periods to avoid the errors because of model instabilities. Final optimized model parameter values are those which yield the best error statistics. Autocalibration was performed against daily groundwater elevations at GW-GC and surface water ﬂows at SW-GC stations. During the calibration processes, the program changes different groundwater (e.g., hydraulic conductivity, speciﬁc yield, initial potential, soil bypass coefﬁcient) and surface water (e.g., manning’s number M (i.e., the inverse of roughness coefﬁcient) for both overland and river channel, and detention storage) parameters and calculate error statistics. The ﬁnal optimized parameters were within the ranges reported in other studies using MIKE SHE (Table 1) [5,14,33,58,69–71]. 2.4. Sensitivity Analysis The sensitivity analyses of model parameters were performed using the Autocalibra- tion Function of MIKE SHE [DHI, 55]. The most sensitive parameters for groundwater and surface water hydrology reported by various previous studies are hydraulic conductivity, speciﬁc yield, initial potential head of aquifer, soil bypass coefﬁcient, manning’s roughness, evapotranspiration parameters, leakage coefﬁcient, and detention storage [9,11,13,66]. The hydraulic conductivity and bypass constant (which refers to the fraction of rainfall inﬁltrates into the soil before water starts to appear as overland/channel ﬂow) were found sensitive for both groundwater and surface water similar in another study [8]. Other surface water sensitive parameters were overland and channel manning’s number M (i.e., the inverse of roughness coefﬁcient), detention storage, ET coefﬁcients (C2 and C3), groundwater initial potential head, and groundwater leakage coefﬁcients. The groundwater and surface water sensitive parameters were similar to other studies for lowland watersheds using the MIKE SHE model [5,8,13,67,68]. 2.3. Input Data The topography of the watershed was represented by a high-resolution (10 m × 10 m) Digital Elevation Model (DEM). Meteorological data, including on precipitation (discrete), were based on global weather data CFSR for SWAT (https://globalweather.tamu.edu/) and NOAA (https://www.ncdc.noaa.gov/cdo-web/). CFSR data are prepared from data- assimilation techniques utilizing conventional meteorological gauge observations, satellite irradiances, and advanced atmospheric, oceanic, and surface-modeling components at ~38 km resolution [61]. The meteorological data used for model calibration and validation were based on CFSR and includes precipitation (mm/day), maximum and minimum temperature (ºC), solar radiation (MJ/m2/d), wind speed (m/s), and average relative humidity (%). The reference evapotranspiration (ET0) was calculated based on the Penman-Monteith method (Zotarelli et al. 2010) using an Excel program [62]. Estimated ET0 (mm/day) was based on the assumption of evapotranspiration from reference vegetation considering canopy resistance, aerodynamic resistance, and soil heat ﬂux intensity. y y Nine major land use types were considered in our study, which collectively comprised more than 90% of the total area (i.e., land, bare soil, deciduous forest, evergreen forest, cultivated crops, hay/pasture, woody wetlands, open water, and herbaceous wetlands). Although there were 128 STATSGO soil types present in the watershed, only ten major soil types were considered by merging similar soil types (i.e., silty clay loam, silt loam, silty clay, sandy loam, urban land, water, clay loam, silt, loam, and sand) [63]. To estimate groundwa- ter ﬂow through the unsaturated soil zone, water retention and hydraulic conductivity of soil were considered, which is based on the Van Genuchten method [13,64,65]. Hydrology 2022, 9, 37 5 of 18 5 of 18 The study area has limited measured surface water ﬂows and groundwater availability for model calibration and validation. Daily averaged surface water data are available from May 2000 to October 2011 for SW-GC (USGS surface water gage station# 05588720) located at the Judy Branch Creek in the city of Glen Carbon, IL (watershed area 21.3 km2). Two USGS gage stations, GW-GC (USGS Groundwater Station# 384656089582001) and GW-PB (USGS Groundwater Station# 384352090054102) located at the city of Glen Carbon and Pontoon Beach, IL, respectively were used to calibrate and validate the groundwater model. 2.6. Model Performance Analysis To test the best ﬁt of a model, the most commonly used statistical performance in- dicators, Coefﬁcient of Correlation (R), Nash-Sutcliffe Efﬁciency (NSE), Mean Absolute Error (MAE), and Bias (BIAS) were estimated in this study [72]. Simulated and measured daily groundwater elevations and surface water ﬂows were compared during the calibra- tion and validation processes to analyze model performance. The R shows the fraction of deviation between modeled and observed data [73], and ranges from 0 to 1, with 0 being the least favored and 1 being the most favored. The NSE describes the predictive power of a hydrological model. The higher the value is, the better representation of model parameters. NSE values less than 0 occur when the observed mean is a better predictor than the model [73]. BIAS measures the average tendency (of the simulated values larger or smaller than their observed ones [71]. The MAE measures an average mean discrepancy between two datasets and is a more natural way of error measurement [74]. Hydrology 2022, 9, 37 6 of 18 Table 1. Final Optimized Calibrated Parameters for Surface water and Groundwater. Model Parameters Initial Range Calibrated MIKE SHE Overland and Unsaturated Zone Manning’s M 17 10–40 26.80 Detention Storage (mm) 2 0–10 5.08 Bypass Constant 0.26 0.15–0.9 0.27 Saturated Zone Horizontal Hydraulic Conductivity (10−6 m/s) 5.6 0.0056–566 96.80 Vertical Hydraulic Conductivity (10−6 m/s) 0.56 56.6 9.68 Speciﬁc Yield 0.2 0.2–0.4 0.20 Initial Potential (−m) 5 1–10 6.32 ET Parameters (Kristensen And Jensen) Canopy Interception (mm) 0.05 0.05–0.4 0.07 C1 0.2 0.05–0.4 0.34 C2 0.2 0.05–0.4 0.06 C3 (mm/day) 20 5–40 6.95 Aroot (/m) 0.3 0.05–0.4 0.31 MIKE Hydro River and Lakes Manning’s M 36 10–40 18.45 Leakage Coefﬁcient (10−6) 5.6 0.0056–566 2.30 Table 1. Final Optimized Calibrated Parameters for Surface water and Groundwater. We further analyzed the model performance by comparing simulated groundwater elevations and surface water ﬂows based on precipitation measured at two NOAA gauge stations and CFSR. All three models have the same calibrated parameters and input datasets except precipitation. We simulated groundwater elevations and surface water ﬂows using models with three different precipitation sources, a. NOAA-Edwardsville, b. NOAA- St. Louis, and c. CFSR. We analyzed differences in model outputs due to precipitation measured at each NOAA gauge station and CFSR. The analysis informs whether NOAA- Edwardsville or NOAA-St. Louis model performs better (groundwater elevations and surface water ﬂows) with reference to the CFSR model. 2.7. Effect of Precipitation Sources on Groundwater and Surface Water Flows 2.7.1. Sensitive Variable (i.e., Groundwater Elevations or Surface Water Flows) 2.7. Effect of Precipitation Sources on Groundwater and Surface Water Flows 2.7.1. Sensitive Variable (i.e., Groundwater Elevations or Surface Water Flows) We quantiﬁed the most sensitive variable (i.e., groundwater elevations or surface water ﬂows) due to the effects of two precipitation sources (i.e., NOAA-St. Louis Vs NOAA-Edwardsville) by estimating R and MAE. The MAEs were normalized with respect to the NOAA-Edwardsville simulated average groundwater elevations or surface water ﬂows at four random stations (Figure 1) and presented in percentages (%). Furthermore, weighted (by surface water ﬂows or groundwater elevations) averages of MAE and R were calculated for comparison. The variable with higher MAE (%) and lower R are deﬁned as the most sensitive. 2.6. Model Performance Analysis The NOAA gauge stations are located at the city of Edwardsville, IL (hereafter NOAA- Edwardsville) and St. Louis, MO (hereafter NOAA-St. Louis). The NOAA-Edwardsville gauge station is located close (less than 1 km) to the watershed boundary, whereas the NOAA-St. Louis is about 6.5 km far away. R and MAE were estimated using simulated groundwater elevations and surface water ﬂows (April 2005–January 2007) at four different random stations (locations) (Figure 1). 2.7.2. Seasonal Variations of Difference in Surface Water Flow with Respect to Change in Precipitation We analyzed the relationships between the difference in simulated surface water ﬂows (%) with respect to difference in precipitation measured at NOAA-Edwardsville and NOAA-St. Louis gauges considering overall and seasonal datasets. Differences (%) in Hydrology 2022, 9, 37 7 of 18 7 of 18 precipitation and surface water ﬂows were calculated relative to the NOAA-Edwardsville gauge precipitation. For the category “All”, we considered entire datasets from April 2005 to January 2007. For “Season”, datasets were classiﬁed into four seasons, Spring (March, April, and May), Summer (June, July, and August), Fall (September, October, and November), and Winter (December, January, and February). Later, we calculated weighted-average surface water ﬂows based on four different random stations for comparison (Figure 1). The analysis will yield if there is a correlation between differences in precipitation and simulated surface water ﬂows considering entire datasets and four seasons. 3.1.1. Model Calibration and Validation 3.1.1. Model Calibration and Validation R, MAE, BIAS, and NSE between simulated and measured groundwater elevations at the GW-GC gage station were 0.74, 0.45 m, 0.08 m, and 0.35, respectively (Figure 2 and Table 2). The average positive BIAS of 0.08 m showed that MIKE SHE underpredicted groundwater elevations during the calibration period (Table 2). The R, MAE, BIAS, and NSE were 0.74, 0.39 m, −0.24 m, and 0.14, respectively for groundwater elevations during the model validation period at the GW-GC gage station. The overall model performance trend during the validation period was similar to the calibration for groundwater elevations, except for BIAS and NSE (Table 2). The BIAS (−0.24 m) was negative for the validation period, which showed overprediction by model, opposite to the outcome from the model calibration (Figure 2b and Table 2). Figure 2. Measured and simulated daily groundwater elevations for (a) Calibration and (b) Validation periods at GW-GC gage station (USGS Groundwater Station# 384656089582001 at the city of Glen Carbon, IL). Figure 2. Measured and simulated daily groundwater elevations for (a) Calibration and (b) Validation periods at GW-GC gage station (USGS Groundwater Station# 384656089582001 at the city of Glen Carbon, IL). Table 2. Model performance statistics for the model calibration and validation. GW, SW, and GC stand for Groundwater, surface water, and Glen Carbon, respectively. Table 2. Model performance statistics for the model calibration and validation. GW, SW, and GC stand for Groundwater, surface water, and Glen Carbon, respectively. Parameters Calibration Validation GW-GC SW-GC GW-GC SW-GC R 0.74 0.80 0.74 0.65 MAE (m, m3/s) 0.45 0.20 0.39 0.22 SD * 0.65 0.44 0.53 0.57 BIAS (m, m3/s) 0.08 −0.14 −0.24 −0.02 NSE 0.35 0.59 0.14 0.42 * Standard deviation of measurement. The R, MAE, BIAS, and NSE for the observed and measured surface water at the SW-GC gage station were 0.80, 0.20 m3/s, −0.14 m3/s, and 0.51, respectively (Figure 3a and Table 2). Unlike for groundwater, negative BIAS (−0.14 m3/s) showed that MIKE SHE overpredicted surface water ﬂow during the calibration period. The surface water ﬂow Hydrology 2022, 9, 37 8 of 18 prediction during the validation period at the SW-GC gage station was R, 0.65, 0.22 m3/s, −0.02 m3/s, and 0.42 for MAE, BIAS, and NSE, respectively (Figure 3b and Table 2). Figure 3. 3.1.1. Model Calibration and Validation Measured and simulated daily surface ﬂow hydrograph at SW-GC gage (USGS surface water station# 05588720 at the city of Glen Carbon, IL) and CFSR precipitation depths for (a) Calibration and (b) Validation. prediction during the validation period at the SW-GC gage station was R, 0.65, 0.22 m3/s −0.02 m3/s, and 0.42 for MAE, BIAS, and NSE, respectively (Figure 3b and Table 2). prediction during the validation period at the SW-GC gage station was R, 0.65, 0.22 m3/s, −0.02 m3/s, and 0.42 for MAE, BIAS, and NSE, respectively (Figure 3b and Table 2). Figure 3. Measured and simulated daily surface ﬂow hydrograph at SW-GC gage (USGS surface water station# 05588720 at the city of Glen Carbon, IL) and CFSR precipitation depths for (a) Calibration and (b) Validation. 3.1.2. NOAA-Edwardsville Vs NOAA-St. Louis The weighted average MAEs for groundwater elevations were 0.09 m (0.07 m to 0.11 m) and 0.15 m (0.03 to 0.21 m) for NOAA-Edwardsville and NOAA-St. Louis models, respectively at all four GW stations. The values reported in parenthesis are a range. Similarly, weighted average R were 0.78 (0.73 to 0.82) and 0.65 (0.58 to 0.70) for NOAA- Edwardsville and NOAA-St. Louis precipitation models, respectively (Table 3). The results showed that the NOAA-Edwardsville model performed better (values close to the CFSR model) than for NOAA-St. Louis considering average R and MAE. The weighted average MAE for surface water ﬂows for the NOAA-Edwardsville and NOAA-St. Louis models were 0.78 m3/s (0.02 to 1.02 m3/s) and 0.68 m3/s (0.02 to 0.91 m3/s), respectively (Table 3). Similarly, weighted average R were 0.64 (0.62 to 0.66) and 0.41 (0.34 to 0.46) for the NOAA-Edwardsville and NOAA-St. Louis models, respectively. The results showed NOAA-St. Louis model performed better (close to the CFSR model) than for the NOAA-Edwardsville considering average MAE, whereas the trend was opposite for R. Hydrology 2022, 9, 37 9 of 18 Table 3. Difference in model performance statistics between NOAA-Edwardsville Vs CFSR and NOAA-St. Louis Vs CFSR precipitation models for groundwater elevations and surface water ﬂows at four random stations. Groundwater Elevation NOAA-Edwardsville Parameters GW1 GW2 GW3 GW4 Average R 0.80 0.73 0.78 0.82 0.78 MAE (m) 0.09 0.09 0.11 0.07 0.09 NOAA-St. Louis R 0.70 0.63 0.58 0.68 0.65 MAE (m) 0.03 0.10 0.23 0.21 0.15 Surface Water NOAA-Edwardsville Parameters SW1 SW2 SW3 SW4 Average R 0.66 0.62 0.63 0.64 0.64 MAE (m3/s) 1.02 0.38 0.02 0.03 0.78 NOAA-St. Louis R 0.46 0.38 0.34 0.45 0.41 MAE (m3/s) 0.91 0.31 0.02 0.02 0.68 3.2. Effect of Precipitation on Groundwater and Surface Water Flows 3.2.1. Sensitive Variable (i.e., Groundwater Elevations or Surface Water Flows) The weighted average MAE (considering all four stations) for simulated groundwater elevations and surface water ﬂows were 0.07% (0.06% to 0.08%) and 6.71% (0.81% to 11.19%) for NOAA-Edwardsville and NOAA-St. Louis models, respectively (Table 4). Similarly, the average R were 0.78 (0.73 to 0.82) and 0.66 (0.63 to 0.72) for simulated groundwater elevations and surface water ﬂows, respectively (Table 4). Therefore, differences were higher for surface water ﬂows considering both MAE and R. Nevertheless, differences in daily average groundwater and surface water ﬂow between NOAA-Edwardsville and NOAA-St. Louis models vary at each station (Figures 4 and 5). 3.1.2. NOAA-Edwardsville Vs NOAA-St. Louis For example, the difference in surface water ﬂows varied from −18% to 10% at station 1, whereas −4% to 1% at station 4 (Figure 5). Negative values indicated higher surface water ﬂows or groundwater elevations for the NOAA-Edwardsville model. Table 4. MAE (%) and R between NOAA-Edwardsville Vs NOAA-St. Louis models for groundwater elevations and surface water ﬂows at four random stations. The difference MAE (%) is calculated relative to the NOAA-Edwardsville model. Table 4. MAE (%) and R between NOAA-Edwardsville Vs NOAA-St. Louis models for groundwater elevations and surface water ﬂows at four random stations. The difference MAE (%) is calculated relative to the NOAA-Edwardsville model. Table 4. MAE (%) and R between NOAA-Edwardsville Vs NOAA-St. Louis models for groundwater elevations and surface water ﬂows at four random stations. The difference MAE (%) is calculated relative to the NOAA-Edwardsville model. Parameters GW/SW1 GW/SW2 GW/SW3 GW/SW4 Average Groundwater R 0.80 0.73 0.78 0.82 0.78 MAE (m) 0.07 0.06 0.08 0.06 0.07 Surface Water R 0.72 0.64 0.65 0.63 0.66 MAE (m3/s) 5.22 9.61 0.81 11.19 6.71 3.2.2. Seasonal Variations of Difference in Surface Water Flow with Respect to Change in Precipitation 3.2.2. Seasonal Variations of Difference in Surface Water Flow with Respect to Change in Precipitation Considering all (April 2005 to April 2007) datasets, the difference in precipitation between NOAA-Edwardsville and NOAA-St. Louis gauge was 12.6%, whereas the dif- ference in surface water ﬂows was 19% (Figure 6). Seasonal differences in precipitation varied between 6% (summer) to 18% (winter), which resulted in the difference in surface water ﬂow between 4.6% (fall) to 36% (winter) (Figure 6). The difference in surface water ﬂows was relatively lower in fall (4.6%) and summer (11.4%) compared to spring (12.9%) and winter (36%). The highest difference for surface water ﬂows was in the winter (36%) between NOAA-Edwardsville and NOAA-St. Louis models. 10 of 18 Hydrology 2022, 9, 37 Figure 4. Distribution of difference (%) in simulated daily groundwater elevations at four different stations for NOAA-Edwardsville and NOAA-St. Louis models. Figure 4. Distribution of difference (%) in simulated daily groundwater elevations at four different Figure 4. Distribution of difference (%) in simulated daily groundwater elevations at four different stations for NOAA-Edwardsville and NOAA-St. Louis models. Figure 4. Distribution of difference (%) in simulated daily groundwater elevations at four different stations for NOAA-Edwardsville and NOAA-St. Louis models. Figure 5. 3.1.2. NOAA-Edwardsville Vs NOAA-St. Louis Distribution of difference in simulated daily surface water ﬂows at four different stations from NOAA-Edwardsville and NOAA-St. Louis models. (a) Station 1; (b) Station 2; (c) Station 3; (d) Station 4. Figure 5. Distribution of difference in simulated daily surface water ﬂows at four different stations from NOAA-Edwardsville and NOAA-St. Louis models. (a) Station 1; (b) Station 2; (c) Station 3; (d) Station 4. Hydrology 2022, 9, 37 11 of 18 Figure 6. Difference in precipitation (Precip.) and surface water flows (SWF) considering all (April 2005 to January 2007) and seasonal datasets based on NOAA-Edwardsville and NOAA-St. Louis models. 4 Discussion Figure 6. Difference in precipitation (Precip.) and surface water flows (SWF) considering all (April 2005 to January 2007) and seasonal datasets based on NOAA-Edwardsville and NOAA-St. Louis models. 4.1. Model Performance We estimated the model performance statistical indicators R, MAE, BIAS, and NSE to analyze model performance against measured groundwater elevations and surface water ﬂows at USGS gauge stations (Figure 1) [75,76]. We observed mixed model performance based on different statistical model performance indicators. The model performed satisfac- torily considering MAE, where the values were generally less than half of SD (standard deviation) of observed ﬂows [76] (Table 2). The model performance was comparable to past studies considering statistical model performance indicators [77,78]. The model per- formance was similar during calibration and validation periods (Table 2). In general, the model overpredicted baseﬂow during non-rainfall events for both calibration and valida- tion periods (Figure 3). Nonetheless, based on the criteria suggested by prior researchers for model performance interpretation, the model performance for a small watershed is deemed satisfactory [67,75,76]. The model performances were analyzed by removing the days when the measured precipitation at CFSR and NOAA-Edwardsville gauge stations differed considerably during calibration and validation periods. Based on our judgement, precipitation measurements had uncertainties (either in CFSR or NOAA-Edwardsville gauges) during those days. We removed 34 (2.3%) and 12 (1.3%) data points out of 1461 and 913, respectively during the calibration and validation processes. Therefore, care should be taken while interpreting the model performance. It is a common practice to remove poorly performing datasets during hydrological model results analyses, however, it may change statistics and data distribution (e.g., mean, SD and range) [79–82]. Furthermore, we calculated the correlation between the measured precipitation (CFSR) and surface water ﬂows at a USGS SW-GC gauge station (April 2005 to March 2012), which was relatively low (R = 0.53) (Figure 7a). The differences were evident during high precipitation and ﬂow events. The lack of good correlations between measured surface ﬂows and precipitations as well extreme events may cause poor model performance. This study used CFSR based precipitation for model calibration, which was interpo- lated on a 38 km grid [35]. Various studies have shown that spatially distributed rainfall at a ﬁne resolution is required for small scales analyses [40,83]. For example, 2 km grid rainfall was suggested by Bell and Moore [41] to model a small watershed (i.e., 132 km2 in size). 12 of 18 12 of 18 Hydrology 2022, 9, 37 Figure 7. 4.1. Model Performance Correlations between (a) CFSR based precipitation and surface water ﬂow (USGS gauge station at Glen Carbon), (b) CFSR and NOAA-Edwardsville precipitation, and (c) CFSR and NOAA-St. Louis precipitation. Figure 7. Correlations between (a) CFSR based precipitation and surface water ﬂow (USGS gauge station at Glen Carbon), (b) CFSR and NOAA-Edwardsville precipitation, and (c) CFSR and NOAA-St. Louis precipitation. A small watershed may have a steep ﬂow slope (ﬂow change faster), high ﬂow variations, and ﬂashy hydrographs compared to a moderate and large watershed that may cause unsatisfactory model performance [46]. Runoff estimation improves as the watershed size increases, despite low rainfall resolution data [40,84]. Cunha, Mandapaka, Krajewski, Mantilla and Bradley [31] investigated the impact of the radar-rainfall error on hydrological model simulated ﬂood magnitude and concluded that uncertainties in simulated peak ﬂow decrease considerably with larger watersheds. Furthermore, as the watershed area increases, the peak ﬂow differences at outlets were practically negligible. The measured surface water ﬂows at the USGS gauge station were comparatively variable (ﬂashy) than measured precipitation in this study, which is a typical trend for a small watershed (Figures 3 and 7a). Furthermore, another study showed that the calibration performance of the model was distinct for two different precipitation datasets (gauge station and NEXRAD). The NEXRAD data performed better than the gauge station for a small watershed [28]. Therefore, we speculated that the model performance was affected by a small drainage area (21.3 km2 at the USGS gauge station) and limited observed data (single gauge station for groundwater and surface water measurements) availability in this study. The GW-GC station had 20 and 11 groundwater elevations measurements during the calibration and validation periods, respectively. Additionally, GW-PB (Figure 1) has only a total of seven data points, which were used to further verify the model performance for groundwater elevation predictions [85]. The time step and the grid size used in the model can affect the model perfor- mances [86–88]. A daily time step was used in this study, but Zhang, Wang, Sun, McNulty, Zhang, Li, Zhang, Klaghofer and Strauss [67] suggested that the daily (24 hours) time step may not be sufﬁcient to capture a quick response of precipitation on surface runoff in overland ﬂow dominant watersheds. The model was not able to predict highly variable surface water ﬂows at the USGS gauge station as a result of rainfall. 4.1. Model Performance Speciﬁcally, during high events, peak ﬂows occur within a few hours (less than 24 hours model time step) in the Canteen-Cahokia watershed, which comprises low stream lengths and channels [89]. Evapotranspiration (ET) had signiﬁcant effects on groundwater and surface water ﬂows [90]. A total of nine different land-use types (developed land, cultivated crops, bare soil, deciduous forest, evergreen forest, hay/pasture, woody wetlands, open water, and herbaceous wetlands) were considered in the analysis. An inaccurate representation of land-use impacts surface runoff, soil moisture, and groundwater recharge spatially and temporally in a distributed hydrological model [9,65,91,92]. Furthermore, because of lack of sufﬁcient vegetation parameters (i.e., LAI and RD), temporal vegetation distribution was assumed to be constant and such a generalization can impact the model performance [93]. Despite some uncertainties in the model, we deemed that the model performance is acceptable to analyze the effects of precipitation on groundwater and surface water ﬂows Hydrology 2022, 9, 37 13 of 18 13 of 18 in a small watershed. Although the calibrated model (with CFSR precipitation) had certain uncertainties in model parameters, it was consistent across all three models. Therefore, the model should not noticeably distort the outcome when comparing the effects of different types of precipitation on hydrological processes. Precipitation has the most critical inﬂuence on hydrological processes [28,39]; therefore, quality data is warranted for better model performance. The lack of ﬁner spatial resolution of precipitation, and a high level of sensitivity of precipitation on a smaller spatial scale watershed inﬂuences the performance of the hydrological model [28]. Despite NOAA gauge network and radar-based precipitation being available for larger areas and different temporal resolutions (e.g., daily), uncertainties remained, which effect simulated hydro- logical processes [31,39]. Therefore, our results showed the importance of spatially and temporally varied high-resolution precipitation and surface water ﬂow measurements for better model performance in small watersheds speciﬁcally to simulate surface water ﬂows. The model performance is also affected by uncertainties in other datasets such as meteoro- logical, land-use, soil type, topography, observed surface water ﬂows and groundwater elevations, and scale (size) of the watershed [16,30,49]. Nonetheless, each of the models can be calibrated using NOAA meteorological and precipitation data. In that case, the effects of precipitation on groundwater elevations and surface water ﬂows may have been different [35]. However, there is a fundamental issue with calibrating the model with different datasets and comparing the results. 4.1. Model Performance For example, calibrations using the two data sources may result in different model parameterizations, although having the same watershed characteristics. This may cause parameters overly inﬂuential in model results, which is also known as an equiﬁnality [15,39]. We did not ﬁnd a consistent pattern that the nearer gauge (i.e., NOAA-Edwardsville) station precipitation model performance is better (close to the CFSR model) than the farther gauge (i. e., NOAA-St. Louis) considering different statistical model performance indicators (MAE and R) for groundwater elevations and surface water ﬂows at individual random stations (Table 3). However, Radcliffe and Mukundan [35], found better results from the model that has precipitation measured at a nearer gauge station than for the farther one. CFSR Vs NOAA-Edwardsville (R = 0.31) measured precipitation had a higher correla- tion than for CFSR Vs NOAA-St. Louis (0.005) (Figure 7b,c). Our results showed a similar pattern (NOAA-Edwardsville performing better) for simulated groundwater elevations and surface water considering R (Table 3). However, NOAA-St. Louis model had a lower average MAE than for the NOAA-Edwardsville, despite NOAA-Edwardsville average precipitation being closer to the CFSR. The average ± SD (standard deviation) precipita- tion (April 2005 to January 2007) at CFSR, NOAA-Edwardsville, and NOAA-St. Louis gauge stations were 2.43 ± 6.34 mm, 2.46 ± 7.33 mm, and 2.17 ± 6.72 mm, respectively. Nevertheless, we just considered two gauge stations located around the watershed, but need to analyze results based on multiple gauges farther apart spatially before drawing any concrete conclusion regarding the model performance based on spatial distances of gauge stations. 4.2. Effects of Precipitation on Groundwater and Surface Water An analysis of the most sensitive variable (groundwater elevation or surface water ﬂow) showed that the effect of precipitation is more considerable in surface water ﬂows (0.81% to 11.19%), than groundwater elevations (0.06% to 0.08%) (Table 4). Effects vary noticeably considering individual stations and daily average ﬂows and groundwater elevations (Figures 4 and 5). Although past studies have shown contradicting results regarding the relationship between precipitation and groundwater elevations [50–52], our results showed minimal (0.06% to 0.08%) effects of precipitation on simulated groundwater elevations. Our results are consistent with Gardner and Heilweil [94], where they concluded groundwater elevations respond slowly to precipitation. Our study did not show a systematic change in surface water ﬂows as a result of the difference in precipitation (Figure 6) but they were season-speciﬁc. We found the lowest Hydrology 2022, 9, 37 14 of 18 difference in surface water ﬂows in summer and fall seasons, which might be due to a higher evapotranspiration rate resulting in lower ﬂows in rivers [95]. Evapotranspiration increases with higher atmospheric temperature and impacts surface runoff balance in river systems. The difference in surface water ﬂow change depends on precipitation, atmospheric temperature, evapotranspiration as well as groundwater contributions [50–53], which support our results. Surface water ﬂows changes were relatively less in summer and fall seasons despite higher differences in precipitation (Figure 6) because of higher evapotranspiration from the system [95]. Decreasing trends in baseﬂow within the US Midwest are attributed to increasing temperatures and evapotranspiration during the summer months [53]. 4.3. Study Application Our study has shown that care should be taken while selecting input datasets (e.g., precipitation) for hydrological model development and forecasting hydrological pro- cesses [16,39]. Many important factors should be considered before selecting precipitation data sources for simulating groundwater elevations and surface water ﬂows, for exam- ple, the spatial and temporal scale of the model, rain gauge data availability, watershed area, input data quality, and model structure and spatial discretization [28]. Based on our results, the effects of precipitation uncertainties on simulated surface water ﬂows vary season by season because of changing atmospheric temperature and evapotranspiration processes [95]. Our results suggested that high temporal and spatial resolution input data and the data used for model calibration (groundwater elevation and surface water ﬂows) would improve hydrological model performance to simulate surface water ﬂows than for groundwater elevations. The results emphasized the importance of the accurate input datasets (e.g., precipitation) for reliable model predictions [39,48]. The model developed in this study can be used to simulate hydrological processes in the future for the Canteen– Cahokia watershed to analyze impacts on future climate change and land cover changes due to urbanization on the hydrological process (groundwater elevations and surface water ﬂows), and subsequent impacts on ecosystems (aquatic and riparian) and urban ﬂooding [96,97]. This study is focused on a small watershed; therefore, further studies are required to transfer the ﬁndings to other watersheds in different geographic regions and large watersheds. 5. Conclusions A spatially distributed MIKE SHE integrated hydrological model was calibrated and validated with daily averaged measured groundwater elevations and surface water ﬂows. The calibrated model was used to analyze the effects of different precipitation on simulated hydrologic processes (i.e., groundwater elevations and surface water ﬂows). The MIKE SHE model was able to predict surface water ﬂows and groundwater elevations with reasonable accuracy at daily time steps for a small watershed. The study did not conclusively show that the model based on precipitation measured at nearer gauge (NOAA-Edwardsville) performs better than the model based on farther gauge (NOAA-St. Louis) precipitation. The analyses for the most sensitive model output variable showed that differences in precipitation had noticeable (0.81% to 11.19%) effect on simulated surface water ﬂows, whereas it had minimal (0.06% to 0.08%) effects on groundwater elevations. We found greater differences in simulated surface water ﬂows in spring (12.9%) and winter (36%) compared to summer (11.4%) and fall (4.6%) due to difference in precipitations (6% to 18%) measured at NOAA-Edwardsville and NOAA-St. Louis gauges. The study showed that uncertainties in precipitation on simulated surface water ﬂows vary with the season in a small watershed. Hydrology 2022, 9, 37 15 of 18 15 of 18 Author Contributions: Conceptualization, R.B.; Data curation, S.P.; Formal analysis, S.P.; Funding acquisition, R.B.; Investigation, R.B.; Methodology, S.P. and R.B.; Software, S.P.; Supervision, R.B.; Validation, S.P.; Writing—original draft, S.P. and R.B.; Writing—review & editing, S.P. and R.B. 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. Data Availability Statement: Please contact corresponding author for data. Data Availability Statement: Please contact corresponding author for data. Acknowledgments: We acknowledge Danish Hydraulic Institute (DHI) for providing MIKE SHE and MIKE Hydro software for the study. We would like to thank SIUE Undergraduate Research and Creative Activities (URCA) student, Abin Paudel for helping to analyze NOAA precipitation data. The comments from the reviewers’ earlier versions helped us to improve the manuscript signiﬁcantly. Sabin Paudel is funded through SIUE Civil Engineering Department and Graduate School during his study period. We also acknowledge Ryan Fries and Yan Qi for constructive comments in an earlier version of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Gordon, L.J.; Finlayson, C.M.; Falkenmark, M. Managing water in agriculture for food production and other ecosystem services. Agric. Water Manag. 2010, 97, 512–519. [CrossRef] 1. 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https://openalex.org/W2058403516	https://bmcinfectdis.biomedcentral.com/counter/pdf/10.1186/1471-2334-13-43	English	null	Identification and characterisation of vaginal lactobacilli from South African women	BMC infectious diseases	2,013	cc-by	6,435	Abstract Background: Bacterial vaginosis (BV), which is highly prevalent in the African population, is one of the most common vaginal syndromes affecting women in their reproductive age placing them at increased risk for sexually transmitted diseases including infection by human immunodeficiency virus-1. The vaginal microbiota of a healthy woman is often dominated by the species belonging to the genus Lactobacillus namely L. crispatus, L. gasseri, L. jensenii and L. iners, which have been extensively studied in European populations, albeit less so in South African women. In this study, we have therefore identified the vaginal Lactobacillus species in a group of 40 African women from Soweto, a township on the outskirts of Johannesburg, South Africa. Methods: Identification was done by cultivating the lactobacilli on Rogosa agar, de Man-Rogosa-Sharpe (MRS) and Blood agar plates with 5% horse blood followed by sequencing of the 16S ribosomal DNA. BV was diagnosed on the basis of Nugent scores. Since some of the previous studies have shown that the lack of vaginal hydrogen peroxide (H2O2) producing lactobacilli is associated with bacterial vaginosis, the Lactobacillus isolates were also characterised for their production of H2O2. Results: Cultivable Lactobacillus species were identified in 19 out of 21 women without BV, in three out of five women with intermediate microbiota and in eight out of 14 women with BV. We observed that L. crispatus, L. iners, L. jensenii, Results: Cultivable Lactobacillus species were identified in 19 out of 21 women without BV, in three out of five women with intermediate microbiota and in eight out of 14 women with BV. We observed that L. crispatus, L. iners, L. jensenii, L. gasseri and L. vaginalis were the predominant species. The presence of L. crispatus was associated with normal vaginal microbiota (P = 0.024). High level of H2O2 producing lactobacilli were more often isolated from women with normal microbiota than from the women with BV, although not to a statistically significant degree (P = 0.064). L. gasseri and L. vaginalis were the predominant species. The presence of L. crispatus was associated with normal vaginal microbiota (P = 0.024). High level of H2O2 producing lactobacilli were more often isolated from women with normal microbiota than from the women with BV, although not to a statistically significant degree (P = 0.064). Conclusion: The vaginal Lactobacillus species isolated from the cohort of South African women are similar to those identified in European populations. * Correspondence: harold.marcotte@ki.se 1Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska University Hospital, Huddinge, Stockholm, Sweden 4Department of Laboratory Medicine, Division of Clinical Immunology, F79, Karolinska Institutet, Stockholm S-141 86, Sweden Full list of author information is available at the end of the article Abstract In accordance with the other published studies, L. crispatus is related to a normal vaginal microbiota. Hydrogen peroxide production was not significantly associated to the BV status which could be attributed to the limited number of samples or to other antimicrobial factors that might be involved. Keywords: Bacterial vaginosis, Lactobacillus, South Africa, Hydrogen peroxide Identification and characterisation of vaginal lactobacilli from South African women Sonal Pendharkar1, Tebogo Magopane2, Per-Göran Larsson3, Guy de Bruyn2, Glenda E Gray2, Lennart Hammarström1 and Harold Marcotte1,4* Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 Open Access © 2013 Pendharkar 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. Identification of lactobacilli Identification of lactobacilli Genomic DNA was extracted from lactobacilli using Qiagen’s DNAeasy Blood & Tissue extraction kit (Qiagen GmbH, Hilden, Germany). The isolates were identified to the species level by sequencing the 16S ribosomal RNA (rRNA) gene as described previously [21]. Briefly, the complete 16S rRNA gene (1.5 kb) was amplified by PCR using the primers P0 (50-GAGAGTTTGATCCTGGCT CAG-30) and P6 (50-CTACGGCTACCTTGTTACGA-30) and sequenced. The obtained sequences were then sub- jected to nucleotide-nucleotide BLAST using BLASTN (http://www.ncbi.nlm.nih.gov/) and subsequently compared to the 16SrRNA sequences of typed strains to validate the results. The identification of Lactobacillus species was con- sidered confirmed if the sequences showed 99-100% hom- ology with the typed strain. The following typed strains and 16S rRNA gene sequence (Genbank accession number) were used for the comparisons: L. mucosae strain CCUG 43179 (AF126738), L. paracasei strain ATCC 25302 (HQ423165), L. ruminis strain ATCC 27782 (CP003032) and NBRC 102161 (AB326354), L. crispatus strain ATCC Background production, by producing various bacteriostatic and bac- tericidal substances such as hydrogen peroxide (H2O2) and through competitive exclusion. Decrease in the number of lactobacilli and overgrowth of diverse anaerobes is asso- ciated with an increased incidence of bacterial vaginosis (BV) [4,5], which is one of the most common vaginal syn- dromes among women in their reproductive age [6-8]. BV is associated with an increased susceptibility to sexually transmitted diseases including infection by human im- munodeficiency virus-1 (HIV-1) [9-11] where the elevated vaginal pH associated with BV may allow the survival of The vaginal microbiota of a healthy woman is often domi- nated by lactobacilli. The most commonly identified Lacto- bacillus species include L. crispatus, L. gasseri, L. jensenii and L. iners [1-3]. Lactobacilli are thought to play an im- portant role in protecting the host from urogenital infec- tion by lowering the environmental pH through lactic acid Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 Page 2 of 7 Page 2 of 7 Human Research Ethics Committee (Medical), University of the Witwatersrand, Johannesburg (Ethical permit num- ber M011138). A regional ethical permit was also granted by Regionala Etikprövningdnämnden, Stockholm, for ana- lysing these samples (Diarienummer: 2012/1362-31/1). HIV. Moreover, BV associated microbiota has been shown to enhance HIV-1 transcription and replication [12]. It has previously been shown that exogenously applied lactobacilli can persist on the mucosal surface of the va- gina, displace BV and restore a normal vaginal microbiota [13,14]. Given the high prevalence of BV in women in many sub-Saharan African countries with HIV epidemics (20%-60%), colonisation by exogenous lactobacilli may help to reduce the risk of HIV-1 transmission [9]. Further- more, delivery of a microbicide by lactobacilli colonising the vagina has previously been suggested as a strategy for prevention of HIV transmission, particularly in developing countries [15]. Cultivation of lactobacilli The swabs were transported at room temperature to the laboratory in Sweden within a maximum of two days. The swabs were directly streaked onto Rogosa agar (BD Difco™Rogosa SL agar, Becton, Dickinson and Company, Spark, MD), Columbia agar (BD Difco™Columbia Blood Agar Base) with 5% horse blood and de Man-Rogosa- Sharpe (MRS) agar (BD Difco™Lactobacilli MRS broth) within 24 hours of arrival. The swabs were then vortexed in 1ml sterile PBS (pH 7.4) to prepare bacterial suspen- sions. The bacterial suspensions were diluted tenfold se- rially (three dilutions per suspension) and each dilution (100 μl) was plated onto the three different agar plates as described above [20]. Plates were incubated for 48 hours at 37°C in an incubator in anaerobic condition using BD GasPack™EZ gaz generating systems (Becton, Dickinson and Company, Sparks, MD). Colonies with different morphology yielding variable rods were re-streaked. Re- spective colonies from re-streaked plates were Gram stained and the colonies with Gram positive rods were used to inoculate the MRS broth medium. Small transpa- rent colonies from blood agar plates yielding Gram positive bacilli not growing in MRS broth were collected directly from the plates and used for genomic DNA isolation. Gly- cerol stocks (15%) were prepared and stored at −80°C. Eight to 12 different colonies were picked per sample. Hence, a better understanding of the species compo- sition and ecology of bacterial ecosystems may help to develop better prophylaxis against BV and HIV. If the Lactobacillus species isolated from African women are similar to those in America and Europe, where probiotic studies have been performed, similar strategies utilising probiotic and engineered lactobacilli could be applied in Africa. Some studies reported differences in the com- position of vaginal microbiota among different ethnic groups in North America, where a higher incidence of vaginal communities not dominated by lactobacilli was observed in black women [16,17]. However, the few studies performed in Africa to date suggest that the va- ginal Lactobacillus species predominant among black women are similar to those dominating in American and European studies [18,19]. In preparation to study the colonisation efficiency of exogenously applied vaginal lactobacilli and to treat BV in South African women, we have therefore identified the cultivable vaginal Lactobacillus species in women with or without BV in Soweto. Hydrogen peroxide (H2O2) production test Hydrogen peroxide (H2O2) production test Lactobacillus isolates were streaked onto a 20 ml MRS agar plate containing 0.25 mg/ml 3,3’, 5,5’-tetramethyl- benzidine (TMB) (Sigma-Aldrich, St-Louis, MO) and 0.01 mg/ml of horseradish peroxidase (HRP) (Sigma- Aldrich). Plates were incubated in anaerobic condition using the BD Gas Pack™anaerobic container system for 72 hours and were exposed to air for 30 minutes before scoring them for blue coloration. HRP generates O2 from any H2O2 produced by the lactobacilli which in turn oxidises the TMB substrate to form a blue pigment. On the basis of the blue coloration, isolates were scored for H2O2 production as 0 for no blue coloration, 1 for light blue, 2 for moderate and 3 for dark blue coloration. Since it was difficult to culture L. iners on MRS agar medium, L. iners isolates were not included in the H2O2 production test. Hydrogen peroxide (H2O2) production and BV status All the isolates but L. iners were tested and scored for H2O2 production on a scale of zero to three on the basis of blue coloration. L. iners was not evaluated for H2O2 production since they do not grow on MRS agar plates. Production of H2O2 was observed in the majority of the L. crispatus (90%), L. jensenii (86%) and L. vaginalis (80%) isolates and in a lower proportion of the L. gasseri isolates (30%). L. ruminis, L. coleohominis, L. paracasei were non-producers. Among the most commonly iso- lated species, the proportion of isolates producing high levels of H2O2 (score 2 and 3) was significantly higher (Fisher test, P<0.001) for L. jensenii (52 %), L. crispatus (33 %) and L. vaginalis (79%) than L. gasseri (0%). 33820 (AF257097) & DSM 20584T (FR683088), L. coleoho- minis DSM 14060 (AM113776), L. vaginalis strain ATCC 49540 (AF243177) and KC19 (AF243154), L. iners strain DSM 13335 (NR_036982) and CCUG 28746T (Y16329), L. gasseri ATCC 33323 (AF519171) and L. jensenii strain ATCC 25258 (AF243176). 33820 (AF257097) & DSM 20584T (FR683088), L. coleoho- minis DSM 14060 (AM113776), L. vaginalis strain ATCC 49540 (AF243177) and KC19 (AF243154), L. iners strain DSM 13335 (NR_036982) and CCUG 28746T (Y16329), L. gasseri ATCC 33323 (AF519171) and L. jensenii strain ATCC 25258 (AF243176). women with BV but not in women with normal or inter- mediate microbiota (Table 1). Lactobacillus colonisation was observed in women both with and without BV, where cultivable Lactobacillus species were identified in 19 out of 21 women with normal microbiota, in three out of five women with intermediate microbiota and in eight out of 14 women with BV. Statistical analysis Fisher’s exact test was performed to test the relation bet- ween BV status (normal microbiota vs. BV) and the pre- sence of any lactobacilli, specific Lactobacillus species and high/low H2O2-producing lactobacilli. Two-tailed P values less than 0.05 were considered statistically significant. All the comparisons were performed with GraphPad Prism 4 software (GraphPad Software, Inc., La Jolla, Ca). The isolation of strong H2O2 producers (either L. jensenii, L. crispatus or L. vaginalis with a score of 2 or 3) was in general more frequent in women with a normal microbiota than in the women with an intermediate microbiota or BV. However H2O2 production as an individual factor did not contribute significantly to the normal vaginal microbiota (Fisher test, normal Vs. BV microbiota, P = 0.064) (Figure 3). Determination of BV A total of 196 isolates were cultured from 40 swabs and 30 women (75%) were identified with cultivable Lactoba- cillus species. L. crispatus, L. iners and L. gasseri were identified in 10 (33%), 8 (27%) and 7 (23%) women re- spectively, whereas L. vaginalis and L. jensenii colonised five (17%) women each. These were the most commonly identified Lactobacillus species. Other Lactobacillus spe- cies identified were L. ruminis, L. mucosae, L. paracasei and L. coleohominis (Figure 1). Among the 30 women har- bouring lactobacilli, nine were colonised by more than one Lactobacillus species (Table 2). L. crispatus was only isolated from women with normal microbiota whereas the other predominant Lactobacillus species (L. jensenii, L. gasseri, L. vaginalis and L. iners) were isolated from women both with and without BV (Figure 2). The two women who were colonised by L. ruminis only and the one woman who was colonized by a mix of L. ruminis, L. mucosae and L. paracasei were identified with BV and intermediate microbiota, respectively. Colonisation by any lactobacilli and in particular L. crispatus was significantly associated with a normal vaginal microbiota (Fisher test, normal vs. BV microbiota, P = 0.0386 and P = 0.024 res- pectively) but not the colonisation by other Lactobacillus species. Vaginal smears were Gram stained and graded on a 10 point scale based on the presence of lactobacilli and other anaerobes as described by Nugent [22] for normal microbiota (score 0–3), intermediate microbiota (score 4–6) and BV (score 7–10). In brief, a score of zero to four is given individually for the presence of lactobaci- lli and for Gardnerella/Bacteroides morphotypes and a score of zero to two for curved Gram variable rods. The sum of all scores is a representative of the BV score. The presence of epithelial cells covered with bacteria (clue cells), a critical component of the Amsel’s clinical criteria for BV, was also noted [23]. Sample collection Forty premenopausal and HIV uninfected black women aged 18–44 years with or without BV were recruited for the study. Study participants were randomly selected among women who visited the Perinatal HIV Research Unit (PHRU) in Chris Hani Baragwanath Hospital, So- weto and received voluntary HIV counseling and testing. Inclusion criteria were their willingness to provide the informed consent, passing the assessment of under- standing, BHCG negative status (non-pregnant) and belonging to a low risk group for HIV acquisition. The women were non-menstruating and not taking antibio- tics at the time of sample collection. Vaginal swabs were collected in Amies agar gel medium with charcoal (Copan Venturi TranssystemW Copan Diagnostics, Italy). Simultaneously, a second similar swab was used to pre- pare a smear on a glass slide for BV scoring. The ethical approval for collection of swab samples was granted by Page 3 of 7 Page 3 of 7 Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 33820 (AF257097) & DSM 20584T (FR683088), L. coleoho- minis DSM 14060 (AM113776), L. vaginalis strain ATCC 49540 (AF243177) and KC19 (AF243154), L. iners strain DSM 13335 (NR_036982) and CCUG 28746T (Y16329), L. gasseri ATCC 33323 (AF519171) and L. jensenii strain ATCC 25258 (AF243176). BV status and Lactobacillus colonisation Nugent scoring of the Gram stained vaginal smears revealed that 21 women had a normal vaginal micro- biota, five had an intermediate microbiota and 14 had BV. The presence of clue cells was observed in all the Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 Page 4 of 7 Table 1 Study participants characteristics Normal Intermediate BV Women Total N=21 N=5 N=14 40 Mean age (range) 27 (19–40) 29 (22–40) 27 (19–44) 27 (18–44) Median (and range) of Nugent scores 1 (0–2) 5 (4–6) 8 (8–10) Number of samples with clue cells 0 0 14 14 Presence of Gardnerella morphotypes on microscopy 0 5 14 19 Presence of Mobiluncus morphotypes on microscopy 0 0 3 3 Presence of any Lactobacillus species on culture 19 3 8 30 Presence of L. crispatus on culture 10 0 0 10 Table 2 Colonising Lactobacillus species in thirty women Colonising species No. of women colonised Nugent score 0-3 4-6 7+ L. crispatus 4 4 L. iners 7 4 1 2 L. gasseri 4 2 1 1 L. jensenii 2 1 1 L. vaginalis 2 1 1 L. ruminis 2 2 L. crispatus, L. jensenii 2 2 L. crispatus, L. gasseri 1 1 L. crispatus, L. vaginalis 1 1 L. crispatus, L. coleohominis 1 1 L. iners, L. vaginalis 1 1 L. crispatus, L. vaginalis, L. gasseri 1 1 L. mucosae, L. paracasei, L. ruminis 1 1 L. gasseri, L. jensenii 1 1 able 1 Study participants characteristics Colonising Lactobacillus species in thirty women Discussion Therefore, our results need to be confirmed in a larger cohort preferably using a longitudinal study design, combined with data on subjects' behaviors. L. crispatus strains were found to be strong H2O2 pro- ducers [5]. On the contrary, L. iners, L. gasseri and L. jensenii were isolated from women with normal microbiota and BV. The presence of these Lactobacillus species in women with BV could be due to their poorer colonisation resistance, thereby allowing overgrowth of other bacteria or due to their better resistance for the environmental conditions associated with BV. Longitu- dinal studies in pregnant women have shown that the women harboring these Lactobacillus species, particu- larly L. gasseri and L. iners, are more susceptible to BV compared to the women colonised by L. crispatus [26]. It has also been suggested that L. iners may become a dominant part of the vaginal microbiota when the microbiota is in a transitional stage between abnormal and normal [27]. L. crispatus strains were found to be strong H2O2 pro- ducers [5]. On the contrary, L. iners, L. gasseri and L. jensenii were isolated from women with normal microbiota and BV. The presence of these Lactobacillus species in women with BV could be due to their poorer colonisation resistance, thereby allowing overgrowth of other bacteria or due to their better resistance for the environmental conditions associated with BV. Longitu- dinal studies in pregnant women have shown that the women harboring these Lactobacillus species, particu- larly L. gasseri and L. iners, are more susceptible to BV compared to the women colonised by L. crispatus [26]. It has also been suggested that L. iners may become a dominant part of the vaginal microbiota when the microbiota is in a transitional stage between abnormal and normal [27]. The vast majority of clinical studies have established an inverse association between BV and the occurrence of vaginal H2O2-producing Lactobacillus species. In a 2- year follow-up study, Hawes et al. documented that the acquisition of bacterial vaginosis was strongly associated with a lack or loss of hydrogen peroxide producing lactobacilli [5]. Although the results of the numerous clinical studies suggest that BV is caused by the lack of The production of H2O2 by L. iners isolates was not evaluated as these strains grow poorly on MRS contai- ning TMB substrate. Most L. Discussion BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 vaginal H2O2-producing Lactobacillus species; the estab- lished correlation may not necessarily indicate causality. Lactobacilli that produce H2O2 have been shown in vitro to inhibit the growth of various microorganisms, including Gardnerella vaginalis, anaerobes, Neisseria gonorrhoeae as well as the survival of HIV [28-31], but the relative contri- bution of the H2O2 produced by the Lactobacillus species to the overall antimicrobial effect is still a matter of de- bate. Under the hypoxic conditions that generally prevail in the vagina, H2O2 production by vaginal lactobacilli is undetectable (detection threshold 10 nM). Additionally, studies have shown that the cervicovaginal fluid and semen have a significant H2O2-blocking activity and that physiological concentrations of H2O2 below 100 μM did not kill any of the tested BV-associated bacteria [32]. 0 10 20 30 40 50 60 70 % women colonised % Normal % Intermediate/BV * * Figure 2 Association between colonising lactobacilli and BV status defined by Nugent Score. * Significantly different (p value < 0.05) according to a two tailed Fisher’s exact test. Figure 2 Association between colonising lactobacilli and BV status defined by Nugent Score. * Significantly different (p value < 0.05) according to a two tailed Fisher’s exact test. In this study, we found that the majority of L. crispatus, L. jensenii and L. vaginalis isolates from South African women were producing H2O2, corroborating previous results [33,34]. Similar to the other reports, we have also observed that a high proportion of L. crispatus, L. jensenii and L. vaginalis isolates were strong H2O2 producers while all the L. gasseri isolates were low producers [24,30,33,35]. The presence of these strong H2O2 produ- cers was higher in women with a normal microbiota al- though not to a significant level. The absence of significant inverse association between BV and the occur- rence of vaginal H2O2-producing Lactobacillus species in the present study could be due to different factors. Our sample size was rather small and a single sampling occa- sion may not properly reflect the status of the vaginal microbiota of a woman as changes in the microflora du- ring the menstrual cycle has been documented previously. Several studies have indicated that Lactobacillus growth increases throughout the menstrual cycle, but decreases during the menses [36,37]. Furthermore, we did not re- cord any behavioral factors that might have affected the vaginal microbiota status during the study. Discussion Akunam et al. [19] in Nigeria and more recently, Damelin et al. in South Africa [18], where the same predominant vaginal Lactobacillus species were isolated. With an increasing number of HIV-1 infected individuals and women being at a higher risk of acquiring HIV infec- tion, it is of great importance to improve women’s vaginal health especially in the high risk population for sexually transmitted diseases. It is estimated that nearly one-third of all new HIV cases in South African population might be prevented if all cases of BV could be cured [9]. With an aim to restore the vaginal health through the adminis- tration of exogenous Lactobacillus among South African women, it was important to identify the Lactobacillus spe- cies that colonise women in this region. Our finding indi- cates that the Lactobacillus species (L. crispatus, L. iners, L. gasseri, L. vaginalis and L. jensenii) that dominate the vaginal microbiota in our cohort are similar to those pre- dominating in European and American women [1,2,24]. Our results are in accordance with studies conducted by As previously reported, the presence of Lactobacillus species was a major determinant of a normal vaginal microbiota [25,26]. More particularly, the isolation of L. crispatus was strongly associated with the normal va- ginal microbiota of women in the study as none of the women colonised by L. crispatus had BV or an inter- mediate microbiota. A number of previous studies have also shown an association between absence of BV and the presence of L. crispatus [1,2,24]. Furthermore, longi- tudinal analysis of the microbiota has shown that the presence of L. crispatus promotes stability of the vaginal microbiota [26]. The ability of L. crispatus to produce H2O2 has been suggested as an important factor in maintaining the normal vaginal mirobiota as most of the L. crispatus L. iners L. gasseri L. jensenii L. vaginalis L. ruminis L. mucosae L. paracasei L. coleohominis (10, 33%) (8, 27%) (7, 23%) (1 each, 3%) (3, 10%) (5, 17%) (5, 17%) Figure 1 Distribution of colonising Lactobacillus species in South African women. The number and percentage of women colonised by the Lactobacillus species is indicated in parenthesis. Figure 1 Distribution of colonising Lactobacillus species in South African women. The number and percentage of women colonised by the Lactobacillus species is indicated in parenthesis. Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 Page 5 of 7 Pendharkar et al. Conclusions The vaginal Lactobacillus species identified in black South African women from Soweto are similar to those identified in women from European populations. Colo- nising Lactobacillus species, in particular L. crispatus, contribute to a normal vaginal microbiota. High H2O2 producers were more frequently isolated from women without BV, although not to a statistically significant degree which might be due to the small sample size. The similarity in the species composition between European and African women suggest that, similar to the studies conducted in North European populations [12,21], ad- ministration of exogenously applied lactobacilli (pro- biotic or designer probiotics) in South African women could potentially be used for treatment of BV and thus reduce the occurrence of infection by sexually transmit- ted pathogens, including HIV-1. 5. Hawes SE, Hillier SL, Benedetti J, Stevens CE, Koutsky LA, Wolner-Hanssen P, Holmes KK: Hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections. J Infect Dis 1996, 174:1058–1063. 6. Spiegel CA: Bacterial vaginosis. Clin Microbiol Rev 1991, 4:485. 6. Spiegel CA: Bacterial vaginosis. Clin Microbiol Rev 1991, 4:485. 7. Koumans EH, Sternberg M, Bruce C, McQuillan G, Kendrick J, Sutton M, Markowitz LE: The prevalence of bacterial vaginosis in the United States, 2001–2004; associations with symptoms, sexual behaviors, and reproductive health. Sex Transm Dis 2007, 34:864–869. 8. Taha TE, Gray RH, Kumwenda NI, Hoover DR, Mtimavalye LA, Liomba GN, Chiphangwi JD, Dallabetta GA, Miotti PG: HIV infection and disturbances of vaginal flora during pregnancy. J Acquir Immune Defic Syndr Hum Retrovirol 1999, 20:52–59. 8. Taha TE, Gray RH, Kumwenda NI, Hoover DR, Mtimavalye LA, Liomba GN, Chiphangwi JD, Dallabetta GA, Miotti PG: HIV infection and disturbances of vaginal flora during pregnancy. J Acquir Immune Defic Syndr Hum Retrovirol 1999, 20:52–59. 9. Myer L, Denny L, Telerant R, de SM, Wright TC Jr, Kuhn L: Bacterial vaginosis and susceptibility to HIV infection in South African women: a nested case–control study. J Infect Dis 2005, 192:1372–1380. 9. Myer L, Denny L, Telerant R, de SM, Wright TC Jr, Kuhn L: Bacterial vaginosis and susceptibility to HIV infection in South African women: a nested case–control study. J Infect Dis 2005, 192:1372–1380. 10. Taha TE, Hoover DR, Dallabetta GA, Kumwenda NI, Mtimavalye LA, Yang LP, Liomba GN, Broadhead RL, Chiphangwi JD, Miotti PG: Bacterial vaginosis and disturbances of vaginal biota: association with increased acquisition of HIV. AIDS 1998, 12:1699–1706. 10. Authors’ contributions GEG GdB HM LH d GEG, GdB, HM, LH designed the research project, SP, TM and PL performed the experiments. Statistical analysis was performed by SP, HM and PG and the paper was written by SP, HM and LH. All authors read and approved the final manuscript. 14. Larsson P-G, Stray-Pedersen B, Ryttig KR, Larsen S: Human lactobacilli as supplementation of clindamycin to patients with bacterial vaginosis reduce the recurrence rate; a 6-month, double-blind, randomized, placebo-controlled study. BMC Womens Health 2008, 8:3. 14. Larsson P-G, Stray-Pedersen B, Ryttig KR, Larsen S: Human lactobacilli as supplementation of clindamycin to patients with bacterial vaginosis reduce the recurrence rate; a 6-month, double-blind, randomized, placebo-controlled study. BMC Womens Health 2008, 8:3. Competing interests Competing interests The authors declare that they have no competing interests. 13. Marcone V, Calzolari E, Bertini M: Effectiveness of vaginal administration of Lactobacillus rhamnosus following conventional metronidazole therapy: how to lower the rate of bacterial vaginosis recurrences. New Microbiol 2008, 31:429–433. 13. Marcone V, Calzolari E, Bertini M: Effectiveness of vaginal administration of Lactobacillus rhamnosus following conventional metronidazole therapy: how to lower the rate of bacterial vaginosis recurrences. New Microbiol 2008, 31:429–433. Discussion iners strains have been found to be non-H2O2 producers which might correlate with our finding that these species was isolated in both BV positive and negative women [33]. The production of H2O2 in this species should be assessed on medium adapted for the growth of L. iners. Furthermore, the pro- portion of H2O2 producing lactobacilli (and not only their presence) might also be important and the relative amount of lactobacilli should be determined by molecu- lar methods. Finally, although the production of H2O2 by lactobacilli maybe an important factor for mainte- nance of the vaginal microbiota, other factors such as 0 2 4 6 8 0 1 2 3 No. of women Scores for H2O2 production Normal Intermediate BV Figure 3 Relation between hydrogen peroxide production by lactobacilli and the Nugent score. Scores for H2O2 production Figure 3 Relation between hydrogen peroxide production by lactobacilli and the Nugent score. Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 Page 6 of 7 competition for adherence and production of other anti- microbial substances may also contribute to vaginal health and might act in synergy with H2O2. Comparative ge- nomic studies of the lactobacilli isolated from the vagina of women with normal microbiota and BV might give a clue of the factors involved in colonisation resistance. 2. Vásquez A, Jakobsson T, Ahrné S, Forsum U, Molin G: Vaginal lactobacillus biota of healthy Swedish women. J Clin Microbiol 2002, 40:2746–2749. 3. Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M: Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microbiota suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis. BMC Microbiol 2004, 4:16. 4. Cherpes TL, Hillier SL, Meyn LA, Busch JL, Krohn MA: A delicate balance: risk factors for acquisition of bacterial vaginosis include sexual activity, absence of hydrogen peroxide-producing lactobacilli, black race, and positive herpes simplex virus type 2 serology. Sex Transm Dis 2008, 35:78–83. Received: 11 July 2012 Accepted: 18 January 2013 Published: 26 January 2013 Received: 11 July 2012 Accepted: 18 January 2013 Published: 26 January 2013 19. Anukam KC, Osazuwa EO, Ahonkhai I, Reid G: Lactobacillus vaginal microbiota of women attending a reproductive health care service in Benin city, Nigeria. Sex Transm Dis 2006, 33:59–62. Acknowledgements 15. Chang TL-Y, Chang C-H, Simpson DA, Xu Q, Martin PK, Lagenaur LA, Schoolnik GK, Ho DD, Hillier SL, Holodniy M, Lewicki JA, Lee PP: Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc Natl Acad Sci USA 2003, 100:11672–11677. 15. Chang TL-Y, Chang C-H, Simpson DA, Xu Q, Martin PK, Lagenaur LA, Schoolnik GK, Ho DD, Hillier SL, Holodniy M, Lewicki JA, Lee PP: Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc Natl Acad Sci USA 2003, 100:11672–11677. The study was supported by an international collaborative research grant funded by the Swedish International Development Cooperation Agency (Sida) (Swedish Research Links Programme) and the National Research Foundation of South Africa. The South African AIDS Vaccine Initiative provided funding for the screening protocol. 16. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SSK, McCulle SL, Karlebach S, Gorle R, Russell J, Tacket CO, Brotman RM, Davis CC, Ault K, Peralta L, Forney LJ: Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci USA 2011, 108(1):4680–4687. 16. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SSK, McCulle SL, Karlebach S, Gorle R, Russell J, Tacket CO, Brotman RM, Davis CC, Ault K, Peralta L, Forney LJ: Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci USA 2011, 108(1):4680–4687. Conclusions Taha TE, Hoover DR, Dallabetta GA, Kumwenda NI, Mtimavalye LA, Yang LP, Liomba GN, Broadhead RL, Chiphangwi JD, Miotti PG: Bacterial vaginosis and disturbances of vaginal biota: association with increased acquisition of HIV. AIDS 1998, 12:1699–1706. 11. van de Wijgert JHHM, Morrison CS, Brown J, Kwok C, Van Der Pol B, Chipato T, Byamugisha JK, Padian N, Salata RA: Disentangling contributions of reproductive tract infections to HIV acquisition in African Women. Sex Transm Dis 2009, 36:357–364. 11. van de Wijgert JHHM, Morrison CS, Brown J, Kwok C, Van Der Pol B, Chipato T, Byamugisha JK, Padian N, Salata RA: Disentangling contributions of reproductive tract infections to HIV acquisition in African Women. Sex Transm Dis 2009, 36:357–364. Abbreviations Abbreviations BV: Bacterial Vaginosis; HIV-1: Human immunodeficiency virus-1; H2O2: Hydrogen peroxide; TMB: 3,3’, 5,5’-tetramethylbenzidine; HRP: Horseradish peroxidase. 12. Al-Harthi L, Roebuck KA, Olinger GG, Landay A, Sha BE, Hashemi FB, Spear GT: Bacterial vaginosis-associated microbiota isolated from the female genital tract activates HIV-1 expression. J Acquir Immune Defic Syndr 1999, 21:194–202. 12. Al-Harthi L, Roebuck KA, Olinger GG, Landay A, Sha BE, Hashemi FB, Spear GT: Bacterial vaginosis-associated microbiota isolated from the female genital tract activates HIV-1 expression. J Acquir Immune Defic Syndr 1999, 21:194–202. Competing interests The authors declare that they have no competing interests. 1. Antonio MA, Hawes SE, Hillier SL: The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis 1999, 180:1950–1956. Author details 1 1Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska University Hospital, Huddinge, Stockholm, Sweden. 2Perinatal HIV Research Unit (PHRU), Chris Hani Baragwanath Hospital, Soweto, Johannesburg, South Africa. 3Department of Obstetrics and Gynaecology Kärnsjukhuset, Skaraborg hospital and University of Skövde, Skövde SE-541 85, Sweden. 4Department of Laboratory Medicine, Division of Clinical Immunology, F79, Karolinska Institutet, Stockholm S-141 86, Sweden. 17. Zhou X, Brown CJ, Abdo Z, Davis CC, Hansmann MA, Joyce P, Foster JA, Forney LJ: Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women. ISME J 2007, 1:121–133. 17. Zhou X, Brown CJ, Abdo Z, Davis CC, Hansmann MA, Joyce P, Foster JA, Forney LJ: Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women. ISME J 2007, 1:121–133. 18. Damelin LH, Paximadis M, Mavri-Damelin D, Birkhead M, Lewis DA, Tiemessen CT: Identification of predominant culturable vaginal Lactobacillus species and associated bacteriophages from women with and without vaginal discharge syndrome in South Africa. J Med Microbiol 2011, 60:180–183. Received: 11 July 2012 Accepted: 18 January 2013 Published: 26 January 2013 Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 21. Kõll-Klais P, Mändar R, Leibur E, Marcotte H, Hammarström L, Mikelsaar M: Oral lactobacilli in chronic periodontitis and periodontal health: species composition and antimicrobial activity. Oral Microbiol Immunol 2005, 20:354–361. 22. Nugent RP, Krohn MA, Hillier SL: Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991, 29:297–301. 23. Lin DP, Pan BJ, Fuh JC, Huang TH: Improving Gram-stained reproducible result by further adding clue cells in diagnosing bacterial vaginosis. Kaohsiung J Med Sci 2002, 18:164–170. 24. Wilks M, Wiggins R, Whiley A, Hennessy E, Warwick S, Porter H, Corfield A, Millar M: Identification and H2O2 production of vaginal lactobacilli from pregnant women at high risk of preterm birth and relation with outcome. J Clin Microbiol 2004, 42:713–717. 25. Boris S, Suárez JE, Vázquez F, Barbés C: Adherence of human vaginal lactobacilli to vaginal epithelial cells and interaction with uropathogens. Infect Immun 1998, 66:1985–1989. 26. Verstraelen H, Verhelst R, Claeys G, De Backer E, Temmerman M, Vaneechoutte M: Longitudinal analysis of the vaginal microbiota in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microbiota and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microbiota. BMC Microbiol 2009, 9:116. 27. Jakobsson T, Forsum U: Lactobacillus iners: a marker of changes in the Vaginal Flora? J Clin Microbiol 2007, 45:3145. 28. Skarin A, Sylwan J: Vaginal lactobacilli inhibiting growth of Gardnerella vaginalis, Mobiluncus and other bacterial species cultured from vaginal content of women with bacterial vaginosis. Acta Pathol Microbiol Immunol Scand B 1986, 94:399–403. 29. Klebanoff S, Hiller S, Eschenbach D, Waltersdorph A: Control of the microbial-flora of the vagina by H2O2-generating lactobacilli. J Infect Dis 1991, 164:94–100. 30. Zheng H, Alcorn T, Cohen M: Effects of H2O2-producing lactobacilli on Neisseria-gonorrhoeae growth and catalase activity. J Infect Dis 1994, 170:1209–1215. 31. Coombs RW, Klebanoff SJ: Viricidal effect of Lactobacillus acidophilus on human immunodeficiency virus type 1: possible role in heterosexual transmission. J Exp Med 1991, 174:289–292. 32. O’Hanlon DE, Moench TR, Cone RA: In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide. BMC Infect Dis 2011, 11:200. 33. Antonio MAD, Rabe LK, Hillier SL: Colonization of the rectum by Lactobacillus species and decreased risk of bacterial vaginosis. J Infect Dis 2005, 192:394–398. 34. References 1. Antonio MA, Hawes SE, Hillier SL: The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis 1999, 180:1950–1956. 20. Ehrström S, Daroczy K, Rylander E, Samuelsson C, Johannesson U, Anzén B, Påhlson C: Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal candidiasis. Microbes Infect 2010, 12:691–699. Page 7 of 7 doi:10.1186/1471-2334-13-43 Cite this article as: Pendharkar et al.: Identification and characterisation of vaginal lactobacilli from South African women. BMC Infectious Diseases 2013 13:43. Pendharkar et al. BMC Infectious Diseases 2013, 13:43 http://www.biomedcentral.com/1471-2334/13/43 Vallor AC, Antonio MA, Hawes SE, Hillier SL: Factors associated with acquisition of, or persistent colonization by, vaginal lactobacilli: role of hydrogen peroxide production. J Infect Dis 2001, 184:1431–1436. 35. Song YL, Kato N, Matsumiya Y, Liu CX, Kato H, Watanabe K: Identification of and hydrogen peroxide production by fecal and vaginal lactobacilli isolated from Japanese women and newborn infants. J Clin Microbiol 1999, 37:3062–3064. 36. Lopes dos Santos Santiago G, Cools P, Verstraelen H, Trog M, Missine G, Aila NE, Verhelst R, Tency I, Claeys G, Temmerman M, Vaneechoutte M: Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles. PLoS One 2011, 6:e28180. 37. Srinivasan S, Liu C, Mitchell CM, Fiedler TL, Thomas KK, Agnew KJ, Marrazzo JM, Fredricks DN: Temporal variability of human vaginal bacteria and relationship with bacterial vaginosis. PLoS One 2010, 5:e10197. 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: Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: doi:10.1186/1471-2334-13-43 Cite this article as: Pendharkar et al.: Identification and characterisation of vaginal lactobacilli from South African women. 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https://openalex.org/W13928782	https://scielo.conicyt.cl/pdf/rfilosof/v76/0718-4360-rfilosof-76-00057.pdf, http://www.scielo.cl/pdf/rfilosof/v76/0718-4360-rfilosof-76-00057.pdf	es		Revisitando la discusión entre Augusto Salazar y Leopoldo Zea. La filosofía latinoamericana: El lugar de un diferendo	Revista de filosofía	2,019	cc-by	8,643	Revista de Filosofía Volumen 76 (2019) 57-74 REVISITANDO LA DISCUSIÓN ENTRE AUGUSTO SALAZAR BONDY Y LEOPOLDO ZEA. LA FILOSOFÍA LATINOAMERICANA: EL LUGAR DE UN DIFERENDO1 Cristóbal Friz Echeverría Universidad de Santiago cristobal.frize@usach.cl Resumen / Abstract El artículo revisita la discusión entre Augusto Salazar Bondy y Leopoldo Zea hacia fines de la década de 1960 sobre la filosofía latinoamericana, con el objetivo de mostrar que la misma es el lugar de un diferendo: de pugna entre posiciones y representaciones disímiles, en algunos casos irreconciliables. Como se propone, lo que está en disputa en tal controversia, es el significado de la filosofía y de lo filosófico, en contextos de enunciación como América Latina. Para ello, se recorren diversos núcleos conceptuales de la disputa, mostrándola como una discusión filosófica aún vigente. Palabras clave: filosofía, América Latina, compromiso político-intelectual, tradición, proyecto, identidad, universalidad, desplazamientos categoriales. Revisiting the discussion between Augusto Salazar and Leopoldo Zea This paper aims the discussion between Augusto Salazar Bondy and Leopoldo Zea about lateinamerican philosophy, that takes place towards the end of the 1960s. The objective is to show that this discussion is the place of a differendum; that means, of conflict between dissimilar positions and representations, in some cases irreconcilable. As proposed, what is discussed in that controversy is the meaning of philosophy and the philosophical, in contexs of enunciation such Latin America. For this, we review some conceptual nuclei of the dispute, showing it as a current philosophical discussion. Keywords: Philosophy, Latin America, Political-intellectual engagement, Tradition, Project, Identity, Universality, Conceptual displacements. 1 Proyecto FONDECYT de Iniciación Nº 11170435. Revista de Filosofía Cristóbal Friz Echeverría A 50 años: Inactualidad y actualidad de una disputa En 2018 se cumplen 50 años de la publicación de ¿Existe una filosofía de nuestra América? (1968) del peruano Augusto Salazar Bondy (1925-1974), libro que generó una serie de respuestas y reacciones entre filósofos y estudiosos del pensamiento latinoamericano, de las cuales la más conocida es la del mexicano Leopoldo Zea (19122004), La filosofía americana como filosofía sin más (1969)2. ¿Por qué volver a esta polémica, la que ha sido profusamente estudiada y comentada 3, y que acaso debiéramos considerar añeja y superada? 4 Son múltiples las respuestas que podríamos esbozar: reparar, por ejemplo, en la inflexión que comportó la disputa para el campo disciplinar de la historia de las ideas 2 3 4 58 En este trabajo referimos la decimotercera edición del libro de Salazar Bondy (1996) y la decimosexta del de Zea (1996). Sobre los comentarios y estudios de la discusión cabe destacar, cercanos cronológicamente a la misma: Cerutti Guldberg (2006, primera edición de 1983), Gallardo (1974), Olmedo (1972), Santos (1977). Posteriormente, Arpini (2003, 2016), además de múltiples obras sobre el pensamiento filosófico latinoamericano en que la misma tiene un lugar señalado; por ejemplo, Santos Herceg (2010). La discusión sobre la existencia de una filosofía latinoamericana, en términos familiares a los planteados por Salazar y Zea en las obras mencionadas se remonta a los seminarios organizados por el hispano-mexicano José Gaos (1900-1969) desde el comienzo de su exilio en México en 1938 –a los que asisten ambos autores–, donde promueve el estudio de la filosofía y el pensamiento en lengua española, en que Gaos incluye al pensamiento latinoamericano. Como puntualiza Dante Ramaglia, los principales ejes de discusión en la filosofía latinoamericana a mediados del siglo XX son, entre otros, la preocupación por la “originalidad”, la “autenticidad” y la “identidad cultural” (Ramaglia 2009, p. 382), los que tienen lugar destacado en las obras de Salazar Bondy y Zea que acá nos ocupan. Debe tenerse presente que, según clarifica Horacio Cerutti, la respuesta de Zea en su libro La filosofía americana como filosofía sin más es solo el inicio de la polémica, la que rebasará a los textos y a los autores. Ambos volverán a la misma en el Simposio de Filosofía Latinoamericana que tiene lugar en San Miguel, Buenos Aires, en la Universidad del Salvador en agosto de 1973, donde, como señala Manuel Ignacio Santos, “se sometió a discusión el problema de una filosofía latinoamericana como filosofía de la liberación” (Santos 1977, p. 19). Tras la muerte de Salazar Bondy en 1974, ese mismo año Zea escribe un artículo en el que, según Cerutti, da por terminada la polémica, respetando el hecho de que Salazar ya no pudiera dar respuesta a sus planteamientos (Cerutti Guldberg 2006, pp. 268-272). No obstante estas importantes indicaciones, aquí nos detendremos exclusivamente en los ejes de la polémica consignados en los libros de 1968 y 1969, por considerar que en ellos queda establecido el ámbito de problematicidad fundamental de la discusión entre ambos autores. José Santos Herceg habla, por ejemplo, de una “añeja y ya desperfilada disputa acerca de si existe o no un pensamiento filosófico latinoamericano” (Santos Herceg 2012, p. 67), lo que se complementa y explica con el parecer de Dante Ramaglia, para quien ya no se discute dicha existencia dado el carácter manifiesto de sus expresiones relevantes (Ramaglia 2009, p. 397). Revisitando la discusión entre Augusto Salazar Bondy y Leopoldo Zea Revista de Filosofía latinoamericanas (Arpini 2003, 2016); o en la importancia decisiva de la misma para la constitución de la “filosofía latinoamericana de la liberación” (Cerutti Guldberg 2006, Santos 1977); o en su impronta para el planteamiento del “problema de la conformación de nuestra conciencia” (Gallardo 1974). No obstante, sin desatender ni desestimar estos y otros motivos posibles, interesa acá prestar atención a la indicación de Arturo Roig respecto de la importancia de estudiar a “nuestros clásicos” latinoamericanos (Roig 2009). Siguiendo esta indicación de Roig, si los planteamientos de Salazar y Zea tienen algo de clásicos, tal disputa tendría alguna relevancia en la actualidad: algo que decirnos a nosotros; alguna pista respecto de algo sobre lo que debiéramos volver, una y otra vez, a pensar. Esto puede ser particularmente válido para Chile, donde la tradición latinoamericana –y de un modo genérico, toda tradición distinta que la europeo-occidental– se halla aún en pugna por su incorporación y reconocimiento en lo que la institucionalidad universitaria sanciona como lo filosófico. En efecto, y como se mostrará a lo largo de este escrito, aquello disputado por los autores es justamente el estatuto de la filosofía y lo filosófico: el significado y sentido de la actividad filosófica, y las posibilidades de la misma en contextos de enunciación distintos a los de Europa occidental. Interesará, por lo tanto, exponer esta disputa como una discusión propiamente filosófica, que en cuanto tal nos conmina a pensar. Y por tratarse de una controversia sobre el estatuto de lo filosófico, interesará revisitarla en sus núcleos conceptuales, poniendo de manifiesto que ella es el lugar de un diferendo: de pugna entre posiciones y representaciones disímiles, en algunos casos irreconciliables. Contexto y coordenadas de la discusión: entre filosofía profesional y compromiso político-intelectual Como toda producción teórica, la discusión de Salazar Bondy y Zea supone un complejo entramado de influencias y discursos, además de ser expresión y respuesta a un determinado ánimo epocal. No me detendré acá en el tema de las influencias, o en la actualidad de las lecturas de los autores, salvo en la medida en que sea necesario para la presente exposición (estas cuestiones, por lo demás, han sido trabajadas en la literatura que ha abordado la disputa). Se tratará, más bien, de inscribir la controversia en una doble coordenada discursiva y epocal, la que, como se procurará mostrar, comporta una serie de tensiones y complejidades que atraviesan sus modos de enunciación, las condiciones de posibilidad de la discusión misma y de lo que en ella se entiende (o puede entenderse) por filosofía, y por filosofía latinoamericana. La coordenada propuesta, como se mostrará, está configurada por el cruce de dos ejes: la profesionalización de la filosofía como actividad universitaria y la asunción del ejercicio filosófico como quehacer comprometido políticamente. El primer eje dice relación con lo que el hispano-argentino Francisco Romero (18911962) denominó, para referir el desarrollo de la filosofía en los países hispanoamericanos desde fines de los 30 y la década del 40, como “normalidad filosófica” (o también 59 Revista de Filosofía Cristóbal Friz Echeverría “normalización filosófica”) (cf. Romero 1952)5. Para Romero, esta nueva etapa comporta, a diferencia de las anteriores, el asentamiento de las condiciones institucionales e intelectuales –es decir, el cultivo de la filosofía como labor profesional universitaria, y una sólida formación filosófica– para el “normal” despliegue del filosofar en la región; es decir, para su incorporación como una “función normal de la cultura”. Normalidad filosófica, como ponen de manifiesto Carlos Ossandón (1984) y José Santos Herceg (2010), es una noción fuertemente normativa y problemática. Por una parte, comporta la identificación de filosofía con filosofía universitaria, según el parámetro de la filosofía occidental contemporánea, lo que con Cecilia Sánchez conduce a la escisión entre un filosofar mundano y uno académico, entre los tiempos del mundo y el tiempo de la teoría (Sánchez 1992)6. Junto con ello, la normalización supone que nuestra “vocación filosófica” adquiere “conciencia de sí”. Esto se pone de manifiesto, además de en la interrogante por su historia, en la pregunta por su estatus y sus posibilidades (su “autenticidad” y “originalidad”, por ejemplo), por sus relaciones con la realidad en que se inserta, con la tradición occidental, etc. El segundo eje atañe a la sensibilidad sesentista en América Latina, la que pone en tensión el eje normativo de la normalidad filosófica, al tiempo que, con él, comparece como trasfondo de la discusión de Salazar Bondy y Zea, y funge de contexto de emergencia del pensamiento liberacionista latinoamericano contemporáneo7. Este ánimo epocal, cuyo hito de apertura es según Eduardo Devés la Revolución cubana del 59, y que tiene por cierre el golpe de Estado del 73 en Chile –razón por la que cabe hablar de unos “largos 60”–, es rubricado por este autor bajo la consigna “todo es política”. 5 6 7 60 La caracterización de la “normalidad filosófica” aquí desarrollada se remite principalmente al estudio de Carlos Ossandón (1984). Para este proceso en el caso argentino, véase Ramaglia (2007). Según Sánchez, en Chile serían Pedro León Loyola (1889-1978) y Enrique Molina (18711964) quienes asentarían las bases de la “institucionalización universitaria de los estudios filosóficos”, en función de la idea de una cierta “autonomía” del ejercicio filosófico respecto de otros campos, particularmente el político. Sumamente esclarecedoras son las declaraciones de Enrique Molina a comienzos de la década de 1950, donde señala que figuras como Andrés Bello, José Victorino Lastarria, Francisco Bilbao o Valentín Letelier no son filósofos propiamente tales. “Todos éstos –comenta Molina– habían dedicado, sin duda, desvelos a la filosofía; pero vivían ante todo absorbidos por los problemas sociales y políticos y por el afán de atender al progreso y reestructuración de la colectividad”. La filosofía, para Molina, comprende exclusivamente “las disciplinas que dicen relación con la indagación del ser y con la actitud del hombre ante él”. Solo quien se ocupa de ellas es, en su opinión, “un especialista de la filosofía (…) lo que se llamaría un filósofo en nuestros días” (Molina 1953, 9-10, 22). En el contexto de este estudio, interesa inscribir la discusión de Salazar Bondy y Zea en los comienzos o detonantes de la “filosofía de la liberación latinoamericana”, únicamente en la medida en que ésta comporta la adopción expresa de un compromiso político-intelectual por parte del quehacer filosófico en la región. Para este tema véase, entre otros, Cerutti Guldberg (2006) y Santos (1977). Revisitando la discusión entre Augusto Salazar Bondy y Leopoldo Zea Revista de Filosofía Dicha sensibilidad –que, en lo que acá interesa, supone que el ejercicio intelectual puede y debe cumplir un rol importante en la comprensión y superación de las condicionantes sociales estructurales que aquejan a los países latinoamericanos, y de modo genérico, a los del denominado tercer mundo– bebe de distintas fuentes. Entre ellas cabe destacar algunas escuelas filosóficas europeas (la fenomenología, el existencialismo, el marxismo; es importante aquí la figura de Sartre como intelectual comprometido) y los planteamientos de Paulo Freire y de la teología de la liberación. Asimismo es importante el influjo de los movimientos de decolonización (bajo la figura central de Frantz Fanon) y de los postulados de las ciencias económico-sociales latinoamericanas; puntualmente, la teoría de la dependencia. Esta última viene a complementar el análisis de clase, de raigambre marxista, con el análisis colonial, postulando que es el propio desarrollo o modernización capitalista –analizado bajo el par categorial metrópoli/satélite– el que ha subdesarrollado a América Latina, comportando un colonialismo e imperialismo no solo económico, sino también cultural y mental: una “ideología de dominación” (Devés 2003-2004, Tomo II, Parte V, cap. I-III). Según lo ya mencionado, considero que esta doble coordenada –profesionalización universitaria del ejercicio filosófico versus compromiso intelectual con la transformación de la realidad– cruza y tensiona los textos de Salazar Bondy y Zea, atravesando no solo los términos y modos de enunciación de la discusión, sino también las posibilidad de la misma, y de lo que en ella se entiende (o puede entenderse) por filosofía, y por filosofía latinoamericana. A esta doble coordenada se refería Horacio Cerutti a comienzos de los 80 cuando afirmaba que “la polémica es uno de los eslabones centrales del pensar filosófico actual en América Latina”; y que la misma trascendía a los autores y se mantenía abierta, por estimar que lo que en buena medida está en juego en ella es “la correlación de filosofía y política que es en definitiva fiel en esto a una larga tradición en nuestra América, el problema central que sigue estando en cuestión” (Cerutti Guldberg 2006, pp. 272-273)8. Dicho lo anterior, el análisis se centrará en tres puntos o momentos fuertemente interrelacionados que parecen particularmente significativos para ponderar la importancia y los alcances (eventualmente actuales) de la querella. En primer lugar, en la pregunta de si la filosofía latinoamericana es una labor que cuenta con una tradición, o si bien es una empresa prospectiva por realizar. En segundo término, en la disyuntiva entre un identitarismo cultural y filosófico al que debiera adscribir la labor filosófica local, y la opción por inscribirla en la presunta universalidad de la cultura y el pensar occidental. Finalmente, en el que es probablemente el punto más delicado –por lo tanto más 8 Igualmente claro al respecto es Helio Gallardo, quien a propósito de la disputa sostiene: “Tras la pregunta por la existencia de una filosofía latinoamericana suele, en ocasiones, esconderse una intención militante”; y que “la pregunta por la posibilidad de nuestra filosofía se inserta dentro del cuadro general de un mundo que lucha por la liberación y la independencia solidaria del hombre”. Es importante destacar que, para Gallardo, dicha intención debe asentarse en un “trabajo filosófico” serio y riguroso. (Gallardo 1974, p. 209) 61 Revista de Filosofía Cristóbal Friz Echeverría sugestivo y productivo– del debate, concerniente a los desplazamientos categoriales presentes en la respuesta de Zea; lo que en un extremo puede conducir a la pregunta de si éste responde propiamente a Salazar Bondy y, por lo tanto, si efectivamente podemos hablar de una discusión o un diálogo entre ambos autores. 1. Filosofía latinoamericana, ¿tradición o proyecto? Según Marcos García de la Huerta, como quiera que se plantee y se entienda, la pregunta por la filosofía latinoamericana debe comprenderse como una interrogante por una identidad cultural en el plano filosófico; como intento de afirmación de una identidad regateada por la conquista, la colonización y la herencia cultural eurocéntrica, que se procura asentar o proyectar en el ámbito del pensamiento (García de la Huerta 2014, p. 77, 1999). Esto se condice con la tesis de Eduardo Devés, según la cual el pensamiento latinoamericano, tanto en el siglo XIX como en el XX, se debate entre la modernización y la identidad (Devés 2003-2004), es decir, entre el ajuste o puesta al día de los países latinoamericanos con los parámetros promovidos por Occidente y la afirmación de lo “propio” o “particular”. Entrando a las obras que aquí nos ocupan, un primer punto en que se puede registrar este intento de vindicación identitaria es la interrogante respecto de si la filosofía latinoamericana es una labor que cuenta con antecedentes asentados en una tradición que podamos asumir y reconocer como “propia”, a la que habría que acudir mediante un ejercicio de memoria reflexiva que apunta a su proyección a futuro; o si bien es una labor que adopta la forma de un proyecto, lo que en una acepción extrema supone entenderla como labor meramente prospectiva, la que solo encontraría en el pasado una fuente de errores que indicarían las vías infecundas por las que no se debe volver a transitar. A este extremo se refería Humberto Giannini al indicar que la historia americana se halla cruzada por un “gesto fundacional”. En sus palabras: “El gesto fundacional se prolonga por la historia americana como un ritual, casi como un automatismo: la obsesión de estos pueblos de empezar cada cierto tiempo de cero, todo da capo; en política, en educación, en economía; la obsesión de lo discontinuo y de no dejar rastros tras sí, como si sólo a través de la destrucción pudiera construirse algo. Un antiplatonismo natural. Diríamos, el olvido como técnica para avanzar. El horror al pasado” (Giannini 2004, p. 74, nota). Con todos los recaudos y matices necesarios, podemos rubricar la perspectiva de Salazar Bondy en este gesto fundacional, toda vez que su diagnóstico sobre la historia y el presente (entiéndase, hacia fines de la década del 60) del filosofar hispanoamericano es fuertemente negativo. Esto se expresa en su afirmación de que son sumamente escasos –en caso de haberlos– los trabajos que dan cuenta de “la existencia de productos filosóficos hispanoamericanos originales, equiparables a los europeos” (Salazar Bondy 1996, p. 74). 62 Revisitando la discusión entre Augusto Salazar Bondy y Leopoldo Zea Revista de Filosofía Este diagnóstico expresa ejemplarmente las tensiones entre profesionalización y compromiso político del quehacer filosófico. El diagnóstico, en efecto, se encuentra fuertemente condicionado por el concepto de filosofía con que opera el peruano, profundamente comprometido a su vez con la categoría romeriana de normalidad, conocida y suscrita por Salazar bajo la noción de “regularización”, según la cual en los países hispanoamericanos ya se ha asentado una “actividad filosófica regular” (Ibíd., p. 19). De hecho, en la introducción de su libro señala: “[S]ólo nos ocuparemos del pensamiento filosófico propiamente tal, y por tanto, no trataremos sino indirectamente de otras modalidades de lo que en forma genérica puede llamarse pensamiento (v.gr. creencias religiosas, programas políticos, ideas artísticas, etcétera)” (Ibíd., p. 8). En virtud de este supuesto de trabajo, filosofía es y puede ser únicamente filosofía académica. Por ello, no es posible reconocer como “pensamiento filosófico propiamente tal” a otras modulaciones del filosofar que las elaboradas en la universidad; esto implica negar el estatuto epistemológico de filosofía, por ejemplo, a las obras de muchos hispanoamericanos del siglo XIX y anteriores o marginales a la normalidad filosófica. El diagnóstico reseñado se monta, asimismo, sobre el supuesto de que únicamente comunidades históricas formadas y maduras, autónomas (como serían contemporáneamente el pueblo alemán, el inglés o el francés) pueden dar frutos culturales propios y legítimos, entre los que se encuentra la filosofía. Tal no sería, para Salazar Bondy, el caso de pueblos alienados, dependientes y subdesarrollados, con una “cultura de dominación”, como los hispanoamericanos. La autoridad con que Salazar refrenda este parecer es Hegel y su ideas de que “la filosofía es la hija de su tiempo”, en base a lo cual afirma: “[L]a filosofía como tal es un producto que expresa la vida de una comunidad, pero que puede fallar en esta función y, en lugar de manifestar lo propio de un ser, puede desvirtuarlo o encubrirlo. Se da según esto el caso de una filosofía inauténtica, de un pensamiento mixtificado” (Ibíd., p. 80). No obstante la negatividad de su diagnóstico –a saber, no hay “productos filosóficos hispanoamericanos originales” arraigados en una tradición que podamos reconocer, en cuanto local, como propia–, para Salazar Bondy sí es posible darle “originalidad” y “autenticidad” a nuestro pensamiento; y es aquí, justamente, donde el eje normativo de la normalidad filosófica queda tensionado por la asunción de un compromiso político-intelectual. Para asentar esta posibilidad, el peruano se torna contra la autoridad de Hegel, rebatiendo la idea de que la filosofía sea –o pueda ser únicamente– como el búho de Minerva que emprende su vuelo al atardecer; es decir, consumación, explicación a posteriori de lo ya logrado en la historia. Contra dicha interpretación de la filosofía, Salazar Bondy postula que ella puede ser también, en tanto reflexión comprometida, “la mensajera del alba”: el principio de un cambio histórico mediante una toma de conciencia radical proyectada al futuro. En virtud de esto, la filosofía no tiene necesariamente que esperar la cancelación del subdesarrollo y la dominación; es capaz, en efecto, de alcanzar su “autenticidad” como parte del movimiento de superación de los mismos. Puede y debe ser –en palabras del peruano– “una conciencia liberadora de las trabas que impiden la expansión 63 Revista de Filosofía Cristóbal Friz Echeverría antropológica del hispanoamericano que es también la expansión antropológica de toda la especie” (Ibíd., p. 90)9. La respuesta de Zea se afinca justamente en este último punto asentado por Salazar Bondy; y en ella se mantiene –aunque desplazada– la tensión entre profesionalización de la filosofía y compromiso político-intelectual10. Su propuesta de “filosofía americana como filosofía sin más” descansa en su interpretación de la filosofía americana –como de toda filosofía posible– en cuanto expresión de la humanidad del hombre: como la capacidad de, mediante el logos, habitar humanamente el mundo (Zea 1996, cap. I). Conforme señala Adriana Arpini, “el planteo zeaniano gana en generalidad lo que pierde en concreción” (Arpini 2003, p. 198), toda vez que al considerar la occidentalización del mundo como un factum, adscribe a una comprensión antropológica, histórica y cultural universal, que prescinde –a contrapelo de la propuesta salazariana– de las diferencias y los conflictos11. 9 10 11 64 Sobre la propuesta de Salazar Bondy, Dante Ramaglia comenta: “Entre los aspectos más debatidos de su posición se encuentra la forma esquemática que representa trasladar directamente los caracteres negativos de la estructura socioeconómica al plano cultural, que se traduce en la idea de que sólo mediante la cancelación del subdesarrollo y la dominación se podría llegar a obtener un pensamiento filosófico propio”. Sin embargo, el filósofo argentino releva que la incorporación de la estructura material al estudio del pensamiento latinoamericano, es un aporte indesmentible para su posterior desarrollo (Ramaglia 2009, pp. 385-386). Complementa y matiza lo comentado por Ramaglia, Manuel Ignacio Santos, quien señala que la perspectiva salazariana supone un “papel determinante, aunque no absolutamente”, de la base económico-social sobre el ámbito cultural (Santos 1977, p. 35). Si así no fuera, sería impensable postular un ejercicio de la filosofía orientado crítico-dialécticamente a la superación del subdesarrollo y la dominación; en términos coloquiales, al filósofo no le restaría más que “cruzarse de brazos” a esperar las condiciones estructurales que posibiliten un adecuado ejercicio intelectual. La idea de un “compromiso de liberación” por parte de la filosofía cruza la extensa producción de Zea, y se pone de manifiesto, por ejemplo, en el título de su libro de 1991 (Zea 1991). La óptica de Zea se encuentra fuertemente influida, entre otras fuentes, por su interpretación del historicismo gaosiano, el que entronca a su vez con el circunstancialismo orteguiano. Arpini señala que en Zea “El ‘hombre’ es definido por su logos universal y colocado en una circunstancia, la americana. Circunstancia –agrega Arpini– (…) histórica, desde luego, pero accidental en relación con la naturaleza universal del logos. Se produce, así, un juego de historización-deshistorización del sujeto de la filosofía”. Por ello, según la autora, en el mexicano prima una concepción de la historia como “la sucesión de mostraciones circunstanciadas de una naturaleza humana idéntica”; una “visión armónica de la historia donde se esfuman los contornos marcados por las diferencias, los conflictos, los momentos de negación, las rupturas movilizadoras del devenir histórico”; lo cual comporta asumir, a fin de cuentas, un “sentido único de la filosofía, que se va manifestando en las diversas concreciones a lo largo de la historia” (Ibíd., pp. 60-61, 65-67). Complementa lo anterior el parecer de Raúl Fornet-Betancourt, para quien los principales desarrollos de la filosofía latinoamericana (entre ellos los de Zea) se encuentran fuertemente Revisitando la discusión entre Augusto Salazar Bondy y Leopoldo Zea Revista de Filosofía Para Zea, por lo tanto, el problema de si tenemos o no filosofía no es otro que el de nuestra humanidad, cuya afirmación constituye en consecuencia el compromiso de los intelectuales y filósofos latinoamericanos con su circunstancia. En virtud de esto, en la óptica de Zea no es casual que nuestra filosofía se inaugure en el siglo XVI, en las figuras de Juan Ginés de Sepúlveda y Bartolomé de Las Casas, con una disputa sobre la condición antropológica de los americanos; humanidad negada por la idea de hombre que tiene el europeo de sí, en cuanto norma o medida de la humanidad o del Hombre. Tampoco es casual, por lo tanto, que desde Juan Bautista Alberdi, los latinoamericanos nos preguntemos casi obsesivamente por nuestra capacidad para hacer filosofía (o por nuestra cultura e identidad, preguntas que para Zea apuntan a lo mismo), entendiendo dicha capacidad como índice de humanidad12. Según Zea, con esta interrogante los latinoamericanos introducen en el ámbito de la filosofía una pregunta extraña, inédita, que el europeo-occidental no se podía plantear en cuanto se consideraba a sí mismo la medida de lo humano, y exigía a los otros rendir cuenta de su estatus antropológico conforme al parámetro occidental. Así, en la perspectiva del mexicano, la pregunta por la filosofía latinoamericana –o lo que es lo mismo, por nuestra capacidad para filosofar– no es sólo una pregunta válida, sino acaso el tema propio de nuestro pensamiento (Zea 1996, 101). Tal pregunta ya supone, según Zea, hacer una filosofía que parte de y se halla comprometida con nuestra problemática y circunstancia (Ibíd., 105); y con ello, un aporte al acervo filosófico mundial, tradicionalmente monopolizado por Occidente, pero que debe abrirse a las distintas expresiones de lo humano. En concordancia con la idea de que hay filosofía latinoamericana desde la incorporación de América a la cultura e historia occidental-europeas (entendidas como cultura e historia universales), cobra particular relevancia el estudio de la historia de las ideas, en cuanto disciplina académica que debe registrar los distintos testimonios –asentados en una tradición remontable a Las Casas– de expresión de humanidad del pueblo latinoamericano. Según Zea, para ser fecunda, esta labor historiográfica debe desprenderse del prejuicio, caro a la “normalidad filosófica”, de que las obras propiamente filosóficas son aquellas que adoptan determinados procedimientos escriturales, literarios y argumentativos, como los del tratado filosófico. Se trata, en consecuencia, de enfocar con otros ojos 12 comprometidos con la idea de “inculturación de la filosofía”; es decir, la afirmación de la historicidad e historización del logos en América Latina, pero considerando como intocable la estructura fundamental de la racionalidad occidental, con valor normativo (FornetBetancourt 2001, p. 62). Véase también Fornet-Betancourt (2004, cap. 2). Como se sabe, Alberdi (1810-1884) fue el primero en plantear, en su texto de 1842 (Alberdi 1993), la pregunta por una “filosofía americana”; situación que lleva a varios filósofos e historiadores de nuestro pensamiento a considerar este texto como “programa fundacional del pensamiento filosófico latinoamericano” (cf. Ramaglia 2009, p. 381, nota). Véase además Roig (2009, cap. XVI). 65 Revista de Filosofía Cristóbal Friz Echeverría nuestro pasado intelectual, en función del futuro que proyectamos realizar; o en otras palabras, de captar lo que de “auténtico” –valga decir, de autoafirmación– hay en la historia de nuestro pensamiento (Ibíd., p. 115). Esto, junto con suponer un compromiso que comporta el rescate de lo positivo de la tradición intelectual latinoamericana –en oposición a la óptica salazariana, para la cual la “autenticidad” y “originalidad” de nuestro pensamiento se encuentran supeditadas a esfuerzos prospectivos–, implica ampliar o descentrar el concepto mismo de filosofía, superando la escisión –tan rotundamente sancionada por Salazar Bondy– entre filosofía y pensamiento. 2. Filosofía americana, entre vindicación identitaria y universalidad El segundo núcleo conceptual que me interesa relevar de la controversia –en gran medida bosquejado en el punto anterior, y que en consecuencia viene a desarrollar y complementar lo ya visto; y en que vuelve a hacerse presente la tensión entre ejercicio filosófico profesional y compromiso político-intelectual–, dice relación con el problema de la identidad cultural latinoamericana, y el papel que le correspondería al ejercicio filosófico en su afirmación o construcción. En términos analíticos, es posible distinguir dos posicionamientos extremos ante esta cuestión, en medio de los cuales habría que ubicar cuidadosamente las posturas de Salazar Bondy y Zea. El primero correspondería a un identitarismo fuerte, para el cual, como respuesta a la herencia cultural eurocéntrica y su filosofía, habría que vindicar lo “propio” y “particular”, aun a costa de postular una suerte de mismidad cultural, substancial (en un límite trans o ahistórica), posición que puede fácilmente recaer en un esencialismo o chovinismo. El segundo, a un universalismo igualmente fuerte, para el cual los procesos de “encuentro” o “choque” de culturas, de colonización, mestizaje, hibridación –o como los queramos llamar– apuntarían a una incorporación de la cultura o las culturas locales (para lo que aquí interesa, la americana) a la cultura europeo-occidental entendida como cultura común, universal. Nuevamente, con todos los resguardos y matices del caso, cabe afirmar que Salazar Bondy coquetea con una suerte de esencialismo cultural, el que se pone de manifiesto en el supuesto de correspondencia entre la madurez de una cultura y la de sus producciones, entre ellas la filosofía. Como veíamos, la asunción de dicho supuesto desempeña un papel destacado en su diagnóstico negativo sobre la tradición filosófica latinoamericana. En sus palabras: “Frente al respaldo con que cuenta la filosofía en Europa, apoyada como está sobre una larga evolución del pensamiento y la cultura tradicionales, en Hispanoamérica no ha encontrado ningún apoyo de la comunidad histórica básica, del fondo popular del espíritu de nuestros pueblos, y vive de la tradición europea, que le es, en este sentido, extraña” (Salazar Bondy 1996, p. 27). Según hemos constatado, para Salazar Bondy la filosofía “expresa la vida de una comunidad”, manifiesta “lo propio de un ser”. Sin embargo, en su opinión, esto falla en nuestra América, en la que prima un “hondo defecto de cultura”, un “ser 66 Revisitando la discusión entre Augusto Salazar Bondy y Leopoldo Zea Revista de Filosofía carencial”, un “existir inauténtico”, por lo que “vivimos alienados respecto a la propia realidad”, en una “representación ilusoria de nuestro ser” (Ibíd., p. 80, 83-84). Los hispanoamericanos, a causa del subdesarrollo, la dependencia y la dominación, nos encontramos alienados, enajenados de la “propia realidad” o del propio “ser”. Nuestra identidad cultural, por lo tanto, nos es igualmente ajena, lo que viene a agravarse con la alienación de nuestra producción filosófica, desfondada como se halla respecto de nuestra realidad, y bebiendo de la tradición occidental cual fuente extraña. A contrapelo de este diagnóstico, según el peruano nuestra filosofía debe, asumiendo un compromiso radical con la superación de nuestra situación deprimida, “sumergirse en la sustancia histórica de nuestra comunidad para buscar en ella el sustento de los valores y categorías que la expresen positivamente y le revelen el mundo” (Ibíd., p. 90). A fin de cuentas, si la filosofía hispanoamericana es inauténtica, esto se debe a la inautenticidad de nuestra cultura –a la distancia al parecer insalvable entre las bases sociales estructurales y nuestras producciones culturales–, por lo que la superación de este estado defectivo demanda una transformación o revolución cultural, a la que puede aportar una filosofía que opte por la liberación. El identitarismo cultural fuerte de Salazar Bondy se expresa sin ambages en su concepto de cultura, definida en ¿Existe una filosofía de nuestra América? como la “articulación orgánica de las modalidades originales diferenciales de una comunidad, susceptibles de servir de pautas para contrastar la obra histórica de los pueblos y las épocas” (Ibíd., p. 83). En esta apelación a una originalidad cultural, relativa a ciertas modalidades “originales” y “diferenciales” de un colectivo –entiéndase, a lo “propio” y “particular” de éste, no confundible con las manifestaciones culturales de otras asociaciones humanas–, las que por de pronto deben arraigar en “la sustancia histórica” de la misma, la identidad parece corresponderse más a un haber sido o a un ser –o a una concordancia de las producciones culturales con aquéllos–, que a un poder o querer llegar a ser. Aun cuando no es la intención de este artículo evaluar ni criticar los postulados de los autores, cuanto más bien ponderarlos como puntos de vista que, con toda su complejidad, apuntan a una problematización del estatuto de lo filosófico y de las posibilidades de la filosofía en contextos de enunciación como América Latina –cuestiones que, como problemas, me parecen de una vigencia indesmentible–, conviene contraponer la noción salazariana de identidad cultural con las reflexiones de Marcos García de la Huerta sobre el particular. Éste menciona: “Identidad significa, por demás, identificación: algo más sutil e indeterminado, más incierto y abierto que un ‘ser’ inmutable. La identidad –continúa el autor– denota una pertenencia y no puede definirse como una esencia, ni siquiera como algo que se encuentra en acto en las obras, instituciones y costumbres. Viene en alguna medida de lo que ha ido sedimentando el pasado, pero viene así mismo del futuro, de lo que se quiere (y no se quiere) ser, de la tarea o proyecto, que define también profundamente lo que uno es. No se puede impedir que la identidad, dentro de ciertos límites, se haga y rehaga” (García de la Huerta 1999, p. 132). 67 Revista de Filosofía Cristóbal Friz Echeverría En virtud de lo previamente señalado sobre la respuesta de Zea, es de esperar que su posición respecto del problema de la identidad latinoamericana y de la tarea que pueda competer a una filosofía comprometida con su vindicación sea de signo contrario de la Salazar Bondy, ¿mas será por ello menos problemática que la del peruano? Según veíamos, para Zea hay filosofía americana desde la incorporación de América a la historia y cultura europeas, entendidas como cultura e historia universales. En su óptica, por lo tanto, la tarea de una filosofía latinoamericana comprometida con su circunstancia no es como en Salazar Bondy una afirmación identitaria opuesta al Occidente, sino una inscripción de lo “local”, “propio” y “particular” en la universalidad del logos y la cultura occidental-universal. Dicha inscripción es la que permite hablar de un universalismo relativamente fuerte en Zea, toda vez que sin negar las peculiaridades culturales, la mentada inscripción se orienta inevitablemente a borronear los rasgos de localidad y particularismo, por apuntar a una construcción mancomunada y ecuménica de una cultura y una filosofía propiamente universales. Este punto tiende a clarificarse al considerar que el rescate de la universalidad de la cultura y la filosofía europeo-occidentales por parte de Zea, comporta una importante crítica a la misma. Para Zea, dicha pretensión reviste la forma de una universalidad abstracta, y en definitiva inconducente, pues ha pretendido erigir lo humano –en cuanto común, universal– negando su condición antropológica a quienes no se corresponden al prototipo de humanidad. En sus expresiones históricas, por lo tanto, el despliegue del logos occidental ha enajenado al no occidental, pero también al europeo-occidental, el que se aliena al deshumanizar a los demás. Contra esta universalidad abstracta y excluyente, la propuesta de Zea apunta a recuperar el sentido positivo del universalismo declarado por Occidente, reconduciéndolo a la construcción de una universalidad concreta; es decir, hacia la confluencia de distintas expresiones de lo humano, que por partir cada una de su circunstancia concreta –justamente, lo que en su opinión habría olvidado el pensar occidental al erigirse en el prototipo de lo humano, en desmedro de las restantes expresiones de humanidad– son igualmente valederas. Se trata, por lo tanto, de una universalidad en que quepan las distintas expresiones de lo humano, sin jerarquizaciones ni exclusiones; lo que conlleva, como señala Arpini, a desentenderse en buena medida de la conflictividad de las construcciones culturales presente en Salazar Bondy. En esta tarea radica la consigna zeaniana de hacer “filosofía sin más”, en la que se puede resumir el compromiso que según el mexicano debe adoptar el filósofo latinoamericano con su circunstancia: “hacer pura y simplemente filosofía”, desde nuestra circunstancia, que no puede ser sino americana; desde nuestros propios problemas, que por ser problemas de hombres no deben sernos ni “propios” ni “particulares”, sino comunes a todos los hombres. Filosofar, a fin de cuentas, bajo la expectativa de que nuestras respuestas son y serán tan valederas como las que se han planteado otros hombres a partir de sus circunstancias. Así, para Zea, no se trata de buscar una verdad americana ni de afirmar una identidad que nos sea propia (lo que en buena medida encontramos en la propuesta salazariana), sino una verdad válida para todos los hombres, una verdad a secas y por 68 Revisitando la discusión entre Augusto Salazar Bondy y Leopoldo Zea Revista de Filosofía tanto universal; hacer filosofía, pues lo americano –si ello tiene alguna importancia– vendrá por añadidura (Zea 1996, p. 44). 3. Desplazamientos categoriales Concluiré refiriéndome al que probablemente sea el punto más controversial –y por ello más sugestivo e interesante– de la discusión de Salazar Bondy y Zea. Conforme hemos podido observar, las posturas de los autores difieren en puntos sustantivos. Nos detuvimos, en primer lugar, en las distintas interpretaciones adoptadas respecto del estatuto de lo que pudiera llamarse filosofía latinoamericana: si es ella una labor remontable a una tradición o si bien es un proyecto prospectivo por realizar. En segunda instancia, en sus posicionamientos respecto del rol que le cabría al ejercicio de la filosofía en el subcontinente en la consolidación o afirmación de una identidad propia y particular, o si más bien ha de conducir sus esfuerzos a una incorporación en la presunta universalidad de la cultura y el pensar occidentales. En tales diferendos procurábamos mostrar, además, que revestía diversas formas la tensión entre el ejercicio filosófico académico normalizado y el compromiso político-intelectual, apuntando este último hacia distintos objetivos. Corresponde ahora señalar que los diferendos registrados se explican y al mismo tiempo se profundizan, al considerar que la respuesta de Zea comporta un desplazamiento de las categorías en que se monta el diagnóstico de Salazar Bondy sobre la historia y la situación del filosofar hispanoamericano hacia fines de la década del 60; a saber, peculiaridad, autenticidad y originalidad 13. Esto, que si bien es propio del trabajo filosófico –y nos permite comprender las reelaboraciones que hacen los autores sobre los postulados de la tradición, al modo de lecturas y deslecturas, propias del interpretar– en un extremo podría ser ponderado como renuncia o elusión de la discusión; pues nada nos asegura que, a fin de cuentas, cada quien se esté refiriendo a alguna cosa distinta, o que en el fondo estemos concurriendo a un “diálogo de sordos”. 13 Estos desplazamientos categoriales –como los hemos querido llamar– están registrados en la bibliografía que ha abordado la polémica. Adriana Arpini, por ejemplo, sostiene que “las cuestiones planteadas por Augusto Salazar Bondy no resultan cabalmente asumidas por Leopoldo Zea”, por cuanto su respuesta parte de supuestos distintos que los del peruano, y de interpretaciones diversas respecto de las categorías centrales de éste (Arpini 2003, p. 67). Helio Gallardo es aun más severo, pues afirma: “En verdad, Zea no asume el cuestionario de Salazar Bondy”; es decir, no da respuesta a las preguntas por él planteadas; o en caso de hacerlo, sus contestaciones suponen desplazamientos tales que difícilmente son reconocibles como réplicas a las preguntas del peruano (Gallardo 1974, p. 195, nota). Dante Ramaglia matiza en alguna medida el asunto, al sostener que no obstante lo mencionado, Salazar Bondy y Zea coinciden en la consideración de la dependencia como fenómeno que interpela a la filosofía latinoamericana, aun cuando difieren, nuevamente, respecto de la importancia de su superación para la conformación de la misma (Ramaglia 2009, p. 387). 69 Revista de Filosofía Cristóbal Friz Echeverría Salazar Bondy procura definir con bastante exactitud los criterios de su evaluación. Afirma que una filosofía es original en cuanto aporta ideas y planteos nuevos, distinguibles como creaciones, como “construcciones conceptuales inéditas de valor reconocido”. Por su parte, sostiene que un producto filosófico es auténtico (o genuino) en cuanto se da propiamente como tal y no como algo falseado o mixtificado. Finalmente, por peculiaridad entiende los rasgos histórico-culturales diferenciales de un producto filosófico, al modo de un tono local o personal, que como tal no implica ni originalidad ni autenticidad (Salazar Bondy 1996, p. 72). Para el peruano, es evidente que el pensamiento hispanoamericano cumple el criterio de peculiaridad, dado que son reconocibles algunas modalidades presentes en nuestra tradición intelectual, que la distinguen de otras. Sin embargo –y he aquí el punto para él decisivo– no es así respecto de la originalidad y la autenticidad. Estos últimos criterios, que constituyen la base de su evaluación negativa de nuestra filosofía, son fuertemente normativos y se hallan íntimamente vinculados, a la vez que se encuentran comprometidos con supuestos fuertes, como las directrices de la filosofía occidental contemporánea de corte académico, y su propia interpretación de las relaciones entre cultura y filosofía. Para Salazar Bondy, un producto es auténtica (o genuinamente) filosófico en cuanto difiere de uno que no lo es, lo que supone una circularidad en la argumentación, cuando no una tautología. En efecto señala: “la filosofía de Kant es genuina y (…) un discurso espiritualista es seudofilosofía” (Ídem.). La aclaración, como vemos, no hace sino oscurecer el asunto, pues sanciona –en base al presuntamente indiscutible carácter filosófico de la obra kantiana, es decir, mediante la apelación a una autoridad consagrada por la filosofía académica– la separación entre auténtica filosofía y una que no lo es. Junto con ello, su concepto de originalidad parece comprometido, al menos parcialmente, con la noción romántica de genio, según la cual solo a algunos insignes pensadores les estaría dado realizar aportes verdaderos, novedosos y reconocibles como tales. Pero solo parcialmente –conviene matizar–, pues tal capacidad radicaría asimismo en que los autores se encuentren adecuadamente asentados en comunidades históricas maduras, autónomas, dueñas de sí, con una identidad consolidada. Según hemos apuntado en momentos anteriores, para el peruano la filosofía hispanoamericana es inauténtica –entiéndase, filosofía solo en apariencia– a causa de la inautenticidad de nuestra cultura, catalogada por el autor, a causa de la estructural situación de subdesarrollo y dependencia social y económica, como una “cultura de dominación”. Ante esta situación defectiva –medida al trasluz de la supuesta autenticidad cultural de los países industriales avanzados–, el pensador local, según Salazar Bondy, construye una imagen ilusoria de sí y de su comunidad, la que no le permite ser original; es decir, expresar con categorías novedosas su propia y peculiar visión del mundo y de la historia. Sin embargo, y como veíamos, para Salazar Bondy nuestra seudofilosofía puede llegar a ser filosofía propiamente tal –auténtica y original–, en la medida en que, comprometiéndose con la superación del estado deprimido de nuestra cultura, opte, cual “mensajera del alba”, por la liberación. 70 Revisitando la discusión entre Augusto Salazar Bondy y Leopoldo Zea Revista de Filosofía Pues bien, cuando sostengo que en La filosofía americana como filosofía sin más Zea lleva a cabo una serie de desplazamientos categoriales, me refiero puntualmente al modo en que resignifica las nociones salazarianas de peculiaridad, autenticidad y originalidad, y sus mutuas relaciones. Estas resignificaciones reposan, según lo visto, en la propia interpretación zeaniana de la filosofía en general, y específicamente de sus posibilidades en contextos de enunciación distintos a los de los países occidentales; vale decir, la filosofía como actividad en que tiene lugar una disputa por el reconocimiento de la condición antropológica de aquellos a quienes el despliegue de la modernidad occidental la había negado. Dicha interpretación supone, al mismo tiempo, un posicionamiento distinto que el del peruano, respecto del problema de si la filosofía latinoamericana es un proyecto o un repertorio que cuenta con una tradición que se debe rescatar, sobre la disyuntiva entre identidad y universalidad, y sobre el compromiso político que corresponde al filosofar en América Latina. Podemos constatar un primer desplazamiento importante en la resignificación llevada a cabo por Zea respecto de las relaciones entre los criterios del diagnóstico de Salazar. Como veíamos, para éste la peculiaridad significa un tono personal o local, que no comporta autenticidad u originalidad de un producto filosófico. Zea resignifica las nociones de peculiaridad, autenticidad y originalidad, al punto de hacerlas convergentes, y haciendo de dicha convergencia el pivote de su enfoque positivo de nuestra filosofía. Según ha destacado Helio Gallardo, Zea reenfoca el tema de la peculiaridad al entenderla, ya no como los rasgos histórico-culturales diferenciales de un producto filosófico, sino poniendo el énfasis en “lo ‘peculiar’ que resulta preguntar por la posibilidad de nuestra filosofía” (Gallardo 1974, p. 194). Una vez obrado este desplazamiento, la peculiaridad deja de ser, como en Salazar Bondy, un aspecto defectivo, por cuanto en sus formas de expresión discursiva nuestros pensadores han tratado de afirmar, a su modo y estilo propios, su condición como sujetos pensantes y, por lo tanto, nuestra condición humana. Así, a contrapelo de la perspectiva del peruano, es esta peculiaridad la que funda la autenticidad y originalidad del filosofar, en la medida en que cada pensador, cada comunidad, encuentra sus modos discursivos de expresar el mundo y expresarse a sí mismo. Ya hemos destacado que el cultivo de la historia de las ideas tiene para Zea, entre otros cometidos, registrar positivamente lo propio de nuestra filosofía en su decurso histórico (lo que Salazar Bondy denomina peculiaridad), desprendiéndose de parámetros impuestos externamente, como los de la filosofía europeo-occidental de corte académico. Dicho campo disciplinar, según Zea, ha de mostrar que nuestro pensamiento sí ha sido, a su modo propio, auténtica filosofía –y no una seudofilosofía, una mixtificación–, en cuanto ha sido el intento del hombre americano por responder a los problemas concretos de su circunstancia histórica. Aquí, como ha señalado Adriana Arpini, autenticidad e inautenticidad dejan de hacer relación al vínculo entre un producto filosófico y las bases materiales de una sociedad, para apuntar más bien a la afirmación o negación de lo humano. En esta nueva perspectiva, inauténtica es, para Zea, una filosofía que adopta una idea de hombre que 71 Revista de Filosofía Cristóbal Friz Echeverría niega a los hombres concretos. Esto le permite afirmar que parte de la filosofía occidental misma ha sido inauténtica, y que la conquista de la autenticidad de nuestra filosofía no depende de una superación del subdesarrollo, sino de una toma de conciencia del propio estatuto antropológico (Arpini 2003, p. 66). Reenmarcada así la cuestión, la filosofía americana ha sido y es, al tiempo que peculiar, auténtica y original. He aquí un nuevo desplazamiento categorial, pues la originalidad ya no radica, como en Salazar Bondy, en la posibilidad de decir algo inédito, sino en la capacidad de incorporar la tradición para resolver nuestros problemas, importando poco si las “herramientas” conceptuales han sido tomadas de Europa o de otro lugar. Esto reposa en el supuesto de que toda asimilación implica un cambio, una traducción, una adopción de lo ajeno –que por ser humano no nos puede ser extraño– a las propias circunstancias, por lo que pierde sentido la disyunción entre copia y originalidad14. Zea postula, en efecto, una “autenticidad de la asimilación”, en la que se juegan la autenticidad y la originalidad de nuestro filosofar (Zea 1996, pp. 34, 39). Es por ello que afirma: “Auténtica ha tenido que ser la filosofía que ha puesto en duda la validez de esta interrogación deshumanizante [de una parte de la filosofía occidental que ha cuestionado nuestra condición antropológica], como la que se ha esforzado por demostrar nuestra humanidad, la que se pregunta por la posibilidad de la existencia de una cultura igualmente nuestra y de una filosofía originada entre nosotros” (Ibíd., p. 115). Para el mexicano, en consecuencia, la originalidad de toda filosofía radica en partir de la propia circunstancia para trascenderla hacia lo plenamente humano, común y universal; partir de lo que se ha sido y de lo que se es para arribar a lo que se quiere ser. En este sentido, a pesar de todos los diferendos, Zea estima que el libro de Salazar Bondy –al igual que todos los que se han preguntado por la filosofía latinoamericana– es expresión de nuestro filosofar, de su peculiaridad, autenticidad y originalidad; del problema, en definitiva, en que nos coloca nuestra condición como subordinados respecto de la cultura occidental (Ibíd., p. 88). Referencias bibliográficas Alberdi, Juan Bautista (1993), “Ideas para un curso de filosofía contemporánea” [1842]. Zea, Leopoldo (comp.), Fuentes de la cultura latinoamericana I. México: FCE; pp. 145-151. Arpini, Adriana (2003), “La polémica entre Augusto Salazar Bondy y Leopoldo Zea. Una revisión crítica del historicismo en América Latina”. Arpini, Adriana 14 72 Podríamos agregar que no solo pierde sentido la disyunción, sino que para Zea la misma no existe. Así parece concebirlo Helio Gallardo, para quien “[e]n el contexto zeaniano, (…) la pregunta por la originalidad del filosofar carece de sentido, no es una pregunta”. (Gallardo 1974, p. 195, nota) Revisitando la discusión entre Augusto Salazar Bondy y Leopoldo Zea Revista de Filosofía (comp.), Otros discursos. Estudios de Historia de las Ideas Latinoamericanas. Mendoza: UNCuyo; pp. 45-70. __________ (2016), “La polémica entre Augusto Salazar Bondy y Leopoldo Zea. Una bisagra en la historia de las ideas latinoamericanas”. Filosofía, crítica y compromiso en Augusto Salazar Bondy. Lima: Fondo Editorial del Congreso del Perú; pp. 179-203. Cerutti Guldberg, Horacio (2006) [1983], “La polémica entre Augusto Salazar Bondy y Leopoldo Zea”. Filosofía de la liberación latinoamericana. México: FCE; pp. 263-273. Devés, Eduardo (2003-2004), El pensamiento latinoamericano en el siglo XX, 3 tomos. Buenos Aires/Santiago: Biblos/DIBAM. Fornet-Betancourt, Raúl (2001), Transformación intercultural de la filosofía. Bilbao: Desclée de Brouwer. __________ (2004), Crítica intercultural de la filosofía actual. Madrid: Trotta. Gallardo, Helio (1974), “El pensar en América Latina. Introducción al problema de la conformación de nuestra conciencia: A. Salazar Bondy y L. Zea”, Revista de Filosofía de la Universidad de Costa Rica, vol. XII, (Nº 35): 183-210. García De La Huerta, Marcos (1999), Reflexiones americanas. Ensayos de IntraHistoria. Santiago: LOM Ediciones. __________ (2014), “Filosofía latinoamericana y ‘diálogo intercultural’”, en García de la Huerta, Marcos y Carlos Ruiz Schneider, Construcción de identidad, creación de sentido. Santiago: Universitaria; pp. 77-95. Giannini, Humberto (2004) [1987], La “reflexión cotidiana”. Hacia una arqueología de la experiencia. Santiago: Universitaria. Molina, Enrique (1953), La filosofía en Chile en la primera mitad del siglo XX. Notas y recuerdos. Santiago: Nascimento. Olmedo, Francisco (1972), “El problema de la filosofía latinoamericana”. El Guacamayo y la Serpiente Nº 5. Ossandón, Carlos (1984), “El concepto de ‘normalidad filosófica’ en Francisco Romero”. Hacia una filosofía latinoamericana. Santiago: Nuestra América; pp. 67-91. Ramaglia, Dante (2007), “Alejandro Korn y la ‘normalización’ de la filosofía”. Jalif, Clara (ed.), Argentina, entre el optimismo y el desencanto. Mendoza: UNCuyo/ Qellqasqa; pp. 63-83. __________ (2009), “La cuestión de la filosofía latinoamericana”. Dussel, Enrique, Eduardo Mendieta, Carmen Bohórquez, ed., El pensamiento filosófico latinoamericano, del Caribe y “latino” (1300-2000): historia, corrientes, temas y filósofos. México: Siglo XXI/CREFAL; pp. 377-398. Roig, Arturo Andrés (2009) [1981], Teoría y crítica del pensamiento latinoamericano. Buenos Aires: Una Ventana. Romero, Francisco (1952), Sobre la filosofía en América. Buenos Aires: Raigal. 73 Revista de Filosofía Cristóbal Friz Echeverría Salazar Bondy, Augusto (1996) [1968], ¿Existe una filosofía de nuestra América? Decimotercera edición. México D.F.: Siglo XXI. Sánchez, Cecilia (1992), Una disciplina de la distancia. Institucionalización universitaria de los estudios filosóficos en Chile. Santiago: CERC/CESOC. Santos, Manuel Ignacio (1977), “La filosofía en la actual coyuntura histórica Latinoamericana. Notas críticas sobre la filosofía latinoamericana como filosofía de la liberación”. Pucará, Nº 2: 13-46. Santos Herceg, José (2010), Conflicto de representaciones. América Latina como lugar para la filosofía. Santiago: FCE. __________ (2012), “¿Qué se dice cuando se dice filosofía latinoamericana?”. Revista de Filosofía, vol. 68: 65-78. Zea, Leopoldo (1991), La filosofía como compromiso de liberación. Caracas: Biblioteca Ayacucho. __________ (1996) [1969], La filosofía americana como filosofía sin más. Decimosexta edición. México D.F.: Siglo XXI. 74
https://openalex.org/W3135521094	https://cadernos.abralin.org/index.php/cadernos/article/download/321/374	Portuguese	null	Tempo verbal, modo verbal e a concepção da realidade em português	Cadernos de Linguística	2,021	cc-by	4,938	cadernos.abralin.org ENSAIO TEÓRICO RESUMO Sempre que pensamos ou falamos sobre situações ou eventos, atribuímos-lhes um estatuto na realidade. Assim sendo, uma questão crucial é: como conceptualizamos a realidade? A resposta para essa questão é essencial para a compreensão do uso que fazemos do tempo verbal e do modo verbal, uma vez que são recursos gramaticalizados que nos permitem localizar a situação conceptualizada numa área específica da (ir)realidade, ou seja: (i) no plano da realidade passada ou imediata, se a situação for conceptualizada como factual; (ii) no plano da realidade não estabelecida, se a existência da situação não for aceita; e (iii) no plano da realidade projetada ou potencial, se a realização de uma situação futura for conceptualizada, respectivamente, como previsível ou apenas possível. Este ensaio, que se baseia teoricamente na Gramática Cognitiva (Langacker, 1991, 1999 e 2003), tem por proposta abordar a relação entre o tempo verbal, modo verbal e a conceptualização da realidade em português. Para tanto, são apresentados e discutidos exemplos de diferentes situações noticiadas em informativos on-line e extraídas da página de resultados da ferramenta de busca Google. ABSTRACT Whenever we think or talk about situations or events, we give them a status in reality. So, a crucial question is: how do we conceptualize reality? The answer to this question is essential for understanding how we use the verbal tense and the verbal mood, since they are TEMPO VERBAL, MODO VERBAL E A CONCEPÇÃO DA REALIDADE EM PORTUGUÊS Jussara ABRAÇADO Universidade Federal Fluminense (UFF) OPEN EDITORES – Miguel Oliveira, Jr. (UFAL) – René Almeida (UFS) ABRAÇADO, Jussara (2021). Tempo verbal, modo verbal e a concepção da realidade em português. Cadernos de Linguística, v. 2, n. 1, p. 01-17. COMO CITAR 1 DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 cadernos.abralin.org grammatical resources that allow us to locate the conceptualized situation in a specific area of (ir) reality, that is: (i) at the level of past reality or immediate reality, if the situation is conceptualized as factual; (ii) at the level of reality not established, if the existence of the situation is not accepted; and (iii) at the level of projected reality or potential reality, if the occurrence of a future situation is conceptualized, respectively, as predictable or only possible. This essay, which is theoretically based on Cognitive Grammar (Langacker, 1991, 1999, 2003), aims to approach the relationship between the verbal tense, the verbal mood and the conceptualization of reality in Portuguese. For that, examples of different situations extracted from online news, obtained in the Google search tool, are presented and discussed. PALAVRAS-CHAVE Tempo Verbal; Modo Verbal; Concepção de Realidade; Português; Gramática Cognitiva. Tempo Verbal; Modo Verbal; Concepção de Realidade; Português; Gramática Cognitiva. DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 INTRODUÇÃO1 Nas diferentes abordagens sobre tempo linguístico, há consenso em torno de sua natureza eminentemente dêitica e de que as situações são ordenadas a partir do presente, mais específicamente, do evento do ato de fala ou do momento da enunciação, conforme descreve Fonseca (1996, p. 44; grifos da autora): Nas diferentes abordagens sobre tempo linguístico, há consenso em torno de sua natureza eminentemente dêitica e de que as situações são ordenadas a partir do presente, mais específicamente, do evento do ato de fala ou do momento da enunciação, conforme descreve Fonseca (1996, p. 44; grifos da autora): O tempo, tal como o concebemos através da linguagem, é de natureza dêitica: presente, passado e futuro não são noções absolutas, são relativas ao momento de enunciação. A interpretação semântica de advérbios temporais como hoje, ontem, amanhã, ou de tempos verbais como estou, estive, estarei, pressupõe uma prévia identificação pragmática do momento de enunciação (...). No que se refere ao modo verbal, também é consensual relacionar seu uso à avaliação que o falante faz sobre a situação a que se refere, considerando-a real, irreal, possível ou não. Neste texto, a abordagem do tempo e do modo verbais não se distancia das demais nos aspectos destacados e tidos como consensuais. Entretanto, na esteira de Langacker (1991, 2002 e 2003), temos como proposta relacionar o emprego do tempo e do modo à concepção de realidade e, para além disso, demonstrar como tal relação se assenta em português, tendo como base diferentes situações ocorridas, ocorrentes e tidas como sujeitas ou não a ocorrem, noticiadas em informativos on-line e extraídas da página de resultados da ferramenta de busca Google com o filtro ‘notícias’ ativado. Para tanto, começamos por apresentar a proposta de Langacker em que nos baseamos. cadernos.abralin.org KEYWORDS Verbal Tense; Verbal Mood; Conception of Reality; Portuguese; Cognitive Grammar. Cognitive Grammar. DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 2 2 1 Este ensaio constitui uma versão ampliada de conferência proferida no evento ABRALIN ao Vivo. 1. TEMPO, MODO E CONCEPÇÃO DE REALIDADE Tendo como fundamento o processo de conceptualização, que corresponde à apreensão, em termos cognitivos, de uma dada entidade ou situação (objeto de conceptualização) por um conceptualizador (sujeito de conceptualização), Langacker (1991) chama a atenção para a forma como conceptualizamos situações que ocorrem ou que estão sujeitas ou não a ocorrerem no mundo. De acordo com o autor, sempre que pensamos ou falamos sobre situações, atribuímos-lhes um estatuto relativo à realidade, situando a situação objeto de conceptualização dentro ou fora da realidade. E, embora se possa entender a realidade como uma entidade objetiva, que independe das pessoas, um dos fundamentos da Linguística Cognitiva é o de que o conhecimento que possuímos da realidade é DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 3 3 cadernos.abralin.org fragmentado, isto é, sabemos que conhecemos certas situações, mas sabemos também que desconhecemos ou ignoramos outras e que, portanto, nosso conhecimento sobre determinados fatos pode ser incompleto ou até mesmo falso. De acordo com Langacker (1991), há perspectivas diferentes e comuns de realidade. Há pessoas com perspectivas de mundo e da realidade diferentes da nossa. No entanto, levando-se em conta que parte importante do nosso conhecimento do mundo é comum à espécie humana e é cultural, podemos supor que haja um grau considerável de concepções semelhantes da realidade. Ademais, conforme explica o autor, nosso conhecimento da realidade não se restringe à apreensão direta das situações: inclui a capacidade que os humanos têm de avaliar as situações em relação à realidade. Em outras palavras, não é com base na real evolução do mundo que uma dada situação é tida como pertencente ou não à realidade. Cabe ao conceptualizador conhecer, ou não, aceitar, ou não, tal situação como parte da sequência evolutiva, o que se dá a partir de uma avaliação epistêmica que se fundamenta no reconhecimento que os humanos têm da natureza dinâmica da realidade. Sob esse viés, o autor postula uma concepção mais abstrata e inclusiva da realidade, dado que ao processo de conceptualização são acrescentados aspectos relacionados ao conhecimento daquilo que não ocorreu, daquilo que poderá ocorrer e daquilo que poderia ter ou não ocorrido. Para Langacker (1991), há então, pelo menos, dois níveis e duas noções de realidade a serem considerados: o nível em que as situações são diretamente apreendidas e o nível das proposições que correspondem, respectivamente, às noções de realidade básica e de realidade elaborada. Para ilustrar esses níveis e noções, tomemos como exemplos manchetes de duas notícias que tratam de uma mesma situação: (1) (2) Disponível em: https://jovempan.com.br/programas/jornal-da-manha/imunidade-rebanho-covid-19-chegar- vacina.html (Acesso em 26/09/2020). DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 4 a percepção do conceptualizador de que a realidade como ele a conhece não é exaustiva do mundo e de sua história evolutiva. O núcleo da realidade conhecida é, portanto, rodeado por uma região muito maior de realidade desconhecida. Incluídas nesta região estão situações de pelo menos dois tipos: aquelas cuja realidade C suspeita ou contempla, mas não aceita como tendo sido estabelecida; e aquelas das quais ele é totalmente ignorante. Observe que a realidade desconhecida é parte da irrealidade (...) (Langacker, 1991, p. 243.)2 2 the conceptualizer's realization that reality as he knows it is not exhaustive of the world and its evolutionary history. The core of known reality is thus surrounded by a much larger region of unknown reality Included in this region are situations of at least two sorts: those whose reality C suspects or contemplates but does not accept as having been established; and those of which he is entirely ignorant. Observe that unknown reality is part of irreality (…). cadernos.abralin.org Em (1) e (2) vemos que, diante de uma mesma situação, a pandemia provocada pela Covid-19, autoridades de saúde se posicionam diferentemente em relação à ‘imunidade de rebanho’, que, segundo especialistas, arrefeceria o contágio pelo referido vírus. Dito de outra forma, temos uma realidade básica, que diz respeito à situação apreendida, e a realidade elaborada, que se evidencia nas proposições, ou seja, nas manchetes dos informativos on-line, que incorporam, conforme destacamos, o conhecimento de mundo e a capacidade que os humanos possuem de avaliar as situações em relação à realidade. Em (1), o uso do presente do indicativo precedido de um advérbio de tempo (‘já temos’) situa a imunidade de rebanho no plano da realidade factual; em (2), por outro lado, o emprego do modal ‘dever’ (‘deve chegar’) sinaliza que a imunidade de rebanho ainda não foi atingida, isto é, não pertence à realidade factual, estando, portanto, no plano da irrealidade. Como podemos perceber, a concepção de realidade é relativa. Conceptualizadores diferentes podem ter concepções diferentes de uma situação, conforme ilustram (1) e (2). Assim sendo, no âmbito da Gramática Cognitiva, entende-se que um dado enunciado corresponde à instanciação de uma perspectivação e não, à ‘realidade’ em si, e é a partir desse entendimento que o enunciado em questão é analisado. Para abordar as relações entre tempo e concepção de realidade, Langacker (1991) recorre a modelos estruturados do mundo (structured world models), como o que se segue, que ilustra a concepção da realidade em seu nível mais básico. Figura 1. Modelo epistêmico básico. Fonte: LANGACKER (1991). Figura 1. Modelo epistêmico básico. Fonte: LANGACKER (1991). No modelo epistêmico básico, visualizamos um cilindro que se divide em realidade conhecida, que corresponde ao passado, e em realidade imediata, que diz respeito ao presente e ao ponto onde se encontra o conceptualizador (C). É do ponto de vista da realidade imediata que o conceptualizador (falante ou escrevente) apreende a situação a que se refere. Tudo mais, como podemos constatar, está no plano da irrealidade. Logo, sob DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 5 cadernos.abralin.org a ótica do modelo epistêmico básico, em (2), a imunidade de rebanho é situada fora do plano da realidade. Contudo, como podemos perceber, essa bipartição é por demais simplista. Não abarca satisfatoriamente o uso do modal em (2), em que a imunidade de rebanho é referida como uma possibilidade futura, além de outros vieses relacionados a nossa concepção de realidade. Langacker (1991) propõe então outros três modelos que incorporam aspectos não capturados pelo modelo epistêmico básico. No Modelo epistêmico elaborado, apresentado a seguir, o núcleo da realidade conhecida é envolvido por uma faixa que abarca situações cuja existência o conceptualizador não aceita como tendo sido estabelecida e ainda aquelas situações que, devido ao seu conhecimento limitado e incompleto da realidade, o conceptualizador desconhece. Figura 2. Modelo epistêmico elaborado adaptado. Adaptação de LANGACKER (1991). Figura 2. Modelo epistêmico elaborado adaptado. Adaptação de LANGACKER (1991). A faixa, que apresentamos aqui com o rótulo ‘realidade não estabelecida’, é denominada ‘realidade desconhecida’ por Langacker (1991). No entanto, em sua explicação sobre o modelo em questão, o autor alega que, em relação ao modelo anterior, o modelo epistêmico elaborado acrescenta: 2 the conceptualizer's realization that reality as he knows it is not exhaustive of the world and its evolutionary history. The core of known reality is thus surrounded by a much larger region of unknown reality Included in this region are situations of at least two sorts: those whose reality C suspects or contemplates but does not accept as having been established; and those of which he is entirely ignorant. Observe that unknown reality is part of irreality (…). DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 6 cadernos.abralin.org Com base em tal explicação, concluímos que, também no caso das situações que o conceptualizador desconhece, verifica-se a não aceitação da existência dessas situações como estabelecida, ou seja, nossa ignorância acerca de situações que possam existir implica não aceitarmos como estabelecida a existência de tais situações. Daí optarmos pelo rótulo ‘realidade não estabelecida’, que entendemos capturar melhor os dois tipos de realidade mencionados por Langacker (1991), em detrimento do rótulo ‘realidade desconhecida’ empregado por ele. No terceiro modelo, o modelo da linha do tempo, o autor incorpora duas noções adicionais: a de tempo (t), ou seja, o eixo ao longo do qual a realidade evolui, e a de ground (G), que se refere ao ato de fala e suas circunstâncias. Figura 6. Modelo da linha do tempo. Fonte: LANGACKER (1991, p. 244). Figura 6. Modelo da linha do tempo. Fonte: LANGACKER (1991, p. 244). No modelo da linha do tempo, evidencia-se que o locus de um evento de fala instaura- se na realidade imediata, onde se encontram falante e ouvinte(s). Ademais, conforme ilustrado neste modelo, Um ato de fala não é pontual. Tem uma duração temporal breve, como indica a linha sinuosa. O tempo é segmentável em passado, presente e futuro e, uma vez que a realidade compreende o passado e o presente, e a realidade imediata constitui o presente, a realidade não imediata corresponde ao passado. (SOUZA; ABRAÇADO; 2020, p. 29) Em seu quarto modelo, denominado modelo evolutivo dinâmico, Langacker (1991) busca dar conta de situações como a exemplificada em (2), com o emprego do modal na manchete ‘Imunidade de rebanho contra Covid-19 só deve chegar com a vacina’. Tais situações cuja ocorrência consideramos possível, somadas àquelas situações que podemos prever, são capturadas pelo modelo em questão. De acordo com Langacker, há situações predispostas a ocorrerem sempre que surgirem condições apropriadas para tal e, a menos que uma DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 7 cadernos.abralin.org certa quantidade de energia seja gasta para se opor à tendência para que ocorram, essas situações de fato ocorrerão. certa quantidade de energia seja gasta para se opor à tendência para que ocorram, essas situações de fato ocorrerão. Figura 4. Modelo evolutivo dinâmico. Fonte: LANGACKER (1991, p. 277). Figura 4. Modelo evolutivo dinâmico. Fonte: LANGACKER (1991, p. 277). O modelo evolutivo dinâmico, por conseguinte, refere-se a características da realidade que tendem a impulsioná-la para certos caminhos no futuro em detrimento de outros. O impulso evolutivo da realidade é representado no modelo pela seta tracejada. Sob essa ótica, as situações cuja realização consideramos apenas possível pertencem à realidade potencial. As situações que, com maior ou menor grau de segurança, podemos prever com base em nosso conhecimento do mundo e em experiências passadas e presentes pertencem à realidade projetada (SOARES DA SILVA, inédito). Vejamos, no exemplo a seguir, notícia publicada meses antes da Copa do Mundo de Futebol de 2018, em que a seleção brasileira é referida como uma das favoritas ao título por comentaristas esportivos e divide com a seleção alemã a liderança nas casas de apostas. (3) cadernos.abralin.org Em (3), vemos que o favoritismo da seleção brasileira - que não se confirma, uma vez que é a seleção francesa que se consagra campeã em final disputada com a seleção da Croácia – tem como respaldo seu desempenho em amistosos com outras seleções, com destaque para sua vitória no jogo contra a seleção alemã, campeã da copa anterior. Entretanto, como não poderia deixar de ser, como pano de fundo de tal favoritismo também está a tradição de vitórias e de bom futebol da seleção do Brasil. Semelhantemente, em nossa referência a um evento futuro, com o respaldo do nosso conhecimento de mundo e de acontecimentos passados e/ou recentes - que correspondem ao impulso evolutivo da realidade, representado pela seta tracejada no modelo evolutivo dinâmico -, atribuímos um estatuto epistêmico ao evento em questão, situando-o na realidade projetada, se podemos prever sua realização, ou na realidade potencial, se julgamos sua realização apenas possível, como ilustram, respectivamente, (4) com ‘vai abrigar’ e (5) com ‘podem estar’: (4) Realidade projetada Disponível em: http://www.gaz.com.br/conteudos/regional/2020/09/25/171105- restaurada_casa_de_cultura_de_vera_cruz_vai_abrigar_museu.html.php (Acesso em 27/09/2020). (5) Realidade potencial Disponível em: https://educacao.estadao.com.br/noticias/geral,clima-e-crise-de-saude-podem-estar-na- redacao,70003516479 (Acesso em 06/12/2020). (4) Realidade projetada (4) Realidade projetada Disponível em: http://www.gaz.com.br/conteudos/regional/2020/09/25/171105- restaurada_casa_de_cultura_de_vera_cruz_vai_abrigar_museu.html.php (Acesso em 27/09/2020). Disponível em: https://www.lance.com.br/odds-copa-do-mundo/saiba-quem-sao-favoritos-nas-casas-apostas- para-vencer-copa.html (Acesso em 27/09/2020). 8 DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 (5) Realidade potencial Disponível em: https://educacao.estadao.com.br/noticias/geral,clima-e-crise-de-saude-podem-estar-na- redacao,70003516479 (Acesso em 06/12/2020). Disponível em: https://educacao.estadao.com.br/noticias/geral,clima-e-crise-de-saude-podem-estar-na- redacao,70003516479 (Acesso em 06/12/2020). Vemos, portanto, que exprimimos nossa avaliação em relação ao estatuto de realidade de uma situação servindo-nos de expressões adverbiais, de expressões modais e, é claro, do modo e do tempo gramaticalizados nas formas verbais, alvos de nosso interesse. Vejamos, então, as relações que se estabelecem em português entre tempo verbal, modo verbal e concepção de realidade. 9 DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 cadernos.abralin.org cadernos.abralin.org (7) Realidade passada (pretérito imperfeito): ‘... quando fazia live...’ Disponível em: https://capitalnews.com.br/colunistas/marco-eusebio/candidato-e-roubado-quando-fazia-live-na- fronteira-de-ms-com-o-paraguai-video/348231 (Acesso em: 19/10/2020). Disponível em: https://capitalnews.com.br/colunistas/marco-eusebio/candidato-e-roubado-quando-fazia-live-na- fronteira-de-ms-com-o-paraguai-video/348231 (Acesso em: 19/10/2020). (8) Realidade passada (pretérito mais que perfeito): ‘... fizera o mesmo percurso...’ Disponível em: https://blogs.oglobo.globo.com/lauro-jardim/post/xp-ja-investiu-r-1-bilhao-com-para-reter-agentes autonomos.html (Acesso em: 04/10/2020). (9) Realidade passada (presente simples): ‘Justiça inaugura...’ Disponível em: https://g1.globo.com/mg/zona-da-mata/noticia/2020/10/16/justica-inaugura-uso-de-plataforma- para-otimizar-servicos-em-comarcas-da-zona-da-mata-e-vertentes.ghtml (Acesso em: 16/10/2020). (10) Realidade imediata (presente simples): ‘Google quer...’ 2. TEMPO VERBAL, MODO VERBAL E CONCEPÇÃO DE REALIDADE EM PORTUGUÊS Considerando os modelos cognitivos postulados por Langacker, quando pensamos ou falamos sobre uma dada situação, atribuímos-lhe um estatuto epistêmico, caracterizando- a como factual, previsível, possível ou não. Dessa caracterização resulta a localização da situação em um dos planos ilustrados no Quadro 1: Quadro 1. Relação entre tempo, modo e concepção de realidade em português. adro 1. Relação entre tempo, modo e concepção de realidade em português. De acordo com o Quadro 1, se a situação é situada no plano da realidade passada ou da realidade imediata e caracterizada como factual, em sua referência empregamos o modo indicativo e as formas verbais, simples ou compostas, referentes ao passado (pretérito perfeito, pretérito imperfeito e pretérito mais que perfeito) e ao presente (presente simples e presente progressivo). Cumpre ressaltar que o presente do indicativo também é utilizado para referência a eventos passados e futuros. No primeiro caso, os eventos referidos são situados na realidade passada e factual e, em geral, excetuando-se os casos abarcados pelo presente histórico, pertencem ao passado recente, como ilustrado em (9); no segundo caso (que ilustraremos mais adiante), os eventos, que tendem a ocorrer num futuro próximo e a carrear um grau de certeza epistêmica mais elevado do que as demais formas de referência ao futuro3, situam-se na realidade projetada. (6) Realidade passada (pretérito perfeito): ‘Chegou a hora...’ (6) Realidade passada (pretérito perfeito): ‘Chegou a hora...’ Disponível em: https://www.portalveneza.com.br/chegou-a-hora-de-tirar-a-ideia-de-startup-do-papel/ (Acesso em: 04/10/2020). 3 Ver em Langacker (2002), Brisard (2002), Abraçado (2020) discussão sobre os usos do tempo presente sob a perspectiva da Gramática Cognitiva. DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 10 DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 (10) Realidade imediata (presente simples): ‘Google quer...’ Disponível em: https://canaltech.com.br/internet/em-busca-da-busca-perfeita-google-refina-mecanismo-de- pesquisa-com-muita-ia-173120/ (Acesso em: 19/10/2020). Disponível em: https://canaltech.com.br/internet/em-busca-da-busca-perfeita-google-refina-mecanismo-de- pesquisa-com-muita-ia-173120/ (Acesso em: 19/10/2020). DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 11 11 11 No caso de situações passadas ou presentes que não são conceptualizadas como factuais, as seguintes combinações entre tempo e modo sinalizam que tal situação pertence ao plano da realidade não estabelecida: (i) futuro do pretérito, no modo indicativo; (ii) pretérito imperfeito, pretérito perfeito e pretérito mais que perfeito, no modo subjuntivo; e (iii) presente, no modo subjuntivo. (12) Realidade não estabelecida (futuro do pretérito do indicativo): ‘...cocaína que seguiria...’ (12) Realidade não estabelecida (futuro do pretérito do indicativo): ‘...cocaína que seguiria...’ Disponível em: https://www.enfoquems.com.br/pmr-apreende-quase-100-kg-de-cocaina-que-seguiria-para-sp/ (Acesso em: 19/10/2020). (13) Realidade não estabelecida (pretérito imperfeito do subjuntivo): ‘...que o cliente tivesse um plano...’ Disponível em: https://g1.globo.com/pr/norte-noroeste/noticia/2020/10/15/advogado-de-andre-do-rap-nega-que- cliente-tinha-plano-de-fuga-essa-historia-e-um-folclore.ghtml (Acesso em: 19/10/2020). cadernos.abralin.org (11) Realidade imediata (presente progressivo): ‘A confiança está voltando...’ Disponível em: https://opiniao.estadao.com.br/noticias/editorial-economico,a-confianca-esta-voltando-ao- comercio-paulista,70003467629 (Acesso em: 19/10/2020). (11) Realidade imediata (presente progressivo): ‘A confiança está voltando...’ (15) Realidade não estabelecida (pretérito mais que perfeito do subjuntivo): ‘Se tivesse visto...’ (15) Realidade não estabelecida (pretérito mais que perfeito do subjuntivo): ‘Se tivesse visto...’ DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 12 cadernos.abralin.org (14) Realidade não estabelecida (pretérito perfeito do subjuntivo): ‘...que essa conversa tenha acontecido’ Disponível em: https://politica.estadao.com.br/blogs/estadao-verifica/postagem-inventa-dialogo-entre-papa- francisco-e-bolsonaro-sobre-a-amazonia/ (Acesso em: 19/10/2020). Disponível em: https://colunadofla.com/2014/09/se-tivesse-visto-o-empurrao-o-arbitro-apitaria-e-nao-teria- gol/(Acesso em: 22/10/2020). (15) Realidade não estabelecida (presente do subjuntivo): ‘Sandra Annenberg nega que esteja...’ Disponível em: https://jmonline.com.br/novo/?paginas/colunas,134,201584 (Acesso em: 19/10/2020). (18) Realidade potencial (futuro do subjuntivo): ‘Quando tudo der...’ (18) Realidade potencial (futuro do subjuntivo): ‘Quando tudo der...’ Disponível em: https://jmonline.com.br/novo/?paginas/colunas,134,201584 (Acesso em: 19/10/2020). No que diz respeito à referência a situações futuras, o quadro é o seguinte: situamos uma dada situação futura na realidade projetada, se podemos prever sua realização, ou na realidade potencial, se julgamos sua realização apenas possível; ao situarmos uma situação no plano da realidade projetada, valemo-nos das formas verbais de futuro e, conforme já mencionamos, também do presente, no modo indicativo; quando situamos uma situação futura no plano da realidade potencial, empregamos o futuro do subjuntivo e também os verbos modais. DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 13 cadernos.abralin.org (16) Realidade projetada (futuro do presente do indicativo): ‘Calor chegará...’ Di í l htt // ib hi / l d /d t lh / ti i / l h f t l d d t Disponível em: https://www.ibahia.com/salvador/detalhe/noticia/calor-chegara-forte-em-salvador-durante-o-mes de-outubro-dizem-especialistas/ (Acesso em: 19/10/2020). (17) Realidade projetada (presente do indicativo): ‘...começa amanhã’. Disponível em: https://www.correiodopovo.com.br/not%C3%ADcias/rural/simp%C3%B3sio-online-de-fruticultura- come%C3%A7a-amanh%C3%A3-1.500961 (Acesso em: 19/10/2020). (20) ( ) Disponível em: https://jornaldoempreendedor.com.br/destaques/inspiracao/8-coisas-para-se-lembrar-quando- tudo-der-errado/ (Acesso em: 19/10/2020). Com base no exposto até então, podemos concluir que a localização temporal de uma dada situação está diretamente relacionada ao estatuto epistêmico que lhe é atribuído, o que envolve não somente a ocorrência da situação em si, mas também o conhecimento de mundo dos conceptualizadores, locutor e interlocutor(es), envolvidos no ato comunicativo. Por conseguinte, quando falamos de predicações temporalmente marcadas, estamos na verdade falando de predicações epistêmicas, dado o estatuto epistêmico da situação que carreiam. Faz-se também necessário ressaltar que, além dos tempos verbais que apontam para intervalos de tempo anteriores, simultâneos ou posteriores ao tempo do ato de fala e, por isso, caracterizados como dêiticos, há os tempos verbais anafóricos que se referem a localizações temporais construídas a partir de pontos de referência discursivamente instituídos, mas que, no entanto, preservam as mesmas relações de anterioridade, simultaneidade e posteridade dos tempos dêiticos (SOARES DA SILVA, inédito,). Em (19), a seguir, há dois pontos de referência discursivamente instituídos (1981 e 2019) e a forma verbal ‘contava’ refere-se anaforicamente ao time do Flamengo do ano de 1981. DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 14 cadernos.abralin.org (19) Disponível em: https://www.terra.com.br/esportes/flamengo/a-diferenca-do-flamengo-de-1981-para-o-vice-de- 2019-zico,769a68ebe9cd63dc2a36752ca8e427a4dlpiqmgk.html (19) Há ainda em (19) um outro aspecto a ser destacado. Enquanto a forma verbal ‘contava’, no pretérito imperfeito do indicativo, indica que o conceptualizador situa o time do Flamengo de 1981 no âmbito de uma realidade factual, a forma verbal ‘fosse’, no pretérito imperfeito do modo subjuntivo, alinhada com o advérbio de dúvida ‘talvez’, sinaliza que a condição de Zico ser o principal jogador do planeta, na ocasião referida, não é concebida pelo conceptualizador como uma situação estabelecida. Vemos, portanto, que também nas localizações temporais discursivamente construídas, a relação entre tempo, modo e concepção de realidade se mantém. Por fim, chamamos a atenção para usos bastante corriqueiros dos tempos e modos verbais, em que a localização temporal e os diferentes níveis de realidade são indicados por recursos outros que não os próprios tempos e modos gramaticalizados nas formas verbais. Além de corriqueiros, são diversos os usos em que isso pode ocorrer. A título de ilustração, primeiramente em relação à localização temporal, consideremos o emprego do pretérito perfeito em referência a um tempo passado precedente a outro tempo passado, conforme ocorre no exemplo a seguir abordado em trabalho anterior (ABRAÇADO 2020, p. 94): (20) Disponível em: https://diariodonordeste.verdesmares.com.br/editorias/verso/online/em-entrevista-fagner-resgata- amizade-e-parceria-musical-com-luiz-gonzaga-1.2130584 (Acesso em: 02/08/2019). Disponível em: https://maisfutebol.iol.pt/internacional/italia/ranieri-o-meu-trabalho-termina-assim-que-acabar-o- campeonato (Acesso em: 19/11/2020). Disponível em: https://maisfutebol.iol.pt/internacional/italia/ranieri-o-meu-trabalho-termina-assim-que-acabar-o campeonato (Acesso em: 19/11/2020). Disponível em: https://diariodonordeste.verdesmares.com.br/editorias/verso/online/em-entrevista-fagner-resgata amizade-e-parceria-musical-com-luiz-gonzaga-1.2130584 (Acesso em: 02/08/2019). Em (20), a sinalização de anterioridade e posterioridade temporal é feita pelos circunstanciais “antes”, “a princípio”, “quando” e “na época”. Situação semelhante se dá concernentemente aos diferentes níveis de realidade. Quando o futuro do subjuntivo, por exemplo, ocorre associado a um circunstancial de tempo, a um evento programado ou previsível, as situações então referidas são remetidas DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 15 cadernos.abralin.org cadernos.abralin.org à realidade projetada (e não, à realidade potencial), como se verifica em (21) com ‘quando acabar o campeonato’: (21) Disponível em: https://maisfutebol.iol.pt/internacional/italia/ranieri-o-meu-trabalho-termina-assim-que-acabar-o- campeonato (Acesso em: 19/11/2020). (21) 3. SUMARIANDO Sempre que pensamos ou nos referirmos a uma situação, atribuímos-lhe um estatuto de realidade e estabelecemos sua localização temporal (relativa ao ato de fala ou a algum ponto de referência discursivamente instituído). Exprimimos nossa avaliação, no que se refere ao estatuto de realidade de uma situação, servindo-nos de expressões adverbiais, de expressões modais, do modo e do tempo gramaticalizados nas formas verbais. Priorizando o modo e o tempo gramaticalizados nas formas do verbo, vimos que, em português, a relação tempo, modo e concepção de realidade se estabelece de tal forma que: (i) Se a situação é conceptualizada no plano da realidade passada ou da realidade imediata, em sua referência empregamos o modo indicativo e as formas verbais, simples ou compostas, referentes ao passado (pretérito perfeito, pretérito imperfeito e pretérito mais que perfeito) e ao presente (presente simples e presente progressivo). (i) Se a situação é conceptualizada no plano da realidade passada ou da realidade imediata, em sua referência empregamos o modo indicativo e as formas verbais, simples ou compostas, referentes ao passado (pretérito perfeito, pretérito imperfeito e pretérito mais que perfeito) e ao presente (presente simples e presente progressivo). (ii) No caso de situações passadas ou presentes que não são conceptualizadas como factuais, nos valemos das seguintes combinações entre tempo e modo para sinalizar que tal situação pertence ao plano da realidade não estabelecida: (a) futuro do pretérito, no modo indicativo; (b) pretérito imperfeito, pretérito perfeito e pretérito mais que perfeito, no modo subjuntivo; e (c) presente, no modo subjuntivo. (ii) No caso de situações passadas ou presentes que não são conceptualizadas como factuais, nos valemos das seguintes combinações entre tempo e modo para sinalizar que tal situação pertence ao plano da realidade não estabelecida: (a) futuro do pretérito, no modo indicativo; (b) pretérito imperfeito, pretérito perfeito e pretérito mais que perfeito, no modo subjuntivo; e (c) presente, no modo subjuntivo. (iii) Quando em referência a uma situação conceptualizada como futura, se pudermos prever sua ocorrência, utilizamos as formas verbais de futuro e do presente simples, no modo indicativo, para situá-la no plano da realidade projetada; se julgamos sua ocorrência apenas possível, empregamos o futuro do subjuntivo e os verbos modais para situarmos tal situação no plano da realidade potencial. cadernos.abralin.org Obviamente, o processo de conceptualização, ou seja, a apreensão, em termos cognitivos, de uma dada entidade ou situação por um conceptualizador, envolve muitos outros aspectos que, justamente por serem muitos, não foram capturados neste texto. Cumpre, contudo, destacar que todo e qualquer processo de conceptualização envolve modos alternativos de se conceptualizar determinada situação (construals, para Langacker 1991, 1999 e 2002), que subjazem, portanto, a relação entre tempo, modo e concepção de realidade aqui focalizada. 4. AGRADECIMENTOS Agradeço à CAPES, pela bolsa de Pós-Doutoramento, e ao CNPq, pela bolsa de Produtividade em Pesquisa, que me foram concedidas e que propiciaram o desenvolvimento de estudos dos quais se originou este texto. 3. SUMARIANDO (iii) Quando em referência a uma situação conceptualizada como futura, se pudermos prever sua ocorrência, utilizamos as formas verbais de futuro e do presente simples, no modo indicativo, para situá-la no plano da realidade projetada; se julgamos sua ocorrência apenas possível, empregamos o futuro do subjuntivo e os verbos modais para situarmos tal situação no plano da realidade potencial. DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021 16 REFERÊNCIAS ABRAÇADO, Jussara. O tempo, o tempo linguístico e o tempo verbal: propriedades e relações. São Paulo: Contexto 2020. ABRAÇADO, Jussara. O tempo, o tempo linguístico e o tempo verbal: propriedades e relações. São Paulo: Contexto 2020. BRISARD, Frank. The English present. BRISARD, Frank (ed.) Grounding: the epistemic footing of deixis and referenc Berlin/New York: Mouton de Gruyter, 2002, p. 251-297. FONSECA, Fernanda I. Dêixis e Pragmática Linguística. FARIA, Isabel Hub; PEDRO, Emília Ribeiro; DUARTE, Inês, Gouveia; Carlos A.M. (Orgs.). Introdução à Linguística Geral e Portuguesa. Lisboa: Caminhos, 1996, p. 437-445. LANGACKER, Ronald W. Foundation of cognitive grammar. Volume II: Descriptive Aplication. California: Stanford University Press, 1991. _____. Losing control: grammaticization, subjectification, and transparency. BLANK, Andreas; KOCH, Peter (Eds.). Historical Semantics and Cognition. Berlin: Mouton de Gruyter, 1999, p.147-175. _____. The English present tense. English Language and Linguistics 5: 251—272. 2002. _____. Extreme subjectification: English tense and modals. CUYCKENS Hubert; BERG, Thomas; DIRVEN, René; PANTHER, Klaus-Uwe (Eds.). Motivation in Language: Studies in honor of Günter Radden. Amsterdam: John Benjamins, 2003, p. 3-26. SOARES DA SILVA, Augusto. O Significado na Linguagem e na Mente. Uma Introdução à Semântica. Braga: Universidade Católica Portuguesa, 320 páginas (inédito). SOUZA; Melina; ABRAÇADO, Jussara. A projeção do tempo futuro em frames de finalidade. DIAS, Nilza; ABRAÇADO, Jussara (orgs.). Estudos sobre o português em uso. Uberlândia: Pangeia, 2020, p.43-77. Disponível em: https://www.academia.edu/43968444/A_PROJE%C3%87%C3%83O_DO_TEMPO_FUTURO_EM_FRAMES_DE_FINALI DADE. Acesso em 20/10/2020. SOUZA; Melina; ABRAÇADO, Jussara. A projeção do tempo futuro em frames de finalidade. DIAS, Nilza; ABRAÇADO, Jussara (orgs.). Estudos sobre o português em uso. Uberlândia: Pangeia, 2020, p.43-77. Disponível em: https://www.academia.edu/43968444/A_PROJE%C3%87%C3%83O_DO_TEMPO_FUTURO_EM_FRAMES_DE_FINALI DADE. Acesso em 20/10/2020. 17 DOI 10.25189/2675-4916.2021.V2.N1.ID321 ISSN: 2675-4916 V. 2, N. 1, 2021
https://openalex.org/W4366439852	https://www.researchsquare.com/article/rs-2640097/latest.pdf	English	null	Prevalence of The Risk Factors Associated with Pre-eclampsia and Eclampsia Disorders in a Tertiary Care Hospital of Lahore, a cross sectional study	Research Square (Research Square)	2,023	cc-by	4,829	Prevalence of The Risk Factors Associated with Pre- eclampsia and Eclampsia Disorders in a Tertiary Care Hospital of Lahore, a cross sectional study Sana Iqbal Mayo Hospital Sibgha Ali Mayo Hospital Mah noor Naeem Mayo Hospital Tooba Fida (  toobafida98@gmail.com ) Mayo Hospital https://orcid.org/0000-0001-9 Zain ul abiddin Mayo Hospital Research Article Keywords: Pre-eclampsia, complication, pregnancy, nulliparity, Gestational diabetes mellitus, Antenatal Posted Date: April 19th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2640097/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License DOI: https://doi.org/10.21203/rs.3.rs-2640097/v1 DOI: https://doi.org/10.21203/rs.3.rs-2640097/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/14 Page 1/14 Page 1/14 Abstract Introduction: Pre-eclampsia (PE) is a multi-system hypertensive condition characterized by a blood pressure of greater than 140/90 mmHg recorded on two separate readings at least 4 hours apart and associated with proteinuria or end organ dysfunction. It is called Eclampsia when a patient with pre- eclampsia develops tonic-clonic seizures as well. It is thought to occur due to disordered trophoblastic invasion that causes uteroplacental hypoperfusion, leading to widespread endothelial dysfunction. Risk factors include PE in a previous pregnancy, Chronic kidney disease, diabetes mellitus, chronic hypertension or autoimmune diseases. Increased maternal age, high BMI and family history also play a role in its pathology. Pakistan has the third highest maternal mortality according to a recent global health analysis, 29% of this is contributed by hypertensive disorders. This study aims to study the predisposing risk factors in pre-eclampsia patients at a tertiary care hospital. Methods: 82 selected cases of pre-eclampsia and eclampsia were included in this cross-sectional study. Informed consent was taken from all the patients and a pre-tested questionnaire was filled out by interviewing them during their antenatal visits. Results: Most (61%) of the patients were in the third decade (20-29) of their life. The mean of the blood pressure checked at the time of interviewing was 153/95 mmHg. The majority (54.90%) of the patients were overweight (BMI=26-34.9). The most commonly identified risk factors include; raised diastolic BP at the booking visit (60%), history of pre-eclampsia in a previous pregnancy (41%), nulliparity (37%), obesity (37%) family history of pre-eclampsia (28%), GDM (21%) and chronic HTN (13%). Other less common risk factors include severe anemia (9%), a gap of more than 10 years (7%), multiple gestations (5%) and maternal age of more than 35 years (4%). Conclusion: Pre-eclampsia is a significant threat to maternal as well as fetal life. And it has a higher incidence in the developing part of the world. Various risk factors are attributed to its pathophysiology. Prompt diagnosis and proper management and monitoring can reduce the morbidity and mortality associated with pre-eclampsia. Introduction Pre-eclampsia (PE) and eclampsia are one of the most dreadful and cryptic complications of pregnancy, which begin after the 20th week of gestation and are cured only by delivery. [1] Pre-eclampsia is a multi- system hypertensive condition characterized by a blood pressure of greater than 140/90 mmHg recorded on two separate readings at least 4 hours apart and associated with proteinuria or end organ dysfunction. It is called Eclampsia when a patient with pre-eclampsia develops tonic-clonic seizures as well. [2] Pre-eclampsia complicates 5–6% of all pregnancies and thus contributes a great share in maternal and fetal morbidity as well as mortality. [3] Pre-eclampsia has a multifactorial etiology, it is said to occur due to disordered trophoblastic invasion that causes uteroplacental hypoperfusion, leading to a widespread endothelial dysfunction due to the secretion of certain angiogenic factors by the placenta. Page 2/14 Page 2/14 Page 2/14 This in turn attacks end-organs during the pregnancy and postpartum period. [4] The risk factors identified for pre-eclampsia are graded as high and moderate. High risk factors include PE in a previous pregnancy, Chronic kidney disease, and diabetes mellitus. chronic hypertension or autoimmune diseases like SLE and antiphospholipid syndrome. Moderate risk factors include primigravida female, maternal age ≥ 40, BMI ≥ 35 or family history of pre-eclampsia. Any female having 1 high or 2 moderate risk factors should undergo early pregnancy screening for pre-eclampsia. [5] Pre-eclampsia has always been a threat to maternal and fetal health worldwide. It gravely burdens the hospitals and the resources of the healthcare department by increasing the hospital stay of patients due to various associated complications, such as placental abruption. [6] Other complications such as fetal growth restriction, intrauterine fetal death, HELLP syndrome are more common. Pre-eclampsia often results in multi-organ dysfunction as it can involve Cardiovascular system to cause heart failure and cardiomyopathy; gastrointestinal system to cause liver failure or liver rupture in eclampsia; renal system to cause kidney failure; central nervous system to cause encephalopathy and stroke. It may also cause generalized edema and pulmonary edema. It can also result in the onset of disseminated intravascular Coagulation (DIC). [7] Pre-eclampsia has a seven times higher incidence in developing countries. A study conducted in Nepal shows the incidence of pre-eclampsia in 2.8% of the live births in contrast to 0.4% in the developed countries. Pre-eclampsia is the second most contributing cause of maternal mortality. Introduction [2] There has been a constant rise in the cases of pre-eclampsia in Indonesia which can also be attributed to the lack of awareness. [8] Pakistan has the third highest maternal mortality according to a recent global health analysis, 34% of this is contributed by hypertensive disorders of pregnancy. [9] This proportion is much less (16%) but still significant in the developed part of the world. [10] In the USA, due to higher standards of antenatal care, prompt screening of high-risk cases and use of low-dose aspirin for the prevention of pre-eclampsia, the overall complications are significantly reduced. [11] Proper antenatal care and awareness about the complications can help decrease the incidence of pre- eclampsia. Scrutinizing the risk factors of Pre-eclampsia on an individual level can help in a better understanding of its etiology. [12] The results of this study may help highlight the importance of prompt screening for pre-eclampsia as a part of regular antenatal checkups in high-risk cases. Objectives: Objectives: To assess the risk factors of pre-eclampsia and eclampsia in a tertiary care hospital in Lahore. To assess the risk factors of pre-eclampsia and eclampsia in a tertiary care hospital in Lahore. To determine the factors majorly attributing to the high incidence of pre-eclampsia and eclampsia in our setting. Where Patients who did not voluntarily participate or patients who were in an altered state of consciousness at the time of data collection were excluded from the study. Where Z2(1-a) = confidence interval = 95%=1.96 Z2(1-a) = confidence interval = 95%=1.96 P = prevalence = 5.6% d = absolute precision = 5% n = 82 n = 82 82 selected cases of pre-eclampsia and eclampsia were included in the study. Informed consent was taken from all the patients and a pre-tested questionnaire was filled out by interviewing them during their antenatal visits. The questionnaire addressed all the common risk factors of eclampsia and pre- eclampsia including; maternal age (> 40 years), nulliparity, an interval of > 10 years since the last pregnancy, multiple gestations, chronic hypertension, type 2 Diabetes Mellitus, gestational diabetes mellitus, obesity, severe anemia (Hb < 7g/dL), history of cardiac or Renal disease, SLE, anti-phospholipid antibody syndrome, previous history of pre-eclampsia and family history. Patients who had been regularly visiting for the antenatal checkup were labelled as booked cases, in contrast to the un-booked cases; defined as the patients visiting for the first time in the given hospital. Inclusion criteria consisted of diagnosed cases of pre-eclampsia and eclampsia at the time of data collection. The diagnostic criteria are the blood pressure of more than 140/90 mmHg taken at two separate events at least 4 hours apart. Patients who did not voluntarily participate or patients who were in an altered state of consciousness at the time of data collection were excluded from the study. 82 selected cases of pre-eclampsia and eclampsia were included in the study. Informed consent was taken from all the patients and a pre-tested questionnaire was filled out by interviewing them during their antenatal visits. The questionnaire addressed all the common risk factors of eclampsia and pre- eclampsia including; maternal age (> 40 years), nulliparity, an interval of > 10 years since the last pregnancy, multiple gestations, chronic hypertension, type 2 Diabetes Mellitus, gestational diabetes mellitus, obesity, severe anemia (Hb < 7g/dL), history of cardiac or Renal disease, SLE, anti-phospholipid antibody syndrome, previous history of pre-eclampsia and family history. Patients who had been regularly visiting for the antenatal checkup were labelled as booked cases, in contrast to the un-booked cases; defined as the patients visiting for the first time in the given hospital. Inclusion criteria consisted of diagnosed cases of pre-eclampsia and eclampsia at the time of data collection. The diagnostic criteria are the blood pressure of more than 140/90 mmHg taken at two separate events at least 4 hours apart. Materials And Methods A cross-sectional study was carried out at the Lady Willingdon hospital Lahore from August 1 to October 15, 2022. The study was approved by the institutional review board. A sample size of 82 subjects was Page 3/14 Page 3/14 calculated by taking a confidence interval of 95%, absolute precision of 5% and expected combined prevalence of preeclampsia and eclampsia as 5.6% [3] calculated by the formula: Results This study included 82 participants (pregnant females with pre-eclampsia diagnosed) with a 100% response rate (due to the study design (analytical cross-sectional study), there was no follow-up and the tool used was an interviewer-administered structured questionnaire). The most common age groups were 20-29 years followed by 30-34 years with a percentage of 61% (n=50) and 22% respectively. While there were 11 (13.40%) pregnant women with age ≥ 35 years. The mean systolic blood pressure (SBP) was 153.05 mm Hg and the mean diastolic blood pressure was 95.55 mm Hg. 54.90% of participants had a body mass index (BMI) value ranging between 26 and 34.9 while 42.7% had BMI between 20-25.90. A significant percentage of study participants (36.60%) had not gone to school at all. 22% of participants had received higher secondary education while 19.50% were educated beyond that. 63.4% were Page 4/14 Page 4/14 Page 4/14 multiparous. The status of antenatal visits was found to be adequate since 62.20% had 4-8 antenatal visits and 23.20% had 1-3 visits.12.20% had >8 visits. The status of antenatal visits was found to be adequate since 62.20% had 4-8 antenatal visits and 23.20% had 1-3 visits.12.20% had >8 visits. ur results can be generalised to the entire region since the study was condu Our results can be generalised to the entire region since the study was conducted in a referral hospital. Risk factors associated with Pre-eclampsia are shown in the following graph: Risk factors associated with Pre-eclampsia are shown in the following graph: Discussion In a study by Ali AA et al [19], the prevalence of pre-eclampsia was found to be higher in women with severe anemia (8.2%). The corrected risk for pre-eclampsia increased in severe anemia (OR = 3.6, 95% CI: 1.4–9.1, P = 0.007). History of multiple pregnancies was not a common risk factor in our study population unlike other studies [3, 4, 5] as only 5% of patients had multiple gestations in our study. Women with multiple pregnancies have a large-sized placenta which results in relatively decreased placental perfusion. The excess of placenta tissues not perfused adequately compared to the women with singleton pregnancy contributes to the risk of pre-eclampsia/eclampsia [3] and increased placental production of soluble factors which antagonizes the VEGF signaling in maternal vasculature [12]. Similarly, the rest of the studied factors, for example, previous medical history of renal disease (5%), antiphospholipid antibody syndrome (4%), and use of assisted reproductive technologies (1%) were uncommon in our study population. In vitro conception was added to the list of moderate-risk factors for preeclampsia in the 2021 guidelines [17]. While none of the participants reported any cardiac disease, systemic lupus erythematosus, untreated HIV, or treatment with HAART therapy. Discussion This institutional-based cross-sectional study was conducted in Lady Willingdon Hospital Lahore to determine the prevalence of the risk factors associated with pre-eclampsia. The pathophysiology of pre- eclampsia, a major contributor to maternal morbidity and mortality, remains elusive but risk factors for pre-eclampsia can be broadly classified into two categories: high-risk factors (pre-eclampsia in a prior pregnancy, multifetal gestation, chronic hypertension, type 2 diabetes, renal disease and autoimmune disease), and moderate risk factors (nulliparity, obesity, family history of pre-eclampsia in first degree relatives, maternal age 35 years or more and inter-pregnancy interval of 10 years) [18]. In our study, risk factors most commonly seen in pre-eclampsia patients were raised diastolic pressure at booking visits (> 80 mm Hg) and a history of pre-eclampsia in previous pregnancies. Gestational hemodynamic adaptations occurring during pregnancy are a decrease in BP from early to mid-gestation (“mid-gestational BP drop”) which benefits placental perfusion. Blood pressure then increases to reach pre-pregnancy levels in late gestation. An absence of mid-gestational blood pressure drop is associated with pre-eclampsia [13]. In our study, more than 60% of patients had raised diastolic blood pressure at booking visits which is consistent with a study by Ayele et al [8] in 2022 in which 68% of participants had diastolic blood pressure between 90–110 mmHg. History of pre-eclampsia in previous pregnancies, a high-risk factor for pre-eclampsia, was seen in 41% of participants which is congruent with [1, 2, 3]. Grum T et al [3] found 4 times higher chances of developing pre-eclampsia among females having a previous history of pre-eclampsia than those who didn’t have a such history which underscores the importance of strict and earlier monitoring of pre-eclampsia patients in their subsequent pregnancies. The next most common factor was nulliparity (37%) since pre-eclampsia is a disease of 1st pregnancy because of exposure of the maternal body to a foreign paternal genome in the form of a fetus. Protective effect in the second pregnancy is found only if the father remains the same, the reason being that the maternal immune system builds tolerance to the paternal antigens with repetitive exposure which would not be the case if the partner changes [20]. Along similar lines, pregnancy with a new partner is a risk factor for pre-eclampsia since changing parents could bring fresh antigens from new parents predisposing females to the risk of PE development due to non-compatibility [6]. Discussion Page 5/14 Page 5/14 Obesity was present in 37% of participants. 54.90% of participants had a BMI value ranging between 26 and 34.9 which is similar to the results of various other studies [8, 10]. The proposed mechanism behind obesity being a risk factor of pre-eclampsia is insulin resistance and systemic inflammation leading to endothelial dysfunction and resulting in multi-organ involvement [10]. History of pre-eclampsia in 1st- degree relatives was another prevalent factor in our study population (28% of participants). Studies show that patients with a strong family history of pre-eclampsia are more likely to develop pre-eclampsia than those who don't have such a history. Obesity was present in 37% of participants. 54.90% of participants had a BMI value ranging between 26 and 34.9 which is similar to the results of various other studies [8, 10]. The proposed mechanism behind obesity being a risk factor of pre-eclampsia is insulin resistance and systemic inflammation leading to endothelial dysfunction and resulting in multi-organ involvement [10]. History of pre-eclampsia in 1st- degree relatives was another prevalent factor in our study population (28% of participants). Studies show that patients with a strong family history of pre-eclampsia are more likely to develop pre-eclampsia than those who don't have such a history. In our study, gestational diabetes mellitus was present in 21% of participants. Various studies have revealed the association of pre-eclampsia complicated by insulin resistance [8, 14–16, 22]. Diabetic pregnant women are 2.37 times more likely to develop pre-eclampsia compared with non-diabetic pregnant women (95% confidence interval: 1.1–3.7). GDM affects the occurrence of PE by inducing placental ischemia, increasing oxidative stress and inflammation [15]. 25% of preeclampsia patients were found to have gestational diabetes in a descriptive cross-sectional study on 108 patients by Nokandeh OM et al [16]. Along similar lines, a history of type 2 diabetes is a risk factor for pre-eclampsia which was seen in 7% of our participants. History of chronic hypertension was present in 13% of participants which is in agreement with other studies [8, 11]. Pre-eclampsia is more common in those with a history of hypertension for the last 5 years [23]. More than 10 years since the last pregnancy as a risk factor for pre-eclampsia was present in only 7% of participants. Severe anemia, defined by hemoglobin levels of less than < 7g/dl, was present in 9% of participants. Conclusion Page 6/14 History of pre-eclampsia in previous pregnancies and raised diastolic blood pressure (> 80 mm Hg) is the most prevalent risk factors for preeclampsia and eclampsia disorders in our population. Other frequent Page 6/14 risk factors are nulliparity,obesity,history of gestational diabetes mellitus and chronic hypertension. In underdeveloped countries already lagging in achieving Sustainable Development Goals regarding maternal health, pre-eclampsia deserves special attention being a major contributor to maternal morbidity and mortality.Health-seeking behaviour in pregnant females should be encouraged for both urban and rural residents to screen the population at risk and prevent the coming complication by ensuring at least 4 antenatal visits as per the focused antenatal care model proposed by WHO. Limitations There are chances of recall bias for some information acquired using the memory of participants. Besides, this institutional study cannot be a true representative of the entire population because of the smaller sample size. Biochemical tests to exclude other related medical conditions were not performed. As the data was collected during pregnancy, we were not able to determine maternal and fetal outcomes. Since a cross-sectional study design was implemented, it can’t establish a cause-effect relationship between risk factors and pre-eclampsia.Since study population was only a subset of the general population,berkson bias is likely to affect our results. CONFLICT OF INTEREST The article and its authors have no conflict of interest. FUNDING This research hasn’t been funded by either internal or external sources. Page 7/14 The research has been conducted only after the ethical approval by the IRB committee of Mayo CONSENT FOR PUBLICATION All the authors mutually agree for the publication in this journal. CONSENT TO PARTICIPATE All the data has been gathered after obtaining a signed written informed consent from the participants. AVAILABILITY OF DATA AND MATERIAL All the data collected from the subjects is available and can be submitted on demand CODE AVAILABILITY The research has been conducted only after the ethical approval by the IRB committee of Mayo CONSENT FOR PUBLICATION All the authors mutually agree for the publication in this journal. The data has been analyzed through the spss 26 version AUTHOR CONTRIBUTION 1 S.I :conception and design of the research, 2 S. A :acquisition of data, 3. M. N: drafting the article 4. T.F: revising it critically and corresponding 5. Z.A: analysis and interpretation of data The data has been analyzed through the spss 26 version AUTHOR CONTRIBUTION 1 S.I :conception and design of the research, 3. M. N: drafting the article 4. T.F: revising it critically and corresponding 5. Z.A: analysis and interpretation of data References 1. 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Wainstock T, Sergienko R, Sheiner E. Who Is at Risk for Preeclampsia? Risk Factors for Developing Initial Preeclampsia in a Subsequent Pregnancy. Vol. 9,Journal of Clinical Medicine. 2020 23. Brumby C, Aherne N, Koh G, Tan YY, Mcmahon L. 292. Risk factors for superimposed preeclampsia in 22. Wainstock T, Sergienko R, Sheiner E. Who Is at Risk for Preeclampsia? Risk Factors for Developing Initial Preeclampsia in a Subsequent Pregnancy. Vol. 9,Journal of Clinical Medicine. 2020 23. Brumby C, Aherne N, Koh G, Tan YY, Mcmahon L. 292. Risk factors for superimposed preeclampsia in women with chronic hypertension. Pregnancy Hypertens [Internet]. 2018;13:S119. Available from: htt // i di t / i / ti l / ii/S2210778918305427 22. Wainstock T, Sergienko R, Sheiner E. Who Is at Risk for Preeclampsia? Risk Factors for Developing Initial Preeclampsia in a Subsequent Pregnancy. Vol. 9,Journal of Clinical Medicine. 2020 22. Wainstock T, Sergienko R, Sheiner E. Who Is at Risk for Preeclampsia? Risk Factors for Developing Initial Preeclampsia in a Subsequent Pregnancy. Vol. 9,Journal of Clinical Medicine. 2020 23. Brumby C, Aherne N, Koh G, Tan YY, Mcmahon L. 292. Risk factors for superimposed preeclampsia in women with chronic hypertension. Pregnancy Hypertens [Internet]. 2018;13:S119. Available from: https://www.sciencedirect.com/science/article/pii/S2210778918305427 p q g y 23. Brumby C, Aherne N, Koh G, Tan YY, Mcmahon L. 292. Risk factors for superimposed preeclampsia in women with chronic hypertension. Pregnancy Hypertens [Internet]. 2018;13:S119. Available from: https://www.sciencedirect.com/science/article/pii/S2210778918305427 23. Brumby C, Aherne N, Koh G, Tan YY, Mcmahon L. 292. Risk factors for superimposed preeclampsia in women with chronic hypertension. Pregnancy Hypertens [Internet]. 2018;13:S119. Available from: https://www.sciencedirect.com/science/article/pii/S2210778918305427 Figures Figure 1 Legend not included with this version. 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Cormick G, Betrán AP, Ciapponi A, Hall DR, Hofmeyr GJ, calcium and Pre-eclampsia Study Group. ;. Inter-pregnancy interval and risk of recurrent pre-eclampsia: systematic review and meta-analysis. Reprod Health. 2016 Jul 18;13(1):83. doi: 10.1186/s12978-016-0197-x. PMID: 27430353; PMCID: PMC4950816. 20. Cormick G, Betrán AP, Ciapponi A, Hall DR, Hofmeyr GJ, calcium and Pre-eclampsia Study Group. ;. Inter-pregnancy interval and risk of recurrent pre-eclampsia: systematic review and meta-analysis. Reprod Health. 2016 Jul 18;13(1):83. doi: 10.1186/s12978-016-0197-x. PMID: 27430353; PMCID: PMC4950816. 21. Njoroge S, Kuriloff M, Mueller A, Lopes Perdigao J, Dhir R, Rana S. The interval between births and the risk of recurrent preeclampsia among predominantly high risk women in urban tertiary care center. Pregnancy Hypertens [Internet]. 2021;25:7–11. Available from: https://www.sciencedirect.com/science/article/pii/S2210778921000416 Page 9/14 Page 9/14 Page 9/14 22. Wainstock T, Sergienko R, Sheiner E. Who Is at Risk for Preeclampsia? Risk Factors for Developing Initial Preeclampsia in a Subsequent Pregnancy. Vol. 9,Journal of Clinical Medicine. 2020 23. Brumby C, Aherne N, Koh G, Tan YY, Mcmahon L. 292. Risk factors for superimposed preeclampsia in women with chronic hypertension. Pregnancy Hypertens [Internet]. 2018;13:S119. Figure 1 Legend not included with this version. Legend not included with this version. Legend not included with this version. Legend not included with this version. Page 10/14 Page 10/14 Figure 2 Legend not included with this version. Legend not included with this version. Page 11/14 Fi 3 Figure 3 Figure 3 Legend not included with this version. Legend not included with this version. Legend not included with this version. Page 12/14 Figure 4 Legend not included with this version. Figure 4 Figure 4 Legend not included with this version. Legend not included with this version. 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https://openalex.org/W3209209809	https://zenodo.org/records/5573625/files/A1404109119.pdf	English	null	Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection	International journal of engineering and advanced technology	2,020	cc-by	13,168	I. 0 V   div (1) ) ( ) ( V k k P grad l V                    (2)  (1) Eringen [1] has presented the theory of micropolar fluids. The theory of micropolar fluids become the very active field of research for the last few decades as this class of fluids represents, mathematically, many industrially important fluids such as paints, body fluids, polymers, colloidal fluids, suspension fluids etc. These fluids display the effects of local rotatory inertia and couple stress and may form suitable non-Newtonian fluid models, which can be used to analyze the behaviour of the exotic lubricants, animal bloods, etc. The study of micropolar fluid mechanics has received the attention of many researchers. A list of published papers for this fluid can be found in Eringen [2] and Ishak et.al [3] . The mathematical theory of equations of micropolar fluids and application of these fluids in the theory of lubrication and in the theory of porous media is presented by G. Lukaszewicz [4] in this book. (3) whereV  is the velocity vector,   is the micro-rotation vector and P is fluid pressure,  and j are the fluid density and micro-gyration parameter, {, k} and {,,} are viscosity and gyroviscosity coefficients. The stress tensor ij and the couple stress tensor mij The stress tensor ij and the couple stress tensor mij re given by are given by are given by ), ( ) 2( m m ijm ij ij ij Q w k e k P           (4) , , , , i j j i ij k k ij m           (5) International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 Ajit Kumar, Preeti Ajit Kumar, Preeti The purpose of the present paper is to study the flow of micropolar fluid between two parallel plates with different periodic suction. The effects of dimensionless parameter pj, pl & pt on the velocity functions f & g have shown graphically and the conclusions have been drawn from the behaviour of the velocity functions with respect to different dimensionless parameters. Abstract: The unsteady stokes flow of incompressible micropolar fluid between two porous plates is considered. The lower plate is subjected to periodic suction and different periodic injection is applied at the upper plate. Stream function for the flow is obtained and the variation of velocity function f  & g with  is shown graphically. The effects of the dimensionless parameters p, frequency parameter pt, micropolarity parameter pl and the microrotation parameter pj on the velocity functions f  and microrotation velocity function g are discussed and shown through the graphs. II. Assuming the flow to be stokesian, neglecting the inertial and gyroinertial terms and ignoring the body force and body couple, the field equations of the micropolar fluid are : Keywords: Unsteady stokes flow, micropolar fluid, porous plates, periodic suction and injection. Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 MICROPOLAR FLUIDS – THEORY AND APPLICATIONS , , , , i j j i ij k k ij m           (5) The problem of steady flow of an incompressible viscous fluid through a porous channel was considered by Berman [5]. He obtained a perturbation solution assuming normal wall velocities to be equal. Sellers [6], Terrill [7] and Yuan [8] have extended the analysis of Berman for various values of suction and injection Reynolds numbers. Terrill and Shretha [9] have examined the same problem, assuming different normal velocities at the walls. Flow of a second- order fluid between torsionally oscillating discs of different permeability was studied by Singh and Agarwal [10]. Asharaf and Bashir [11] have obtained the numerical solution of MHD stagnation point flow and heat transfer of a micropolar fluid towards a heated shrinking sheet. Srinivasacharya, D. et.al [12] have solved the problem of stokes flow of micropolar fluid between two parallel porous plates. They have studied the effect of pj, pl & pt on the skin friction. where w is the vorticity vector δij is kronecker delta ijm is the alternating symbol eij is the strain rate tensor. Consider the two dimensional flow of micropolar fluid through two porous parallel plates y=0 and y =h along the direction of x axis. Since the flow is along the x- direction, all the variables are independent of z. Let us take a periodic suction of velocity voeiwt at the lower plate and periodic injection of velocity nvoeiwt at the upper plate. Hence we choose the velocity vector V  , micro-rotation vector   and the pressure P in the form iwt iwt iwt e y x N e k y x N e j y x i y x u V ) , ( ,0,0 ( ) , ( , ] ) , ( ) , ( [            and iwt e y x p y x P ) , ( ) , (  (6) (6) We introduce the stream function (x,y) satisfying the continuity equation (1) through Revised Manuscript Received on February, 10 2020. ropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection can be obtain as follows : a2 + b2 = 1+i2 , a2b2 = 3+i4 (25) here The values of a2 + b2, a2b2 etc. can be obtain as follows : The values of a2 + b2, a2b2 etc. can be obtain as follows : a2 + b2 +i a2b2 +i (25) ) ( )) ( ( ) 2 ( 2 2 k k j w i k k b a              (15) ) ( ) 2 ( 2 2 k jw i k w i b a        (16) , a2 + b2 = 1+i2 , a2b2 = 3+i4 h (25) j t j t l p p h p p h p h 2 4 3 2 2 2 1 1 ), 1( 1 , 1         and . 2 4 4 K p h t   (26) (16) (26) III. We assume The boundary conditions on the velocity components u, v and microrotation component N are : j 4 2 1 6 3 2 2 2 1 5 4 2 , 4              , 2 ) ( , 7 7 5 8 2 6 2 5 7           B , 2 ) ( , 2 ) ( 8 7 1 10 7 5 7 9             , 2 ) ( , 2 ) ( 8 7 1 12 9 7 2 11             , ,2 /) ( 2 11 2 10 14 9 7 2 13            , 2 ) ( , 2 ) ( 14 10 14 16 14 10 14 15             p y = 0 u = 0, v = v0, N = 0 y = h u = 0, v = nv0, N = 0 (17) Following Terrill [8], we introduce f(ζ) and g(ζ) as: ) ( 1 ), ( 0 0 0 0      g x h nv U h N f x h nv U h             p y = 0 u = 0, v = v0, N = 0 y = h u = 0, v = nv0, N = 0 (17) F ll i T ill [8] i t d f(ζ) d (ζ) p y = 0 u = 0, v = v0, N = 0 y = h u = 0, v = nv0, N = 0 y = 0 u = 0, v = v0, N = 0 y = h u = 0, v = nv0, N = 0 (17) Following Terrill [8], we introduce f(ζ) and g(ζ) as: ) ( 1 ), ( 0 0 0 0      g x h nv U h N f x h nv U h             (18) (18) where 0 0 0 0 0 \| \| \| \| 0 , 1 , U and nv v nv h y         is the average entrance velocity. III. h To calculate the value of a, b, a2 b2 a4 and b4 in terms of the dimensionless parameter p, pt, pl, pj etc. We assume To calculate the value of a, b, a2 b2 a4 and b4 in terms of the dimensionless parameter p, pt, pl, pj etc. ropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection The boundary conditions on f(ζ) and g (ζ) become: x y x y y x u           ) , ( , ) , ( (7) (7) f(0)=1-= n 1 , f(1)=1, f'(0)=f'(1)=0, g(0)=g(1)=0 Using expression (6), (7) in equation (2) and (3) comparing the i,jth component of equation (2) and kth components (which is only existing non zero component) of equation (3) respectively, we get; (21) n=2, 3, 4 , , The solution of equation (19) is : f(ζ) = C1 + C2 ζ + C3eahζ + C4e-ahζ + C5ebhζ + C6e-bhζ (22) and hence g(ζ)=A(C3eahζ + C4e-ahζ) + B(C5ebhζ + C6e-bhζ) (23) where The solution of equation (19) is : f(ζ) = C1 + C2 ζ + C3eahζ + C4e-ahζ + C5ebhζ + C6e-bhζ (22) and hence g(ζ)=A(C3eahζ + C4e-ahζ) + B(C5ebhζ + C6e-bhζ) (23) where ) 2 ( } ) ( { 2 2 2 2 2 4 2 jwk i k a wh i k a a k h A           ) 2 ( } ) ( { 2 2 2 2 2 4 2 jwk i k b wh i k b b k h B           (24)     2 ) (               y k y N k x p y w i (8)     2 ) (              x k x N k y p x w i (9) N k kN wjN i 2 2 2          (10) (22) where ) 2 ( } ) ( { 2 2 2 2 2 4 2 jwk i k a wh i k a a k h A           ) 2 ( } ) ( { 2 2 2 2 2 4 2 jwk i k b wh i k b b k h B           (24) On differentiating to equation (8) w.r.t. y and to equation (9) w.r.t. ropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection x and then adding, the equations thus obtained we get: g q g iw 2  = k 2 N+(+k) 4  (11) multiply to equation (10) by k and to (11) by ν and then subtracting to the equations thus obtained we get: iw 2  = k 2 N+(+k) 4  (11) multiply to equation (10) by k and to (11) by ν and then subtracting to the equations thus obtained we get: (11) ) ( j (24) ing the dimensionless parameter         k k p   ; (24)         k k p   ; k(ijw+2k)N=-(+k) 4 +(iw-k2) 2  (12) 2 Using the dimensionless parameter         k k p   ; on operating 2  on both sides of the equation (12) and then substituting the value of 2 N in equation (11) we get: 2 ( 2 -a2)( 2 -b2)=0 (13) and k(ijw+2k)N=-(+k) 4 +(iw-k2) 2  (14) 2 2 on operating 2  on both sides of the equation (12) and then on operating 2  on both sides of the equation (12) and then substituting the value of 2 N in equation (11) we get: 2 ( 2 -a2)( 2 -b2)=0 (13) and k(ijw+2k)N=-(+k) 4 +(iw-k2) 2  (14) 2 2 frequency parameter         k wh pt   2 ; micro-polarity on operating  on both sides of the equation (12) and then substituting the value of 2 N in equation (11) we get: substituting the value of 2 N in equation (11) we get: g q ( ) g 2 ( 2 -a2)( 2 -b2)=0 (13) and k(ijw+2k)N=-(+k) 4 +(iw-k2) 2  (14) 2 2   and parameter          ) ( ) 2( 2 k h k k pl    ; micro-rotation parameter parameter          ) ( ) 2( 2 k h k k pl    ; micro-rotation parameter parameter          ) ( ) 2( 2 k h k k pl    ; micro-rotation parameter k(ijw+2k)N=-(+k) 4 +(iw-k2) 2  (14) 2 2 k(ijw+2k)N=-(+k) 4 +(iw-k2) 2  (14)           ) ( k j p j and the fluid parameter       2 kh K where 2 2 2 2 2 y x        is the Laplacian operator and where 2 2 2 2 2 y x        is the Laplacian operator and The values of a2 + b2, a2b2 etc. Revised Manuscript Received on February, 10 2020. ng 78 Published By: Blue Eyes Intelligence Engineering & Sciences Publication continuity equation (1) through Dr. AJIT KUMAR, Assistant Professor , Department of Mathematics Teerthanker Mahaveer University Moradabad, Preeti, Assistant Professor , Department of Mathematics Noida Institute Of Engineering of Technology Greater Noida 78 Published By: Blue Eyes Intelligence Engineering & Sciences Publication 078 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1078 Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection . 2 2 2  d d D  III. 9 we assume 18 17 3 16 14 1 2 15 14 1 , ,       h d h d h d   and 19 17 4 B B h d  and 28 = 2d1-2cosd2 sinhd1, 29 = 2d2 - 2Sind2 Coshd1 30 = (26 22 + 27 23) / ( 2 22  + 2 23  ) 31 = (27 22 - 26 23) / ( 2 22  + 2 23  ) 32 = d130 – d231, 33 = d230 + d131 34 = 30 1 de Cosd2 - 31 1 de Sind2 35 = 30 1 de Sind2 + 31 1 de Cosd2 36 = 3 de Cosd4, 37 = 3 d e Sind4 38 = -1+30 + 32 - d3 - 34 + 36 39 = 31 + 33 – d4 - 35 + 37 40 = 3 d e Cosd4, B41 = 3 d e Sind4 42 = -1+30 + 32 + d3 - 34 + 40 43 = 31 + 33 + d4 - 35 - 41 44 = 2d1 – 2d1 coshd1 cosd2 + 2d2 sinhd1 sind2, 45 = 2d2 – 2d2 coshd1 cosd2 +2d1 sinhd1 sind2, 46 = 32 – d1 34 + d2 35 – d3 + d3 36 - d4 37, 47 = 33 - d1 35 - d2 34 - d4 + d3 37 + d4 36, 48= 32 - d134 + d235 + d3-(d336 + d437)/( 2 36  + 2 37  ), 49 = 33 -d234- d135 + d4-(d436 – d337)/( 2 36  + 2 37  ), 50 = 2[22 cosd2 Sinhd1 - 23 Sind2 Coshd1] 51 = 2[23 cosd2 sinhd1 + 22 sind2 coshd1) 52 = 26 (36 – 1 de cosd2)- 27 (37 - 1 de sind2) 53 = 26 (37 – 1 de sind2) + 27 (36 - 1 de cosd2) 54=26 ( 1 de cosd2 – 3 d e cosd4) - 27( 1 de sind2 + 3 d e sind4) 55= 27 ( 1 de cosd2- 3 d e cosd4)+26 ( 1 de Sind2+ 3 d e Sind4) (45) , 1 , 2 2 1 bh ah e e A B bh ah A B A B e e ah                            , 1 3 bh ah e e A B bh ah A B A B                           Then , 16 14 15 14     i a   , 19 17 18 17     i b   , , 13 12 2 11 10 2     i b i a     ), 2 ( 11 10 2 11 2 10 4     i a    ) 2 ( 13 12 2 13 2 12 4     i b    (28) 4 = 2ah – ah eah – ah e-ah = 2ah - 2ah cosh(ah), , 5 bh ah e bh bh e A B ah ah A B                  , 6 bh ah e bh bh e A B ah ah A B                   7 = 2A Now to calculate the value of A and B we assume Now to calculate the value of A and B we assume     sinh(ah), 8 = B (ebh - eah), sinh(ah), 8 = B (ebh - eah), 11 2 10 2 2 11 2 10 4 20 ) ) (        h p p Kh h p t      11 2 11 10 4 10 2 21 2        Kh h p h p pt    ) 4 /( ) 2 ( 2 2 2 21 20 22 j t j t p p k p p k       ) 4 /( ) 2 ( 2 2 2 20 21 23 j t j t p p K p p K       , ) ( 13 2 12 2 2 13 2 12 4 24        h p p Kh h p t      , 2 13 2 13 12 4 12 2 25        Kh h p h p p     ), 4 /( ) 2 ( 2 2 2 25 24 26 j t j t p p K p p K       ) 4 /( ) 2( 2 2 2 24 25 27 j t j t p p K p p K       (29) Then A = 22 + i 23 (30) and B = 26 + i 27 (31) 9 = B(eah – e-bh) 9 = B(eah – e-bh) (44) (44) For calculating 1, 2, 3, ……………. III. 2 2 2  d d D  079 Published By: Blue Eyes Intelligence Engineering & Sciences Publication 079 Published By: Blue Eyes Intelligence Engineering & Sciences Publication 1079 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 9 Published By: Blue Eyes Intelligence Engineering & Sciences Publication 1079 Published By: Blue Eyes Intelligence Engineering & Sciences Publication eval Number: A1404109119/2020©BEIESP : 10.35940/ijeat.A1404.029320 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1079 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 17 12 17 18 2 13 2 12 17 2 ) ( ,            , and 17 12 17 19 2 ) (       (27) Then , 16 14 15 14     i a   , 19 17 18 17     i b   , , 13 12 2 11 10 2     i b i a     ), 2 ( 11 10 2 11 2 10 4     i a    ) 2 ( 13 12 2 13 2 12 4     i b    (28) Now to calculate the value of A and B we assume , 0 6 9 5 8 4 7 C C C        (43) where , 1 , 2 2 1 bh ah ah ah e e A B bh ah A B A B e e ah                               , 1 3 bh ah e e A B bh ah A B A B                           4 = 2ah – ah eah – ah e-ah = 2ah - 2ah cosh(ah), , 5 bh ah e bh bh e A B ah ah A B                  , 6 bh ah e bh bh e A B ah ah A B                   7 = 2A 17 12 17 18 2 13 2 12 17 2 ) ( ,            , and 17 12 17 19 2 ) (       (27) Then , 0 6 9 5 8 4 7 C C C        (43) where , 1 , 2 2 1 bh ah ah ah e e A B bh ah A B A B e e ah                                     , 0 6 9 5 8 4 7 C C C        (43) where , 1 , 2 2 1 bh ah ah ah e e A B bh ah A B A B e e ah                               , 1 3 bh ah e e A B bh ah A B A B                           4 = 2ah – ah eah – ah e-ah = 2ah - 2ah cosh(ah), , 5 bh ah e bh bh e A B ah ah A B                  , 6 bh ah e bh bh e A B ah ah A B                   7 = 2A bh h (43) 17 12 17 2 ) (     (27) , 16 14 15 14     i   , 19 17 18 17     i   , , 13 12 2 11 10     i b i     ), 2 ( 11 10 2 11 2 10     i    ) 2 ( 13 12 2 13 2 12     i    (28) ulate the value of A and B we assume 11 2 10 2 2 11 2 10 4 0 ) ) (       h p p Kh h p t      11 2 11 10 4 10 2 2       Kh h p h   ) 4 /( ) 2 2 2 21 20 j t j t p p k p p    ) 4 /( ) 2 2 2 20 21 j t j t p p K p p    , ) ( 13 2 12 2 2 13 2 12 4     h p p Kh t    , 2 13 2 13 12 4 12 2       Kh h p h p   ), 4 /( ) 2 2 2 25 24 j t j t p p K p p    ) 4 /( ) 2 2 2 24 25 j t j t p p K p p   (29) 22 + i 23 (30) 26 + i 27 (31) of the constants C1, C2, C3, C4, C5 and C6 are m equations (22) and (23) subjected to the onditions (21) for this from (21), (22) and (23) 6 5 4 3 C C C C    (32) +C3eah +C4 e-ah +C5ebh + C6 e-bh (33) ah – C4 ah +C5 bh - C6bh (34) h eah –C4 ah e-ah +C5 bh ebh bh (35) C4) + B (C5+C6) (36) h + C4 e-ah) + B (C5ebh + C6 e-bh) quations (32), (34) and (36) we get 6 5 1 1 C A B C A B               (38) 6 5 4 2 C bh ah A B C bh ah A B ahC                              (39)   6 5 C C A B         (40) he values of C1, C2 and C3 in equations no. III. (33), 7) we get; , 1 , 2 2 1 bh ah e e A B bh ah A B A B e e ah                            , 1 3 bh ah e e A B bh ah A B A B                           4 = 2ah – ah eah – ah e-ah = 2ah - 2ah cosh(ah), , 5 bh ah e bh bh e A B ah ah A B                  , 6 bh ah e bh bh e A B ah ah A B                   7 = 2A sinh(ah), 8 = B (ebh - eah), 9 = B(eah – e-bh) (44) For calculating 1, 2, 3, ……………. III. 9 we assume 18 17 3 16 14 1 2 15 14 1 , ,       h d h d h d   and 19 17 4 B B h d  and 28 = 2d1-2cosd2 sinhd1, 29 = 2d2 - 2Sind2 Coshd1 The values of the constants C1, C2, C3, C4, C5 and C6 are obtained from equations (22) and (23) subjected to the boundary conditions (21) for this from (21), (22) and (23) we get 6 5 4 3 1 1 C C C C C n      (32) 1 = C1+C2 +C3eah +C4 e-ah +C5ebh + C6 e-bh (33) 0 = C2 + C3 ah – C4 ah +C5 bh - C6bh (34) 0 = C2 +C3ah eah –C4 ah e-ah +C5 bh ebh -C6bh e-bh (35) 0 = A (C3 + C4) + B (C5+C6) (36) 0 = A (C3 eah + C4 e-ah) + B (C5ebh + C6 e-bh) (37) From equations (32), (34) and (36) we get 6 5 1 1 1 1 C A B C A B n C                  (38) 5 4 2 2 ah A B C bh ah A B ahC C                           (39)   6 5 4 3 C C A B C C           (40) i h l f C C d C i i 6 5 4 3 1 1 C C C C C n      (32) 1 = C1+C2 +C3eah +C4 e-ah +C5ebh + C6 e-bh (33) 0 = C2 + C3 ah – C4 ah +C5 bh - C6bh (34) 0 = C2 +C3ah eah –C4 ah e-ah +C5 bh ebh -C6bh e-bh (35) 0 = A (C3 + C4) + B (C5+C6) (36) 0 = A (C3 eah + C4 e-ah) + B (C5ebh + C6 e-bh) (37) From equations (32), (34) and (36) we get 6 5 1 1 1 1 C A B C A B n C                  (38) 6 5 4 3 1 1 C C C C C n      (32) 1 = C1+C2 +C3eah +C4 e-ah +C5ebh + C6 e-bh (33) 0 = C2 + C3 ah – C4 ah +C5 bh - C6bh (34) 0 = C2 +C3ah eah –C4 ah e-ah +C5 bh ebh -C6bh e-bh (35) 0 = A (C3 + C4) + B (C5+C6) (36) 0 = A (C3 eah + C4 e-ah) + B (C5ebh + C6 e-bh) (37) From equations (32), (34) and (36) we get 6 5 1 1 1 1 C A B C A B n C                  (38) 6 5 4 2 2 C bh ah A B C bh ah A B ahC C                              (39)   6 5 4 3 C C A B C C           (40) 39 31 33 4 35 37 40 = 3 d e Cosd4, B41 = 3 d e Sind4 42 = -1+30 + 32 + d3 - 34 + 40 43 = 31 + 33 + d4 - 35 - 41 44 = 2d1 – 2d1 coshd1 cosd2 + 2d2 sinhd1 sind2, 45 = 2d2 – 2d2 coshd1 cosd2 +2d1 sinhd1 sind2, 46 = 32 – d1 34 + d2 35 – d3 + d3 36 - d4 37, 47 = 33 - d1 35 - d2 34 - d4 + d3 37 + d4 36, 48= 32 - d134 + d235 + d3-(d336 + d437)/( 2 36  + 3  49 = 33 -d234- d135 + d4-(d436 – d337)/( 2 36  + 2 37  50 = 2[22 cosd2 Sinhd1 - 23 Sind2 Coshd1] 51 = 2[23 cosd2 sinhd1 + 22 sind2 coshd1) 52 = 26 (36 – 1 de cosd2)- 27 (37 - 1 de sind2) 53 = 26 (37 – 1 de sind2) + 27 (36 - 1 de cosd2) 54=26 ( 1 de cosd2 – 3 d e cosd4) - 27( 1 de si 3 d e sind4) 55= 27 ( 1 de cosd2- 3 d e cosd4)+26 ( 1 de Sind2+ Si d ) (45) 6 ( ) 0 = A (C3 + C4) + B (C5+C6) (36) h h bh bh (37) From equations (32), (34) and (36) we get 6 5 1 1 1 1 C A B C A B n C                  (38) 6 5 4 2 2 C bh ah A B C bh ah A B ahC C                              (39)   6 5 4 3 C C A B C C           (40) 55= 27 ( 1 de cosd2- 3 d e cosd4)+26 ( 1 de Sind2+ 3 d e Sind4) (45) 55= 27 ( 1 de cosd2- 3 d e cosd4)+26 ( 1 de Sind2+ 3 d e Sind4) (45) on putting the values of C1, C2 and C3 in equations no. III. the average entrance velocity. Using expressions (18) in equations (12) and (13) we get: D2(D2-a2h2) (D2-b2h2)f(ζ)=0, (19) Using expressions (18) in equations (12) and (13) we get D2(D2-a2h2) (D2-b2h2)f(ζ)=0, (19) ζ kh2(ijw+2k)g(ζ)=-(+k)D4f(ζ) +h2(iρwk2)D2f(ζ) (20) h ζ kh2(ijw+2k)g(ζ)=-(+k)D4f(ζ) +h2(iρwk2)D2f(ζ) h kh2(ijw+2k)g(ζ)=-(+k)D4f(ζ) +h2(iρwk2)D2f(ζ) h where where g . III. 69, let us take d5 = 2854 - 2955 - 4250 + 4351 d6 = 2855 + 2954 - 4251 - 4350 d7 = 2 54  + 2 55  d8 = 3854 - 3935 + 4252 - 4353 d9 = 3855 + 3954 + 4253 + 4352 d10 = 4454 - 4555 - 4850 + 4951 d11 = 4455 + 4554 - 4851 - 4950 66 = (62 d16 - 63 d17)/d18 67 = -(63 d16 + 62 d17)/d18 d19 = 5266 - 5367 - 5064 + 5165 d20 = 5267 + 5366 - 5065 - 5164 68 = (54 d19 + 55 d20)/d7 69 = (54 d20 - 55 d19)/d7 d21 = 6523 - 6422 - 26 (66 + 68) + 27 (67 + 69) d22 = 6522 + 6423 + 27 (66 + 68) + 26 (67 + 69) d23 = 2 22  + 2 23  70 = (22 d21 - 23 d22)/d23 71 = - (22 d22 + 23 d21)/d23 72 = n 1 -70 - 64 - 66 - 68 73 = 71 + 65 + 67 + 69 74 =2(d164 –d2 65) 75 =2(d165 + d2 64) 76 =d3(68 – 66) + d4(67 – 69) 77 =d4(68 – 66) + d3(69 – 67) 78 =(d126 – d2 27)/d23 79 =(d127 + d2 26)/d23 80 = 22 (66 + 68) + 23(67 + 69) 81 = 22 (67 + 69) - 23(66 + 68) 82=74 + 76 + (7880 - 7981) 83=75 + 77 + (7980 + 7881) (51) Then C4 = 14 = 64 + i65 C5 = 15 = 66 + i67 C6 = 16 = 68 + i69 (52) On putting the values of C4, C5, C6 in equations (38), (3 and (40) we get Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection Hence 1 = 28 + i 29, 2 = 38 + i 39, 3 = 42 + i 43, 4 = 44 + i 45, 5 = 46 + i 47, 6 = 48 + i 49, 7 = 50 + i 51, 8 = 52 + i 53, 9 = 54 + i 55, (46) 66 = (62 d16 - 63 d17)/d18 67 = -(63 d16 + 62 d17)/d18 d19 = 5266 - 5367 - 5064 + 5165 d20 = 5267 + 5366 - 5065 - 5164 68 = (54 d19 + 55 d20)/d7 69 = (54 d20 - 55 d19)/d7 Hence 66 = (62 d16 - 63 d17)/d18 1 = 28 + i 29, 2 = 38 + i 39, 3 = 42 + i 43, 4 = 44 + i 45, 5 = 46 + i 47, 6 = 48 + i 49, 7 = 50 + i 51, 8 = 52 + i 53, 9 = 54 + i 55,   On solving equations (41), (42), (43) we get the values of C4, C5 and C6 as ) ( ) ( 1 1 14 12 11 13 10 13 4 say n C                ) ( 15 13 14 12 5 say C        ) ( ) ( 16 9 14 7 15 8 6 say C          (47) d22 = 6522 + 6423 + 27 (66 +  26 (67 + 69) d23 = 2 22  + 2 23  70 = (22 d21 - 23 d22)/d23 71 = - (22 d22 + 23 d21)/d23 72 = n 1 -70 - 64 - 66 - 68 73 = 71 + 65 + 67 + 69 74 =2(d164 –d2 65) ) ( ) ( 1 1 14 12 11 13 10 13 4 say n C                ) ( 15 13 14 12 5 say C        ) (      ) ( ) ( 1 1 14 12 11 13 10 13 4 say n C                ) ( 15 13 14 12 5 say C        ) ( ) ( 16 9 14 7 15 8 6 say C          (47) Where W e e 9 8 3 9 2 11 9 7 3 9 1 10 ) ( , ) (                 ) 48 ( ) ( , ) ( 9 9 5 8 6 13 9 7 6 9 4 12                 The values of 10, 11, …………., 16 are calculated in terms of 56, 57, ………….. III. 69, let us take    d5 = 2854 - 2955 - 4250 + 4351 d6 = 2855 + 2954 - 4251 - 4350 d7 = 2 54  + 2 55  (51) C4 = 14 = 64 + i65 C5 = 15 = 66 + i67 C6 = 16 = 68 + i69 (52) d8 = 3854 - 3935 + 4252 - 4353 d9 = 3855 + 3954 + 4253 + 4352 d10 = 4454 - 4555 - 4850 + 4951 d11 = 4455 + 4554 - 4851 - 4950 d12 = 4852 - 4953 + 4654 - 4755 d13 = 4952 + 4853 + 4655 + 4754 (49) Then (52) On putting the values of C4, C5, C6 in equations (38), (39) and (40) we get (49) (53) On substituting the values of the constants C1, C2, C3, C4, C5, C6 in equations (22) ; (23) and thereafter using the values of f(ζ) and g(ζ) thus obtained in the expressions (18) we get the value of the stream function ψ and the micro-rotation N 56 = (54 d5 + 55 d6)/d7 57 = (54 d6 - 55 d5)/d7 10 = 56 + i 57 58 = (54 d8 + 55 d9)/d7 59 = (54 d9 - 55 d8)/d7 11 = 58 + i 59 60 = (54 10 + 55 11)/d7 61 = (54 11 + 55 10)/d7 12 = 60 + i 61 62 = (54 d12 + 55 d13)/d7 63 = (54 d13 - 55 d12)/d7 (50) 13 = 62 + i 63 Now if take in terms of the variable ζ and ξ=      h x nv U 0 0  . On taking in terms of the variable ζ and ξ=      h x nv U 0 0  . III. On taking h=1, ξ=1 the expressions (6) and (7) provides us h=1, ξ=1 the expressions (6) and (7) provides us 1081 Published By: Blue Eyes Intelligence Engineering & Sciences Publication U= Real part of u(x, y)eiwt =[82+  1 de {70(d1cos(d2ζ)-d2sin(d2ζ))- 71(d1sin(d2ζ)+d2cos(d2ζ))}-  1 -d e {64(d1cos(d2ζ)+d2sin(d2ζ))-65(d2cos(d2ζ)- d1sin(d2ζ))}+  3 d e {66(d3cos(d4ζ)-d4sin(d4ζ))- 67(d3sin(d4ζ)+d4cos(d4ζ))}-  3 -d e {68(d3cos(d4ζ)+d4sin(d4ζ))-69(d4cos(d4ζ)- d3sin(d4ζ))}].cosτ-[83+  1 de {71(d1cos(d2ζ)- d2sin(d2ζ))+70(d1sin(d2ζ)+d2cos(d2ζ))}-  1 -d e {65(d1cos(d2ζ)+d2sin(d2ζ))+64(d2cos(d2ζ)- (50) d14 = 5662 - 5763 - 5860 + 5961 d15 = 5762 + 5663 - 5861 - 5960 64 = (1- n 1 )(62 d14 + 63 d15)/( 2 14 d + 2 15 d ) 65 = (1- n 1 )(63 d14 - 62 d15)/( 2 14 d + 2 15 d ) d16 = 6165 - 6064 d17 = 6065 + 6164 d18 = 2 62  + 2 63  d16 = 6165 - 6064 d17 = 6065 + 6164 d18 = 2 62  + 2 63  1081 Published By: Blue Eyes Intelligence Engineering & Sciences Publication III. (33), (35) and (37) we get; on putting the values of C1, C2 and C3 in equations no. III. (33), (35) and (37) we get; 1080 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 , 1 1 6 3 5 2 4 1 C C C n        (41) , 0 6 6 5 5 4 4 C C C       (42) , 1 1 6 3 5 2 4 1 C C C n        (41) , 0 6 6 5 5 4 4 C C C       (42) 1080 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 , 1 1 6 3 5 2 4 1 C C C n        (41) , 0 6 6 5 5 4 4 C C C       (42) , 1 1 6 3 5 2 4 1 C C C n        (41) , 0 6 6 5 5 4 4 C C C       (42) (42) 1080 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1080 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1080 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 ween Two Parallel Plates with Different Periodic Suction and Injection (46) we get the values of ) y (47) 9 8 3 9 2 ) (       ) 48 ( ) 9 9  16 are calculated in take 66 = (62 d16 - 63 d17)/d18 67 = -(63 d16 + 62 d17)/d18 d19 = 5266 - 5367 - 5064 + 5165 d20 = 5267 + 5366 - 5065 - 5164 68 = (54 d19 + 55 d20)/d7 69 = (54 d20 - 55 d19)/d7 d21 = 6523 - 6422 - 26 (66 + 68) + 27 (67 + 69) d22 = 6522 + 6423 + 27 (66 + 68) + 26 (67 + 69) d23 = 2 22  + 2 23  70 = (22 d21 - 23 d22)/d23 71 = - (22 d22 + 23 d21)/d23 72 = n 1 -70 - 64 - 66 - 68 73 = 71 + 65 + 67 + 69 74 =2(d164 –d2 65) 75 =2(d165 + d2 64) 76 =d3(68 – 66) + d4(67 – 69) 77 =d4(68 – 66) + d3(69 – 67) 78 =(d126 – d2 27)/d23 79 =(d127 + d2 26)/d23 80 = 22 (66 + 68) + 23(67 + 69) 81 = 22 (67 + 69) - 23(66 + 68) 82=74 + 76 + (7880 - 7981) 83=75 + 77 + (7980 + 7881) (51) Then Plates with Different Periodic Suction and Injection 66 = (62 d16 - 63 d17)/d18 67 = -(63 d16 + 62 d17)/d18 d19 = 5266 - 5367 - 5064 + 5165 d20 = 5267 + 5366 - 5065 - 5164 68 = (54 d19 + 55 d20)/d7 69 = (54 d20 - 55 d19)/d7 d21 = 6523 - 6422 - 26 (66 + 68) + 27 (67 + 69) d22 = 6522 + 6423 + 27 (66 + 68) + 26 (67 + 69) d23 = 2 22  + 2 23  70 = (22 d21 - 23 d22)/d23 71 = - (22 d22 + 23 d21)/d23 72 = n 1 -70 - 64 - 66 - 68 73 = 71 + 65 + 67 + 69 74 =2(d164 –d2 65) 75 =2(d165 + d2 64) 76 =d3(68 – 66) + d4(67 – 69) 77 =d4(68 – 66) + d3(69 – 67) 78 =(d126 – d2 27)/d23 79 =(d127 + d2 26)/d23 80 = 22 (66 + 68) + 23(67 + 69) 81 = 22 (67 + 69) - 23(66 + 68) 82=74 + 76 + (7880 - 7981) 83=75 + 77 + (7980 + 7881) (51) Then Plates with Different Periodic Suction and Inject 66 = (62 d16 - 63 d17)/d18 67 = -(63 d16 + 62 d17)/d18 d19 = 5266 - 5367 - 5064 + 5165 d20 = 5267 + 5366 - 5065 - 5164 68 = (54 d19 + 55 d20)/d7 69 = (54 d20 - 55 d19)/d7 d21 = 6523 - 6422 - 26 (66 + 68) + 27 (67 + 69) d22 = 6522 + 6423 + 27 (66 + 68) + 26 (67 + 69) d23 = 2 22  + 2 23  70 = (22 d21 - 23 d22)/d23 71 = - (22 d22 + 23 d21)/d23 72 = n 1 -70 - 64 - 66 - 68 73 = 71 + 65 + 67 + 69 74 =2(d164 –d2 65) 75 =2(d165 + d2 64) 76 =d3(68 – 66) + d4(67 – 69) 77 =d4(68 – 66) + d3(69 – 67) 78 =(d126 – d2 27)/d23 79 =(d127 + d2 26)/d23 80 = 22 (66 + 68) + 23(67 + 69) 81 = 22 (67 + 69) - 23(66 + 68) 82=74 + 76 + (7880 - 7981) 83=75 + 77 + (7980 + 7881) (51) Then Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection Hence 1 = 28 + i 29, 2 = 38 + i 39, 3 = 42 + i 43, 4 = 44 + i 45, 5 = 46 + i 47, 6 = 48 + i 49, 7 = 50 + i 51, 8 = 52 + i 53, 9 = 54 + i 55, (46) On solving equations (41), (42), (43) we get the values of C4, C5 and C6 as ) ( ) ( 1 1 14 12 11 13 10 13 4 say n C                ) ( 15 13 14 12 5 say C        ) ( ) ( 16 9 14 7 15 8 6 say C          (47) Where 9 8 3 9 2 11 9 7 3 9 1 10 ) ( , ) (                 ) 48 ( ) ( , ) ( 9 9 5 8 6 13 9 7 6 9 4 12                 The values of 10, 11, …………., 16 are calculated in terms of 56, 57, ………….. RESULTS AND DISCUSSION IV. The variation of the velocity U with ζ at n=2.0, h=1.0 K=0.5, pj=5.0, p=4.0, pt=3.0, =1 for different values pl =1,5, 10 in case of =0, /3 is represented through Fig. (2.1). It is clear from the figure and numerical values that the velocity U decreases in the region 0ζ 0.2 and 0.8ζ1 and increases in the region 0.3ζ0.7 with an increase in the micro-polarity parameter pl in case of =0 whenever the behaviour of the velocity U in case of =/3 is just reversed to its behaviour in case of =0. The velocity is zero at the wall of the channel. ) 58 ( ) (" 0 2  f nv h k u   ) 58 ( From equation (57), we get From equation (57), we get From equation (57), we get                ) (' ) ('" ) (' 2 2 0 0      wf i f h k u g h k h x nv U x p ) ( 0 0   f h x nv U       (59)                ) (' ) ('" ) (' 2 2 0 0      wf i f h k u g h k h x nv U x p ) ( 0 0   f h x nv U       (59) (59) Fig. (2.2) represent the variation of the dimensionless velocity U with ζ at n=2, h=1, K=0.5, pl=2, pt=3, =1 for different values of pj=1,5,10 in case of =0, /3. It is seen from this figure and obtained numerical values that there is no much difference in the values of U w.r.t. different values of pj in case of =0, /3 but the values and branches of graph of U have significant difference in case of =0, /3. The branch of the velocity U in case of =0 is lying above the branch of U in case of =/3 throughout the gaplength. All the branches in the graphs of U are being overlapped in case of =0 and /3 separately. The velocity attains its maxima in the middle of the gaplenth. Fig. 3.1 From equations (8), (9) and (18), we get q ( ), ( ) ( ), g ) ( ) (' 0 0 2 0 0      g h x nv U h k x p wf i h x nv U                 ) ('" 0 0 2   f h x nv U h k u        (57) ) ( 1 ) ( 2 0 0     g h knv p h f wnv i      Hence the coefficient of skin friction on the lower and upper plates is given by Hence the coefficient of skin friction on the lower and upper plates is given by .1 0 2 2 0        and at u C yx f (57) Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1081 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 x h x nv U p x p          2 ) ,0 ( ) , ( 0 0           ) (' ) (''' ) (' 2 2     wf i f h k u g h k (62) d1sin(d2ζ))}+  3 d e {67(d3cos(d4ζ)- d4sin(d4ζ))+66(d3sin(d4ζ)+d4cos(d4ζ))}-  3 -d e {69(d3cos(d4ζ)+d4sin(d4ζ))+68(d4cos(d4ζ)- d3sin(d4ζ))}].sinτ (54) V=Real part of v(x, y)eiwt =[(72+82ζ)+  1 de {70cos(d2ζ)-71sin(d2ζ)}+  1 -d e {64cos(d2ζ)+65sin(d2ζ)}+  3 d e {66cos(d4ζ)- 67sin(d4ζ)}+  3 -d e {68cos(d4ζ)+69sin(d4ζ)}].cosτ- [(83ζ-73)+  1 de {71cos(d2ζ)+70sin(d2ζ)}+  1 -d e {65cos(d2ζ)- 64sin(d2ζ)}+  3 de {67cos(d4ζ)+66sin(d4ζ)}+  3 -d e {69cos(d4ζ)-68sin(d4ζ)}].sinτ (55) On putting =0 in both sides of the equation (62), we get On putting =0 in both sides of the equation (62), we get 7 0 0 2 )0,0 ( )0, ( xC h x nv U p x p           7 0 0 2 )0,0 ( )0, ( xC h x nv U p x p           (63) On substituting the value of p (x,0) in equation (61) we get On substituting the value of p (x,0) in equation (61) we get   ) (' 2 )0,0( ) , ( 0 7 0 0    f nv h k u xC h x nv U p x p            ) (' 2 )0,0( ) , ( 0 7 0 0    f nv h k u xC h x nv U p x p                        0 2 0 ) ( ) ( d wf i g h k hnv (64) 3 2 Skin Friction N = Real part of N(x, y)eiwt=Real part of N(cosτ + i sinτ) ={22(84+85)-23(88+89)+26(86+87)- 27(90+91}cosτ -{23(84+85)+22(88+89)+ 27(86+87)+26(90+91}sinτ (56) N = Real part of N(x, y)eiwt=Real part of N(cosτ + i sinτ) ={22(84+85)-23(88+89)+26(86+87)- 27(90+91}cosτ -{23(84+85)+22(88+89)+ 27(86+87)+26(90+91}sinτ (56) N = Real part of N(x, y)eiwt=Real part of N(cosτ + i sinτ) ={22(84+85)-23(88+89)+26(86+87)- 27(90+91}cosτ             0 2 0 ) ( ) ( d wf i g h k hnv (64)   } -{23(84+85)+22(88+89)+ 27(86+87)+26(90+91}sinτ (56) -{23(84+85)+22(88+89)+ 27(86+87)+26(90+91}sinτ (56) 3.2 Skin Friction Taking i = 1, j = 2 in equation (4) and converting into physical components x and y the shearing stress yx becomes where   wt is dimensionless phase parameter.             ) ( ) ('' 2 0 0    g f h k u h x nv a U h k yx (65) RESULTS AND DISCUSSION (2.3) represents the variation of U with ζ for different values of pt=1,5,10. ) ( ) (' ) (''' ) (' 2 2      F wf i f h k u g h k     Then at = 0 we get )0 (''' )0 (' )0 ( 2 2 7 f h k u g h k C F     (60) )0 (''' )0 (' )0 ( 2 2 7 f h k u g h k C F     (60) On integrating to equation (58) with respect  and taking the limit from  = 0 to  we obtain :   ) (' ) 0, ( ) , ( 1 0 2   f nv h k u x p x p h                  0 2 0 ) ( ) ( d wf i g h k nv (61) 1082 Published By: Blue Eyes Intelligence Engineering & Sciences Publication val Number: A1404109119/2020©BEIESP 10.35940/ijeat.A1404.029320             2 ) ( ) ( d wf i g h k (61) ating (59) w.r.t.x, we get 1082 Published By: Blue Eyes Intelligence Engineering & Sciences Publication (61) on integrating (59) w.r.t.x, we get 1082 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1082 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 V. Fig.5 exhibits the behaviour of the velocity V with ζ at n=2, h=1, K=0.5, pj=5, p=4, pt=3, for different values of pl=1,5,10 in case of =0, /3. Two separated curve are seeing in this figure, the above one is the curve of the case =0 (formed from the overlapped branches of pl=1,5,10) and the lower one is of the case =/3(for pl=1,5,10). It is also observed that the velocity V is decreasing with an increase in pl up to the middle and start increasing thereafter upto the wall ζ=1 in case of =0, /3 both. The velocity is minimum at the lower wall and maximum at the upper wall. In fig.6 the curves of the velocity V with different pj are similar to the curves of V with pl (fig.5) in case of =0, /3. The variation of V with pj has no definite trend in case of =0 whenever in case of =/3 the velocity V increases with an increase in pj up to the middle with its reversed behaviour thereafter. In fig.7 the velocity increases with an increase pt with up to the middle and decreases thereafter up to the wall ζ=1 in both the cases =0 and /3. In fig.8 the curves of the velocity V for different values of n= 2, 5, 8 are similar in shape as it’s curves in fig. 5,6,7. The velocity is being decrease with an increase in n throughout the gaplength in both the cases =0, /3. It is evident from the graphs of the dimensionless velocity U that the velocity U is maximum at the middle point of the gap length and vanishes at the surface of the plates. The velocity curves are parabolic with vertex upward. The values of the velocity at the phase =0 is maximum than its values at phase =/3. It is also concluded thus the dimensionless velocity V is also maximum at phase =0 than its value at =/3. The difference in the values of V w.r.t. different values of pl, pt and pj respectively is not significant at both the phases separately. Hence the branches of the velocity V w.r.t. pt, p1 and pj at the separate phase =0 and /3 are being overlapped whenever in case of the variation of V with n the branches are not being overlapped. ropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection The behaviour of the velocity U in this figure is similar to its behaviour seen in fig 2.2. Here the velocity U increase with an increase in pt in the regions 0ζ0.2 ; 0.8ζ1 and decreases in the region 0.3ζ0.7 in case =0. The behaviour U with pl in case =/3 is just reversed to that of its behaviour with pl in case =0. the cases =0, =/3. Fig.11 shows that the behaviour of microrotation velocity N with pt at =0 is similar to it’s behaviour with pj in fig.10. In case of =/3 no clear trend of micro-rotation is detected as it increases from pt=1 to 5 and decrease at pt=10 in the first half and shows reversed behaviour in the second half of the gaplength. The variation The variation of the velocity U with ζ at h=1 K=0.5, pj=5, p=4, pt=3, pl=2 for different values of n =2,5, 8 in case of =0, /3 is represented through Fig. 4. It is clear from this figure that in case of =0, value of U is maximum than its values in case =/3 for same n throughout the gaplength. It is also clear from obtained numerical values that the velocity U is increasing with an increase in n throughout the gaplength with its maximum value at the middle point of the gaplength. of micro-rotation velocity N with ζ for different values of n is represented through fig.12. The shape of the curves of N is similar approximately to the shape of figure.9, 10, 11. The micro-rotation velocity N increases with an increase in n in the first half and decreases in the second half of the gapelength in case of =0, /3 both. Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 V. It is also observed that the micro rotation N decreases with an increase in pj in the first half and increases in the second half of the second half of the gaplength in both 1083 Published By: Blue Eyes Intelligence Engineering & Sciences Publication 1083 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1083 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 Fig.2: Variation of U with ζ at τ =0 and τ=π/3 for different values of pj. Fig.3: Variation of U with ζ at τ =0 and τ=π/3 for different values of pt. Fig.4: Variation of U with ζ at τ =0 and τ=π/3for different values of n. Fig.5: Variation of V with ζ at τ =0 and τ=π/3 for different values of pl. Fig.6: Variation of V with ζ at τ =0 and τ=π/3 for different values of pj. International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 Fig.2: Variation of U with ζ at τ =0 and τ=π/3 for different values of pj. Fig.3: Variation of U with ζ at τ =0 and τ=π/3 for different values of pt. Fig.4: Variation of U with ζ at τ =0 and τ=π/3for different values of n. Fig.5: Variation of V with ζ at τ =0 and τ=π/3 for different values of pl. Fig.2: Variation of U with ζ at τ =0 and τ=π/3 for different values of pj. Fig.2: Variation of U with ζ at τ =0 and τ=π/3 for different values of pj. Fig.5: Variation of V with ζ at τ =0 and τ=π/3 for different values of pl. Fig.5: Variation of V with ζ at τ =0 and τ=π/3 for different values of pl. Fig.6: Variation of V with ζ at τ =0 and τ=π/3 for different values of pj. Fig.3: Variation of U with ζ at τ =0 and τ=π/3 for different values of pt. Fig.3: Variation of U with ζ at τ =0 and τ=π/3 for different values of pt. Fig.6: Variation of V with ζ at τ =0 and τ=π/3 for different values of pj. Fig.6: Variation of V with ζ at τ =0 and τ=π/3 for different values of pj. V. It is also observed that there is no microrotation at the surface of the plates and in the middle of the gaplength whenever the microrotation is maximum in the middle of the first half and maximum in magnitude in the middle of the second half of the gap length. Fig.1: Variation of U with ζ at τ =0 and τ=π/3 for different values of pl. Fig.9 represents the behaviour of the micro-rotation velocity function N at n=2, h=1, K=0.5 pj=5, p=4, pt=3 for different values of pl=1,5,10 in case of  = 0 and /3. It is evident from this figure that N is zero at ζ=0, 0.5 and 1.0 in both the cases =0 and /3. It is also evident from the figure that the micro-rotation function N increases with an increase in pl in the first half of the gaplength and start decreasing in the second half in both the case =0 and /3. The value of the function N is maximum in the middle of the first half and minimum in the middle of the second half of the gaplenth in both the cases =0 (except pl=1)and =/3 (except pl=1). The result of maxima and minima at pl=1 is reverse to that if pl=5,10 in both the cases. Fig.1: Variation of U with ζ at τ =0 and τ=π/3 for different values of pl. Fig.10 depicts the behaviour of the micro-rotation velocity N with ζ at n=2, h=1, ξ=1, K=0.5, pl=2, pt=3 for different values of pj=1,5,10. It is evident from this figure that the velocity is zero at lower (ζ=0), middle (ζ=0.5) and upper (ζ=1) wall with it’s maximum in the middle of the first half and minimum in the middle of the second half of 1083 Published By: Blue Eyes Intelligence Engineering & Sciences Publication of N s h the gaplength. Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 V. Fig.4: Variation of U with ζ at τ =0 and τ=π/3for different values of n. 084 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Published By: Blue Eyes Intelligence Engineering & Sciences Publication Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1084 Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection Fig.7: Variation of V with ζ at τ =0 and τ=π/3 for different values of pt. Fig.8: Variation of V with ζ at τ =0 and τ=π/3 for different values of n . Fig.9: Variation of N with ζ at τ =0 and τ=π/3for different values of pl. Fig.10: Variation of N with ζ at τ =0 and τ=π/3 for different values of pj. Fig.11: Variation of N with ζ at τ =0 and τ=π/3 for different values of pt. Fig.12: Variation of N with ζ at τ =0 and τ=π/3 for tes with Different Periodic Suction and Injection Fig 10: Variation of N with ζ at τ =0 and τ=π/3 for Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection Fig.7: Variation of V with ζ at τ =0 and τ=π/3 for different values of pt. Fig.8: Variation of V with ζ at τ =0 and τ=π/3 for different values of n . Fig.9: Variation of N with ζ at τ =0 and τ=π/3for different values of pl. Published By: Blue Eyes Intelligence Engineering & Sciences Publication Fig.10: Variation of N with ζ at τ =0 and τ=π/3 for different values of pj. Fig.11: Variation of N with ζ at τ =0 and τ=π/3 for different values of pt. Fig.12: Variation of N with ζ at τ =0 and τ=π/3 for different values of n . Fig.7: Variation of V with ζ at τ =0 and τ=π/3 for different values of pt. Fig.7: Variation of V with ζ at τ =0 and τ=π/3 for different values of pt. Fig.10: Variation of N with ζ at τ =0 and τ=π/3 for different values of pj. Fig.10: Variation of N with ζ at τ =0 and τ=π/3 for different values of pj. Fig.8: Variation of V with ζ at τ =0 and τ=π/3 for different values of n . Fig.9: Variation of N with ζ at τ =0 and τ=π/3for different values of pl. REFERENCE 3, No.1, January-June (2012). 1. Eringen, A.C., Theory of micropolar fluids. J Math. Anal. Appl. 38(1972) 480-496. 1. Eringen, A.C., Theory of micropolar fluids. J Math. Anal. Appl. 38(1972) 480-496. y  Thin Film Flow of an Electrically Conducting Micropolar Fluid Down an Inclined Plane. K.R. Singh, Preeti, Ajit Kumar International Journal of Mathematics & Computing Applications (IJMCA), Vol. 4, No.1, June(2012), P:1-12.  Thin Film Flow of an Electrically Conducting Micropolar Fluid Down an Inclined Plane. K.R. Singh, Preeti, Ajit Kumar International Journal of Mathematics & Computing Applications (IJMCA), Vol. 4, No.1, June(2012), P:1-12. 2. Eringen, A.C., Theory of microcontinunm field theories II: Fluent Media, Springer, New York,2001. 3. Ishak, A., R. Nazar, I. Pop, Moving wedge and flat plate in a micropolar fluid, Int. J. Eng. Sci. 44(2006) 1225-1236. ( ) ( )  Flow of a Micropolar Fluid Between Two Porous Discs with Uniform Suction. Preeti, Ajit Kumar, K.R. Singh International Journal of Emerging Research in Management & Technology (IJERMT), Vol .5 , Issue 4, April (2016). 4. Lulaszewicz, G., Micropolar fluids-theory and application, Birkhauser, Basel,1999. Preeti, Ajit Kumar, K.R. Singh International Journal of Emerging Research in Management & Technology (IJERMT), Vol .5 , Issue 4, April (2016). 5. Berman, A.S., Laminar flow in channels with porous walls, J. Appl. Phys., 24(1953) 1232. 6. Sellers, J.R., Laminar flow in channels with porous walls at high suction Reynolds number, J. Appl. Phys., 26(1955) 489.  Flow of an Electrically Conducting Micropolar Fluid Down a Vertical Cylinder. Ajit Kumar, Preeti, K.R. Singh International Research Journal of Mathematics, Engineering and IT (IRJMEIT),Vol. 3 , Issue 4, April ( 2016). 7. Terrill, R.M., Laminar flow in uniformly porous channels, Aero. Quart., 15(1964) 299. 8. Yuan, S.W., Further investigation of Laminar flow in channels with porous walls , J. Appl. Phys., 27(1956) 267. Experiences: 13 years of teaching ❖ ❖ Worked as Lecturer at RGEC, Meerut from 28- 08-2006 to 21-07-2008.  M.Sc. Mathematics from Meerut College, Meerut (C.C.S. University Meerut) in 2004 with 1st division. ❖ Worked as Lecturer at IEC Engg. College, Greater Noida from 31-08-2008 to 17-11- 2009. Educational Qualification: p ❖ M.Sc. (Mathematics) (2004), from Meerut College, Meerut affiliated to Chaudhary Charan Singh University, Meerut in first division with 63.7%.  Ph.D. on “Study of some flow and heat transfer problems in non-Newtonian Reiner-Rivlin fluid” from department of fluid” from department of mathematics; Meerut College, Meerut affiliated to C.C.S. University Meerut in 2011. . Preeti Contact: +91- 7042744487 Present Address Email preeti14julyster@gmail.com C-103 Stellar MI City Homes Greater Noida Contact: +91- 7042744487 Present Address Email preeti14julyster@gmail.com C-103 Stellar MI City Homes Greater Noida International Journal of Engineering and Advanced Technology (IJEAT) ISSN: 2249 – 8958, Volume-9 Issue-3, February, 2020 International Journal of Mathematics & Applied Statistics, Vol. 3 N 1 J J (2012) International Journal of Mathematics & Applied Statistics, Vol. 3, No.1, January-June (2012). International Journal of Mathematics & Applied Statistics, Vol. 3, No.1, January-June (2012). Research Papers: ❖ Worked as Senior Lecturer at Sunderdeep Engg College, Ghaziabad from 18-11-2009 to 15-07-2012. ❖ Worked as Senior Lecturer at Sunderdeep Engg College, Ghaziabad from 18-11-2009 to 15-07-2012.  Flow of a Non-Newtonian Reiner-Rivlin Fluid Over an Enclosed Torsionally Oscillating Disc with Uniform Suction and Injection. K.R. Singh, Ajit Kumar Journal of Reflection des ERA-JMS, Vol. 4, Issue 1, (2009) P: 41-64. ❖ Worked as Assistant Professor at IIMT Engg. College, Meerut Since 18 July 2012 to 25 January 2019. ❖ Worked as Assistant Professor at IIMT Engg. College, Meerut Since 18 July 2012 to 25 January 2019. ❖ Working as Assistant Professor at Noida Institute Of Engineering of Technology Greater Noida from 28 January 2019 to till date.  Flow of a Non-Newtonian Reiner-Rivlin Fluid Between Two Enclosed Torsionally Oscillating Discs. K.R. Singh, Ajit Kumar Journal of The Mathematics Education, Vol. XLIII, No.1, March (2009) P: 32. V. Fig.11: Variation of N with ζ at τ =0 and τ=π/3 for different values of pt. Fig.11: Variation of N with ζ at τ =0 and τ=π/3 for different values of pt. N Fig.9: Variation of N with ζ at τ =0 and τ=π/3for different values of pl. Fig.9: Variation of N with ζ at τ =0 and τ=π/3for different values of pl. 1085 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Fig.12: Variation of N with ζ at τ =0 and τ=π/3 for different values of n . Fig.12: Variation of N with ζ at τ =0 and τ=π/3 for different values of n . Fig.12: Variation of N with ζ at τ =0 and τ=π/3 for different values of n . Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 Educational Qualification: ❖ Ph.D. awarded from Meerut College, Meerut affiliated to Chaudhary Charan Singh University, Meerut on entitled “Computational Techniques for flows of a micropolar fluid”. AUTHOR PROFILE Dr. AJIT KUMAR E-Mail: ajit.chauhan79@gmail.com, ajit.computers@tmu.ac.in Mob No: 9837942422 Educational Qualification:  Ph.D. on “Study of some flow and heat transfer problems in non-Newtonian Reiner-Rivlin fluid” from department of mathematics; Meerut College, Meerut affiliated to C.C.S. University Meerut in 2011. Work Experience: 9. Terrill, R.M. and G.M. Shrestha, Laminar flow in uniformly porous channel with an applied transverse magnetic field, Appl. Sci. Res. B12(1965) 203.  I have four years teaching experience, worked as tutor in Meerut College, Meerut, from 31-10-2007 to 05-02-2011 till. Meerut College, Meerut, from 31-10-2007 to 05-02-2011 till. 10. Singh K.R. and V.K. Agarwal, Flow of a second-order fluid between torsionally oscillating discs of different permeability, Int. J.M.C.S. & I.T., 1, 2008, 103-122.  I have also one year teaching experience, worked as Assistant Professor in S.V.S. Engineering College, Mawana, Meerut from 01-08-2011 to 18-08-2012 till.  Presently working as Assistant Professor in Teerthanker Mahaveer University Moradabad, from 24-08-2012 to till date. 11. Asharaf, M. and S. Bashir, Numerical solution of MHD stagnation point flow and heat transfer of a micropolar fluid towards a heated shrinking sheet. Int. J. Number. Math. Fluid, 69 (2012) 384. 12. Srinivasacharya, D. et.al., Unsteady stokes flow of micropolar fluid between two parallel porous plates Int. J. Sci. 39(2001) 1557-1563. . Preeti Contact: +91- 7042744487 Present Address Email preeti14julyster@gmail.com C-103 Stellar MI City Homes Greater Noida Journal Published Journal Published ❖ Preeti ,Kavi Raj Singh,Ajit kumar “Thin film flow of an electrically conducting micropolar fluid down an inclined plane”, International Journal of mathematics and computing applications.(ISSN :0976-6790). IJMCA,Vol 4 ,No1 January-June 2012 (page 1- 12) Published. ❖ Preeti ,Kavi Raj Singh,Ajit kumar “Thin film flow of an electrically conducting micropolar fluid down an inclined plane”, International Journal of mathematics and computing applications.(ISSN :0976-6790). IJMCA,Vol 4 ,No1 January-June 2012 (page 1- 12) Published. ( )  Heat Transfer in the Forced Flow of a Non-Newtonian Reiner- Rivlin Fluid Between Two Porous Discs of Different Permeability. K.R. Singh, Shubhi Singhal, Ajit Kumar International Journal of Fluid Mechanics, Vol. 1 (2), July- December (2009), P: 167-180. International Journal of Fluid Mechanics, Vol. 1 (2), July- December (2009), P: 167-180.  Heat Transfer in the Flow of Reiner-Rivlin Fluid Between Two Enclosed Counter Rotating Discs. K.R. Singh, Ajit Kumar International Journal of Mathematics & Applications, Vol. 2, No. 1-2, December (2009), P: 103-111. Published By: Blue Eyes Intelligence Engineering & Sciences Publication International Journal of Mathematics & Applications, Vol. 2, No. 1-2, December (2009), P: 103-111.  Flow of an Electrically Conducting Micropolar Fluid Flow on a Moving Belt. K.R. Singh, Preeti, Ajit Kumar K.R. Singh, Preeti, Ajit Kumar Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 Dr. AJIT KUMAR E-Mail: ajit.chauhan79@gmail.com, ajit.computers@tmu.ac.in Mob No: 9837942422 Dr. AJIT KUMAR E-Mail: ajit.chauhan79@gmail.com, ajit.computers@tmu.ac.in Mob No: 9837942422 Educational Qualification: Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320 1086 Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection ❖ Preeti ,Kavi Raj Singh,Ajit kumar “Flow of an l t i ll d ti i l fl id fl Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection Flow of Micropolar Fluid Between Two Parallel Plates with Different Periodic Suction and Injection ❖ Preeti ,Kavi Raj Singh,Ajit kumar “Flow of an electrically conducting micropolar fluid flow on a moving belt”. International Journal of mathematics and applied statistics”(ISSN:0973-5739). IJMAS, Vol 3 ,No1(2012) Published. ❖ Preeti, Kavi Raj Singh,Ajit Kumar “Flow of a micropolar fluid between two porous discs with uniform suction. " International Journal of Emerging Research in Management & Technology. " International Journal of Emerging Research in Management & Technology. gy (ISSN :2278-9359), Volume-5, Issue-4, April 2016. (Published) 4 . Ajit Kumar, Preeti , Kavi Raj Singh" Flow of an electrically conducting micropolar fluid down a vertical cylinder. "International Research Journal of mathematics, Engineering and 4 . Ajit Kumar, Preeti , Kavi Raj Singh" Flow of an electrically conducting micropolar fluid down a vertical cylinder. "International Research Journal of mathematics, Engineering and IT, Vol.3, Issue 4, "International Research Journal of mathematics, Engineering and IT, Vol.3, Issue 4, IF-3 563 (ISSN: 2349-0322) April 2016 ( Published) IF-3.563, (ISSN: 2349-0322), April 2016. ( Published). Associated Asia Research Foundation (AARF). 1087 Published By: Blue Eyes Intelligence Engineering & Sciences Publication 1087 Published By: Blue Eyes Intelligence Engineering & Sciences Publication 1087 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: A1404109119/2020©BEIESP DOI: 10.35940/ijeat.A1404.029320
https://openalex.org/W4387409025	https://link.springer.com/content/pdf/10.1007/s00382-023-06981-7.pdf	English	null	Correction to: Characteristics of clustered heavy precipitation events at Northeast China and associated atmospheric circulations	Climate dynamics	2,023	cc-by	469	Correction to: Characteristics of clustered heavy precipitation events at Northeast China and associated atmospheric circulations Shunli Jiang1 · Tingting Han1,2,3 · Botao Zhou1,2 · Qiushi Zhang4 · Xin Hao1,2,3 · Huixin Li1,2,3 Published online: 6 October 2023 © The Author(s) 2023 Published online: 6 October 2023 © The Author(s) 2023 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/. Climate Dynamics (2023) 61:5935 https://doi.org/10.1007/s00382-023-06981-7 Climate Dynamics (2023) 61:5935 https://doi.org/10.1007/s00382-023-06981-7 CORRECTION CORRECTION Correction to: Climate Dynamics https://doi.org/10.1007/s00382-023-06944-y In this article the statement in the Funding information sec tion was incorrectly given as “This work was jointly supported by Guangdong Major Project of Basic and Applied Basic Research (Grant No. 2020B0301030004) and the National Natural Science Foun dation of China (Grants Nos. 42025502 and 41875119)” Publisher’s Note Springer Nature remains neutral with regard to juris dictional claims in published maps and institutional affiliations. “This work was jointly supported by Guangdong Major Project of Basic and Applied Basic Research (Grant No. 2020B0301030004) and the National Natural Science Foun dation of China (Grant No. 41875119)” The original article has been corrected. 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 The online version of the original article can be found at https://doi. org/10.1007/s00382-023-06944-y. The online version of the original article can be found at https://doi. org/10.1007/s00382-023-06944-y. Tingting Han hantt08@126.com Tingting Han hantt08@126.com hantt08@126.com 1 School of Atmospheric Sciences, Nanjing University of Information Science and Technology, Nanjing, China 2 Collaborative Innovation Center on Forecast and Evaluation of Meteorological Disasters/Key Laboratory of Meteorological Disaster, Ministry of Education, Nanjing University for Information Science and Technology, Nanjing, China 3 Nansen-Zhu International Research Centre, Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing, China 4 Heilongjiang Sub-Bureau of Northeast Air Traffic Management Bureau, Ha’erbin, China 1 School of Atmospheric Sciences, Nanjing University of Information Science and Technology, Nanjing, China 2 Collaborative Innovation Center on Forecast and Evaluation of Meteorological Disasters/Key Laboratory of Meteorological Disaster, Ministry of Education, Nanjing University for Information Science and Technology, Nanjing, China 3 Nansen-Zhu International Research Centre, Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing, China 4 Heilongjiang Sub-Bureau of Northeast Air Traffic Management Bureau, Ha’erbin, China 1 3 1 3
W4386895652.txt	https://journals.wlb-stuttgart.de/ojs/index.php/wfr/article/download/7777/7659	de		Rezension von: Mattern, Hans; Wolf, Reinhard, Die Haller Landheg	Württembergisch Franken	2,023	cc-by	1,119	Einzelne Orte 12. Gymnasium bei Walter St. Michael i. A. Hampele chael e. V. - Einzelne Hall. Schwäbisch 14. Juli am Orte Geschichte und Geschichten / hrsg. des Freundeskreises alter Schwäbisch Hall: Mahl, 1990. Schulleiterwechsel - 323 Haller Pennäler Gymnasium bei von St. Mi- 127 S., 111. 1990 Gymnasium bei St. Michael Schwäbisch Hall. 68 S. - (Synthesis 1990; Sonderh.) Walter langjähriger erfolgreicher, zum Ende des Schuljahrs 1990 aus dem geschiedener Leiter des Haller Gymnasiums bei St. Michael, hat im Auftrag Hampele, aktiven Dienst Freundeskreises des Arbeit Hampeles 1980« alter erschienen - »Schola latina und reizvolle Weise schaulicht der 1980 in Gymnasium illustre Anhand einen illustre schmucken Band bei Gymnasium zum mit Verbindung dem Faksimiledruck in Schwäbisch Hall« dem aus 1811 Wilhelm von Text- und sorgfältig ausgewählter der herausgegeben, St. Michael Jahr bis Kolbs 1916 Bildquellen Herausgeber die Entwicklung der renommierten Anstalt seit - auf veran- ihrer Wieder- 1868. »Lehrerconvents-Protokollen« haltensregeln für die Schüler« Schüler Pennäler Gymnasium ergänzt. herstellung im Jahr Aus den Haller »Vom werden wichtige Beschlüsse zitiert; aus den Jahren 1894 bzw. aus verpflichtet war, »nur dann zu reden und 1901 antworten, zu den »Ver- geht u.a. hervor, daß jeder wenn er von dem Lehrer Nachmurmelns oder sonsti- dazu aufgefordert wird und insbesondere sich des Einblasens, drakonische Karzerstrafen konnten zahlreiche Schüler gen Geflüsters zu enthalten«. Auch vom allseits beliebten Kneipenbesuch abhalten. Auf die Entwicklung der Schule in nicht der ersten Hälfte Schüler, etwa des von 20. Jahrhunderts Rudolf Kraiß bunte Schlaglichter. dem Hall 1990 die Erinnerungen ehemaliger Lehrer Dürr oder von Hans Taxis Wagner, und andere schulische Ereig- Sonderheft erfolgte Schulleiterwechsel am der Schulzeitschrift Gymnasium bei »Synthesis« ist St. Michael Mattern; Sigmaringen: Reinhard am M. Akermann Wolff: Die 1990. Thorbecke, der Schwäbisch in erschöpfend dokumentiert. Hans und und Carl gute Bildauswahl bereicherte Quellenkunde ab. erschienenen gleichzeitig 14. Juli werfen Ilse Zeitungsberichte über Schulschlußfeiern nisse runden die durch eine In und - 175 S.: Haller Landheg. 154 111., 3Ktn. Ihr Verlauf und (Forschungen ihre aus Reste. - Württember- gisch Franken; Bd. 35) Seit im Jahr 1936 in unserer Zeitschrift der Aufsatz große Karl von »Die Schümm Hällische Landheg« erschienen ist, hat man sich kaum mehr mit dieser rund 200 Kilometer das Territorium der Reichsstadt langen Grenzmarkierung rund um jetzt erschienene Monographie vernachlässigten Thema eine Hans Mattern von neue und Wendung. Beide Reinhard Hall Wolf Autoren haben sich beschäftigt. gibt dem Die etwas im Rahmen ihrer Tätigkeit an der Bezirksstelle für Naturschutz in Stuttgart der aufwendigen und mühsamen Arbeit unterzogen, den tatsächlichen Verlauf der Haller Landheg zu erforschen. Archivalische Studien, aber ausgedehnte Wanderungen, Forschen gen und Profilaufnahmen lückenlos zu Flügelhegen, und die aller Sichtung im erfassen. machen Die es Autoren Kartenwerke im möglich, können und Luftaufnahmen, sichtbaren Reste vor allem Gelände, Streckenvermessungen, Grabundas den bedeutende komplizierten Kulturdenkmal Verlauf Sperrvorrichtungen an Straßen und Wegen die Türme und Gelände einschlägigen im einzelnen schildern. der nahezu Heg, der genau darstellen Instruktive Abbildungen, Skizzen und Kartenausschnitte geben ein anschauliches Bild der jeweiligen Situation. Die Streitfrage, möchten die entscheiden. gebaut ob die Heg Autoren, Sie worden als die Rechtsgrenze oder als Verteidigungslinie zu verstehen sei, Historiker verstehen, nicht als Geographen, nicht als sich die Landheg neigen der Auffassung zu, daß daß ihre Bedeutung sich jedoch allmählich ist, schließlich die Territorialgrenze bezeichnete. Das Buch von als Verteidigungsanlage verschoben hat und sie Hans Mattern und Reinhard 324 Neue Bücher Wolf über die Haller Landheg ist ein Glanzstück geographischer Spurensuche. Es will kann eine neue grundlegende Arbeit zur Geschichte des hällischen Gebiets und der Landheg nicht ersetzen. E. Göpfert Gerhard Fritz: Stadt und Kloster Murrhardt im zeit. - Nach 1990. Sigmaringen: Thorbecke, gisch Franken; seiner Hochmittelalter Murrhardt im - Spätmittelalter und in der Reformations- 421 S.: zahlr. 111. (Forschungen Württember- aus Bd. 34) 1982 erschienenen Früh- und Monographie Gerhard legt Spätmittelalter und in der der Geschichte des Fritz den nun Klosters Murrhardt im Folgeband »Stadt Reformationszeit« vor: angelegter Auswertung aller einschlägigen Quellen Ein erarbeitetes und in und zum Kloster intensiver, breit entsprechend umfang- reiches wissenschaftliches Werk, das erschöpfend und wohl auch abschließend was und damit auch alles bietet, Thema zu sagen ist. Ein ausführliches Inhaltsverzeichnis und ein Register erschlie- ßen die Stoff- und Ereignisfülle, die Gerhard Fritz in sicher entsagungsvoller Forschungsarbeit zu bewältigen hatte und die hier im Mit- und Gegeneinander ein nur angedeutet werden kann. Stadt und Kloster teilen wechselvolles Schicksal: Löwensteinische Herrschaft, wir- tembergische Vogtei, österreichische Statthalterregierung, Bauernkrieg und Reformation, Zeiten der Blüte und Zeiten des Niedergangs. Neben die treten wirtschafts-, Grundlagen der Patronate und sozial- und klösterlichen kulturhistorische Herrschaft, ihre Darstellung der Ereignisgeschichte Untersuchungen: Rechte, Die wirtschaftlichen Besitzungen und Erwerbungen, Inkorporationen, Religiosität und kulturelles Leben und ein Kapitel, das man als eine Art Buch im Buch bezeichnen kann, eine eigenständige Sozial- und Wirtschaftsgeschichte der Stadt Murrhardt. Hinzu kommen der Stadt im Rahmen Klosterpfarreien und Pfründen. Die Forschungsleistung Sorgfalt und Genauigkeit, ihre 15. und 16. Jahrhundert, weiterhin der Äbte und Konventualen des Klosters und kommentierte Listen durch Untersuchungen zum militärischen Aufgebot des wirtembergischen Heeres im durch das der Geistlichen auf den Gerhard Fritz beeindruckt von Beharren geduldige auf der konkreten Einzelheit und auf der sorgfältigen Analyse. Alles wird in nahezu gleicher Ausführlichkeit festgehalten und berichtet. Dem geduldigen Leser drängt sich alles, was schreiben den Akten und unsere Urkunden zu entnehmen ist, Historiker? Für ihresgleichen, also scher? Oder wollen sie auch ein größeres oder müßte man dann Geschichte schreiben? gar Frage auf, ob denn auch berichtenswert ist. da die Für wen für andere Wissenschaftler und For- großes Publikum erreichen? Spätmittelalter und in der Reformationszeit« jedenfalls ist wohl kein am historischen belehrendes Buch Fachbuch auch zum das Lesevergnügen schätzt; Nachschlagen und es Benutzen. ist Als Buch für den Leser, der ein sehr solches unentbehrlich. Stadt - Bild - Und wie Das Buch über »Stadt und Kloster Murrhardt im ist gelehrtes und es in der Tat E. Göpfert Geschichte. Reutlingen in Ansichten aus fünf Jahrhunderten / hrsg. Stadt Reutlingen. - Reutlingen: Oertel und Spörer, 1990. - von der 288 S.: zahlr., z.T. färb. 111. Im Rahmen des Stadtjubiläums »900Jahre Reutlingen« veranstalteten Heimatmuseum und Stadtarchiv Reutlingen im Sommer 1990 eine Ausstellung, in der der reiche Vedutenschatz, mit dem die Stadt am Fuß der Achalm aufwarten kann, ausgebreitet wurde. Ähnlich wie der Katalog der etwa zur gezeigten Ausstellung und tadelsfrei Betrachtung Dr. Heinz selben Zeit »Ansichten im aus Hällisch-Fränkischen Museum in Schwäbisch Hohenlohe« enthält die gedruckte Veröffentlichung weit mehr als nur Hall hervorragend aufgemachte nützliche Hinweise für die der Exponate. Der Katalog ist vielmehr, was dem Willen der beiden Bearbeiter, Alfred Gemeinhardt (Stadtarchiv) und Dr. entspricht, eine »Stadt-Bild-Geschichte« Bilddokumentationen zum alten und im besten neuen Werner Ströbele (Heimatmuseum), Sinne, mit der bereits vorliegende reine Reutlingen hervorragend ergänzt werden. Ein sechsköpfiges Autorenteam gliederte das umfangreiche Material in lockerer chronologischer Folge nach besonderen historischen Ereignissen, etwa dem Stadtbrand von 1726 oder dem
https://openalex.org/W2751884883	https://www.frontiersin.org/articles/10.3389/fcell.2017.00076/pdf	English	null	Editorial: Signaling Pathways in Embryonic Development	Frontiers in cell and developmental biology	2,017	cc-by	2,162	Juan J. Sanz-Ezquerro 1, Andrea E. Münsterberg 2 and Sigmar Stricker 3 1 Department of Molecular and Cellular Biology, National Center for Biotechnology (CSIC), Madrid, Spain, 2 School of Biological Sciences, University of East Anglia, Norwich, United Kingdom, 3 Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany Keywords: signaling pathways, embryo development, mechanotransduction, notch signaling pathway, Wnt Signaling Pathway, Shh signaling Signaling Pathways in Embryonic Development The formation of a complex multicellular organism from a single cell is one of the most amazing processes of biology. Embryonic development is characterized by the careful regulation of cellular behaviors such that cells proliferate, migrate, diﬀerentiate, and form tissues at the correct place and time. These processes are genetically controlled and depend both on the history of cells, their lineage, and on the activities of signaling pathways, which coordinate the cell interactions leading to organogenesis. g g A limited number of key signaling pathways—Fgf, Hedgehog, Wnt, TGFß, Notch among the most important—operate during development, acting repeatedly at diﬀerent times and in diﬀerent regions in the embryo and eliciting diverse cellular responses. This raises the question of how cells integrate all the information they receive and can respond in cell type-speciﬁc ways to the same signals. Classical concepts in embryology such as organizers (groups of cells producing instructive signals) and competence (ability of cells to respond) can now be analyzed in molecular terms. In recent years many advances have been made in identifying the signals acting during embryo development and understanding their properties and functions, which is equally of relevance for human pathology and evolution. An important discovery is the conservation of signals and mechanisms, not only in evolutionary terms (similar genes and signals acting in distant organisms), but also in the repeated use of the same signaling pathways at diﬀerent times and places in the embryos. Moreover, many of those mechanisms are involved in adult tissue homeostasis and regeneration. EDITORIAL EDITORIAL published: 31 August 2017 doi: 10.3389/fcell.2017.00076 published: 31 August 2017 doi: 10.3389/fcell.2017.00076 Edited and reviewed by: Pedro M. Fernández-Salguero, University of Extremadura, Spain Correspondence: Juan J. Sanz-Ezquerro jjsanz@cnb.csic.es Edited and reviewed by: Pedro M. Fernández-Salguero, University of Extremadura, Spain Edited and reviewed by: Pedro M. Fernández-Salguero, University of Extremadura, Spain *Correspondence: Juan J. Sanz-Ezquerro jjsanz@cnb.csic.es g Understanding developmental signaling pathways is important for several reasons. It gives us information about basic mechanisms of cell function and interactions needed for morphogenesis and organogenesis. It uncovers the basis of congenital malformations, since errors at any step of cell signaling during development are a major cause of defects. Fundamental insight also gives us clues to understand the mechanisms operating in evolution that explain diversity in form and function. And ﬁnally, it allows the identiﬁcation of possible causes of disease in the adult organism (such as cancer or degenerative diseases) pinpointing possible targets for therapeutic approaches. Specialty section: This article was submitted to Signaling, a section of the journal Frontiers in Cell and Developmental Biology Received: 02 August 2017 Accepted: 11 August 2017 Published: 31 August 2017 Specialty section: This article was submitted to Signaling, a section of the journal Frontiers in Cell and Developmental Biology Received: 02 August 2017 Accepted: 11 August 2017 Published: 31 August 2017 Citation: Sanz-Ezquerro JJ, Münsterberg AE and Stricker S (2017) Editorial: Signaling Pathways in Embryonic Development. Front. Cell Dev. Biol. 5:76. doi: 10.3389/fcell.2017.00076 In this context, the aim of the Frontiers research topic “Signaling pathways in embryonic development” has been to provide a forum for experts in cell and developmental biology to share recent advances in the ﬁeld of signaling during embryonic development. Sixteen articles in a variety of formats are united in this Topic, oﬀering a valuable collection for researchers looking for an update in the knowledge of signaling pathways operating during embryogenesis. The works, focused mainly on vertebrates, explore diﬀerent aspects of this theme from cell communication to organ formation and have implications for areas as distant as evolution or pathology. Citation: Among the signaling pathways with important and widespread roles in development is the Wnt pathway, comprising a family of ligands with homology to wingless in Drosophila. Wnts can bind August 2017 \| Volume 5 \| Article 76 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 1 Editorial: Signaling Pathways in Embryonic Development Sanz-Ezquerro et al. to multiple receptor complexes and trigger several downstream signaling cascades [including the so-called canonical WNT/β- catenin dependent signaling pathway, the non-canonical WNT/planar cell polarity (PCP), and the WNT/Ca2+ pathways], illustrating how the same signal can elicit diverse cellular responses depending on the cell type, context, and developmental timing. Fujimura reviews the role of canonical Wnt signaling in eye development, highlighting the important roles it plays in patterning of ocular tissue, diﬀerentiation of retinal pigment epithelium, and morphogenesis of the optic cup. Importantly, mis-regulation of the signaling cascade can lead to eye malformations and disease. Gentzel and Schambony review a group of core intracellular eﬀectors of the Wnt pathway, disheveled (DVL) proteins, which comprise three members in vertebrates. Although all DVLs share a common basic function in Wnt signaling, the expression patterns, and functions of the diﬀerent isoforms are not totally redundant and have also diverged between diﬀerent species, suggesting they play speciﬁc roles depending on the tissue distribution and speciﬁc interactions. Again, mutations in DVL genes can cause human congenital disease, highlighting their important role in development. Additionally, Berger et al. review the role of PTK7 (protein tyrosine kinase 7, a transmembrane receptor) in the ﬁne-tuning of the Wnt signaling network. Its functions in establishing cell polarity, regulation of cell movements, and migration are also essential for development and disease, particularly in cancer and metastasis. neuromesodermal population located at the caudal part of the embryo. Extension of this region is coupled to spinal cord formation and several essential processes such as neurogenesis, ventral patterning or neural crest speciﬁcation are controlled by Fgf signaling. These embryonic functions of Fgfs could be related to its ability to promote regeneration in the injured spinal cord of adults. Signalling pathways often converge on controlling the expression of transcription factors, which regulate cell fate speciﬁcation. The integration of Notch signaling and bHLH transcription factors during inner ear development is analyzed by Gálvez et al. which also highlight that these same mechanisms are involved in hair cell regeneration, opening avenues for possible therapeutic approaches in hearing impairment. Citation: Ear development is also the topic reviewed by Magariños et al. They present evidence for a crucial role of autophagy, the regulated process of degradation, and recycling of cellular components, in vertebrate inner ear formation. The limb is a classic model in embryology and some of the most important discoveries related to the roles of signaling pathways in pattern formation, growth, and diﬀerentiation have been made studying limb development. Tickle and Towers review the role of Shh in this process, a paradigm of how signals control and integrate tissue pattern and growth. They also discuss the implications of this important pathway for congenital malformations in humans and for the generation of limb morphological diversity during evolution. Montero et al. also treat this evolutionary aspect in their perspective article. They present a detailed analysis of Sox9 expression in developing digits of several species. This transcription factor, regulated by signaling pathways such as BMPs, Tgfßs, or Fgfs is involved in formation of the chondrogenic template of the skeleton. Diﬀerences in Sox9 expression patterns among species that have speciﬁc morphologies may reﬂect diﬀerences in signaling pathways controlling its expression. Also related to skeletal development, Amara et al. show that the eﬀects of Calcium/Calmodulin dependent kinase II (CAMKII), an eﬀector for Ca+2 -dependent signal transduction, in promoting chondrogenic diﬀerentiation seems to be speciﬁc for chicken embryos. This function is not observed in the mouse, thus highlighting the existence of diﬀerences in signaling functions and regulation among diﬀerent species. Another important signaling pathway is Notch, a transmembrane protein that mediates juxtacrine cell-cell communication. Notch has many functions in organ formation and adult homeostasis, including cell determination and stem cell maintenance. Carrieri and Dale review the particularly well-studied function of Notch in somitogenesis and also present recent data on the role of FBXW7 protein in regulating the turnover of Notch intracellular domain (NICD, the eﬀector of the pathway), in development and cancer. This relates to an often-overlooked essential point in signaling, which is the termination of activation and resetting of the components, allowing the cells to become competent again. Multiple mechanisms of regulation exist (positive and negative feedback loops) that allow a ﬁne control of signaling pathways at diﬀerent steps of the intracellular cascades. Frontiers in Cell and Developmental Biology \| www.frontiersin.org Citation: Crosstalk between the limited numbers of signaling pathways is a mechanism that allows cells to respond diﬀerently to the same signal, producing the diverse cellular behaviors that are needed to build tissues and organs. A new example of this is provided by Bernatik et al. reporting on the role of the BMP antagonist Noggin in sensitizing cells and potentiating the activation of non-canonical Wnt signaling in skeletal development. They also provide evidence for a genetic interaction between these two pathways, which are involved in human congenital malformations. p Integration of extrinsic and intrinsic regulatory cues is essential for organ formation. Dueñas et al. review the role of signals, transcription factors and cellular processes in the formation of the epicardium. This is the external-most layer of the heart that serves not only as the outer cover for this organ, but also seems to play a role in regeneration. Thus, understanding the basis of its development may have important therapeutic implications. Two articles deal with muscle development. Hernandez-Torres et al. review the role of Pitx2 in embryonic and adult myogenesis. A hierarchy of transcription factors controls skeletal muscle diﬀerentiation and Pitx2 plays an important role in the regulation of this process. Importantly, it also seems to be involved in the establishment and function of satellite The role of speciﬁc signaling pathways in the formation of particular organs is discussed in other articles. Díez del Corral and Morales review the multiple roles of Fgf signaling in the developing spinal cord. This important structure of the nervous system arises from neural derivatives of an early August 2017 \| Volume 5 \| Article 76 2 Editorial: Signaling Pathways in Embryonic Development Sanz-Ezquerro et al. cells, the stem cells resident in adult muscle, thus opening new avenues for development of regenerative therapies. Additionally, Nassari et al. review the role of connective tissues in muscle development. Apart from the intrinsic molecular signals mentioned above, the interaction of muscle cells with surrounding tissues (bone, cartilage, tendon, and ligament) is critical for the correct assembly of the musculoskeletal system during development and for maintaining adult homeostasis. discussion on this area and opening new avenues for thinking and investigation. AUTHOR CONTRIBUTIONS JS was the Guest editor of this Research Topic, inviting co-editors AM and SS and working with them to deﬁne the subjects to be treated. They identiﬁed and invited leaders in speciﬁc research ﬁelds to contribute their work to the Research Topic. They acted as handling editors of manuscripts in the topic. JS wrote the Editorial with input from the other co-editors. An emerging theme in developmental biology is the control of tissue morphogenesis by physical forces (mechanotransduction). Valdivia et al. review the mechanical control of myotendinous junction formation and tendon diﬀerentiation, highlighting again the importance of the interplay between chemical and mechanical signaling during embryogenesis. In the same line, Stricker et al. provide a timely discussion reminding us that cells in embryos and adult organisms are not present in isolation, but embedded in extracellular matrices into complex tissues. Cells attach to the ECM and sense its mechanical properties. Typically, experimental in vitro conditions do not fully reproduce this environment, which is however critical for the physiological cellular responses to signaling cascades. The challenge for the future is to try and integrate as many interactions as possible when designing experiments. Frontiers in Cell and Developmental Biology \| www.frontiersin.org FUNDING Research in AM laboratory is supported by BBSRC grants BB/N007034, BB/N002970 and BHF grant PG/15/77/31761. Research in JS laboratory is supported by grants SAF2013-45331- R and SAF2016-79792-R from spanish MINECO. 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. Copyright © 2017 Sanz-Ezquerro, Münsterberg and Stricker. 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. We hope that the articles in this topic will be of interest to researchers working in development and cell biology, fuelling August 2017 \| Volume 5 \| Article 76 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 3
https://openalex.org/W2586657640	https://europepmc.org/articles/pmc5689957?pdf=render	English	null	The spatial accuracy of two frameless, linear accelerator‐based systems for single‐isocenter, multitarget cranial radiosurgery	Journal of applied clinical medical physics	2,017	cc-by	5,505	R A D I A T I O N O N C O L O G Y P H Y S I C S R A D I A T I O N O N C O L O G Y P H Y S I C S Abstract Abstract Single-isocenter, multitarget cranial stereotactic radiosurgery (SRS) is more efﬁcient than using an isocenter for each target, but spatial positioning uncertainties can be magniﬁed at locations away from the isocenter. This study reports on the spatial accuracy of two frameless, linac-based SRS systems for multitarget, single-isocenter SRS as a function of distance from the isocenter. One system uses the ExacTrac platform for image guidance and the other localizes with cone beam computed tomography (CBCT). For each platform, a phantom with 12 target BBs distributed up to 13.8 cm from the isocenter was aligned starting from ﬁve different initial off- sets and then imaged with the treatment beam at seven different gantry and couch angles. The distribution of the resulting positioning errors demonstrated the value of adding a 1-mm PTV margin for targets up to about 7–8 cm from the isocenter. For distances 10 cm or more, the CBCT-based alignment remained within 1.1 mm while the ExacTrac-based alignment differed by up to 2.2 mm. Author to whom correspondence should be addressed. Gary A. Ezzell E-mail: ezzell.gary@mayo.edu. P A C S 87.53.Ly, 87.55.D- K E Y W O R D S margins, multitarget, single-isocenter, stereotactic radiosurgery P A C S 87.53.Ly, 87.55.D- Received: 4 September 2016 \| Accepted: 14 December 2016 DOI: 10.1002/acm2.12044 Received: 4 September 2016 \| Accepted: 14 December 2016 DOI: 10.1002/acm2.12044 Received: 4 September 2016 \| Accepted: 14 December 2016 DOI: 10.1002/acm2.12044 The spatial accuracy of two frameless, linear accelerator- based systems for single-isocenter, multitarget cranial radiosurgery Gary A. Ezzell Department of Radiation Oncology, Mayo Clinic Arizona, Phoenix, AZ, USA wileyonlinelibrary.com/journal/jacmp \| 37 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors. Journal of Applied Clinical Medical Physics published by Wiley Periodicals, Inc. on behalf of American Association of Physicists in Medicine. 1 \| INTRODUCTION spatial accuracy of a clinic’s SRS system as a function of distance from the isocenter is needed as part of commissioning multitarget, single-isocenter SRS. Stereotactic radiosurgery (SRS) for patients with multiple brain metastases is becoming more common.1 Linear accelerator-based treatment using a single isocenter to treat multiple targets improves the efﬁciency of the delivery and has been the subject of numerous recent publications.2–8 Spatial accuracy is fundamental to SRS, and rotational spatial uncertainties in image-guided frameless SRS mag- nify spatial errors when the target is not at the isocenter. Few stud- ies9–12 have systematically investigated how targeting errors may be manifested in these treatments. Experimental validation of the This study reports on the spatial accuracy of two frameless, linac-based SRS systems within our department for multitarget, sin- gle-isocenter SRS as a function of distance from the isocenter. The testing was conducted using a home-made phantom with twelve tar- get BBs distributed spatially over a volume representative of a cra- nium. The purpose was to determine if the single isocenter approach could be used safely and whether any additional margin should be considered for targets off axis. J Appl Clin Med Phys 2017; 18:2:37–43 J Appl Clin Med Phys 2017; 18:2:37–43 F I G. 1. Photograph of the “Blockhead” phantom (a), anterior MV radiograph (b), and lateral MV radiograph (c). Target T0 is at the center. In the anterior radiograph, targets T1–T6 run top to bottom, targets T7–T9 are on the image right, top to bottom, and targets T10–T11 are on the image left, top to bottom. 2 \| METHODS AND MATERIALS 38 \| EZZELL EZZELL 38 little to the overall information used to drive the image guidance. This is more like the clinical situation in which one aligns to the bony anatomy and cannot visualize the actual targets with the imaging used for alignment. 2.A \| Phantom design When this study was initiated, there was no commercially available head phantom with multiple radio-opaque targets distributed throughout the bony structure of a skull. We therefore constructed a phantom from readily available materials: three sections of wood beam of nominal commercial cross-section 4″ 9 4″ (actual dimen- sions approximately 8.7 9 8.7 cm2) were cut to lengths of approxi- mately 20 cm and glued together after ﬁrst embedding in them 12 chrome steel ball bearings of diameter 4.8 mm (3/16″). Figure 1 shows a photograph of the phantom and orthogonal radiographs demonstrating the distribution of the targets. The most central target was designed to be at the isocenter for the subsequent plans. The radial distance of the targets from the isocenter ranged from 3.1 to 13.8 cm. This range was chosen because in some cases one might choose to put the isocenter on a target near a critical structure, such as the brainstem or the optic chiasm, instead of in the center of the distribution of targets. 2.B.1 \| Varian iX accelerator with ExacTrac Our current process for treating frameless SRS employs a Varian iX accelerator (Varian Medical Systems, Palo Alto, CA, USA) combined with the BrainLAB ExacTrac system (BrainLAB, Feldkirchen, Ger- many) and the BrainLAB six degree of freedom robotic couch. This system has been in place since early 2012 and to date has been used with approximately 245 patients for various indications, employing either single fraction or fractionated treatments. Our stan- dard process is to do the ExacTrac localization and adjustment after each couch rotation, correcting for imperfections in the couch isocentricity and patient motion. We usually apply ExacTrac toler- ances of 0.5 mm and 0.5 degree if we use the BrainLAB mask and associated localization frame but will sometimes use tolerances of 0.7 mm and 0.7 degrees if we need to use a different masking sys- tem. For this study, the phantom was placed on the couch without using a mask and the BrainLAB “reference star” was attached to the couch to supply the reﬂective markers needed by the ExacTrac sys- tem. Note that ExacTrac system uses the reﬂective markers only to Although the internal structure of the phantom is not anthropo- morphic, the wood grain permeates the phantom and is used in the image guidance process (Fig. 2). This has some advantages over phantoms that embed BBs in plastic cubes, because in this phantom, while the target BBs appear in the images, they contribute relatively (a) (b) (c) F I G. 1. Photograph of the “Blockhead” phantom (a), anterior MV radiograph (b), and lateral MV radiograph (c). Target T0 is at the center. In the anterior radiograph, targets T1–T6 run top to bottom, targets T7–T9 are on the image right, top to bottom, and targets T10–T11 are on the image left, top to bottom. (a) (a) (b) (c) F I G. 1. Photograph of the “Blockhead” phantom (a), anterior MV radiograph (b), and lateral MV radiograph (c). Target T0 is at the center. In the anterior radiograph, targets T1–T6 run top to bottom, targets T7–T9 are on the image right, top to bottom, and targets T10–T11 are on the image left, top to bottom. \| 39 EZZELL 39 39 (a) (b) F I G 2. Example alignment image on the ExacTrac system (a) and on the CBCT system (b). The planned image is in the spyglass window in each. (a) (b) (a) (b) (a) (b) (b) F I G 2. 2.C \| Treatment plan The phantom was scanned on a GE Lightspeed RT CT simulator (GE Healthcare, Chicago, IL, USA) at 1.25 mm spacing with axial scans, following our SRS scanning protocol. Small external BBs were placed at the desired isocenter, which was placed on the central target. The laser lines were marked on the phantom surface to facilitate setup in the treatment room. 2.B.1 \| Varian iX accelerator with ExacTrac Example alignment image on the ExacTrac system (a) and on the CBCT system (b). The planned image is in the spyglass window in each. systems was determined to have a 0.59-mm radius at the time of acceptance. The coincidence of the kV imaging system and MV treat- ment beam was within 0.3 mm per the Varian IsoLock procedure dur- ing this period. Monitoring of patient positioning can be accomplished via an optical surface-monitoring system, but that system was not employed for this study. The phantom was initially aligned using CBCT and no adjustment was made after each couch rotation. control the motion of the robotic couch. The assessment of the accuracy of the localization depends on the radiographic imaging and image guidance software. For this study, we used ExacTrac toler- ances of 0.7 mm and 0.7 degrees because those are the largest tol- erances that we employ clinically. A Winston–Lutz test is performed weekly and also daily when- ever an SRS treatment is scheduled in order to test and maintain the alignment of the ExacTrac imaging system to the radiation isocenter. When these test images were obtained, the agreement between the ExacTrac system’s location of the Winston-Lutz pointer and that measured with MV imaging was within 0.3 mm. 3 \| RESULTS For each iteration of the testing process, the phantom was placed on the linac couch, initially aligned to the external BBs and laser lines, and then displaced in translation and rotation. It was then imaged and aligned using the available image guidance system, either ExacTrac or CBCT. Automatic alignment was used, and the couch performed its six degrees of freedom motion. After the initial align- ment at the nominal couch 0, the four MV ﬁelds at the cardinal angles were imaged. The couch was then moved to the next posi- tion, either nominal 45, 90, or 315 degrees. For the iX/ExacTrac sys- tem, the phantom was re-imaged and aligned, following our current clinical practice. For the TrueBeam system, no additional corrections were applied. The MV image at gantry 0 was taken for each of the three non-zero couch angles. Collision with the image receptor pre- vented imaging at the lateral angles. After all the imaging, the couch was returned to angle 0, the phantom moved to a different starting position, and then the alignment and imaging process was repeated for a total of ﬁve trials. Table 1 shows the range of the initial correc- tions applied in the ﬁve iterations of each process. Table 2 shows the average offset, maximum offset, standard devia- tion and 95% conﬁdence limit as a function of distance from the isocenter for the two linacs and image guidance processes. Figure 3 plots the average and maximum offsets, and the aver- age plus two standard deviations as a function of distance from the isocenter for the two linacs and image guidance processes: (a) for the iX with ExacTrac and (b) for the TrueBeam with CBCT. Figure 4 shows the 95% conﬁdence limits for the two processes on a single graph. For the target planned to be at the isocenter, the 95% conﬁ- dence limit was 1.0 mm for the iX with ExacTrac and 0.6 mm for the TrueBeam with CBCT. At 7 cm from the isocenter, both systems demonstrated a variation of 0.9–1.1 mm. From 10 cm to 13.8 cm from the isocenter, the TrueBeam system’s variation remained at 1.1 mm or less while the iX with ExacTrac varied up to 2.2 mm. For the one ﬁeld that was measured on three occasions to test the reproducibility of the measurements, the maximum difference observed for each of the 84 target and ﬁeld combinations was tabu- lated. 3 \| RESULTS These differences ranged from 0 to 0.4 mm with an average of 0.12 mm and standard deviation of 0.09 mm. For the target at the isocenter, the maximum difference was 0.2 mm with an average of 0.1 mm and standard deviation of 0.08 mm. The standard devia- tion results shown in Table 2 range from 0.2 to 0.5 mm, so one can conclude that the uncertainty associated with this manual measure- ment technique contributed to but did not dominate the variability in the observations. 2.E Each of the acquired MV images was opened in the Aria Ofﬂine Review module (Varian Medical System, Palo Alto, CA, USA) as an T A B L E 1 Range of initial corrections applied to the phantom. Maximum absolute values of the initial corrections of the phantom position for the ﬁve iterations on each of the two SRS platforms. ExacTrac shifts (mm) ExacTrac angles (degrees) Vert Long Lat Yaw Pitch Roll (A) iX/ExacTrac platform 6.9 7.6 1.3 1.0 2.0 2.0 CBCT shifts (mm) CBCT angles (degrees) Vert Long Lat Yaw Pitch Roll (B) TrueBeam/CBCT platform 7.2 4.5 3.3 1.8 2.2 1.9 2.B.2 We recently installed a Varian TrueBeam accelerator (Varian Medical Systems, Palo Alto, CA, USA) with SRS capability. This system uses cone beam computed tomography (CBCT) as the method of image guidance combined with Varian’s PerfectPitch, six degree of freedom couch. The isocentricity of the combined couch, gantry, and collimator A treatment plan was created on the Varian Eclipse (Varian Medical System, Palo Alto, CA, USA) planning system using seven 25 9 25 cm ﬁelds: the four cardinal angles at couch 0 and three at gantry 0 with EZZELL EZZELL 40 overlay to its associated DRR. Using the distance measuring tool, the offset between the center of the target in the DRR and that in the MV image was measured and recorded. The 12 targets were mea- sured for the seven ﬁelds for the ﬁve repetitions on the two linacs for a total of 840 measurements. One ﬁeld was measured on three occasions to test the reproducibility of this manual process. couch angles 45, 90, and 315. These are the couch angles most typically used for our single-isocenter, multitarget treatments. High-quality digi- tally reconstructed radiographs (DRRs) were created for each beam with the Hounsfeld units (HU) range clipped so that only the BBs appeared on the DRR. This facilitated later analysis. The HU of the wood grain varied between approximately 800 and 250. While that structure showed up clearly on the planning CT, CBCT, and Eclipse DRRs, it entirely disappeared on the DRRs con- structed by the ExacTrac system’s algorithm during its alignment pro- cess. For that reason, a second CT study was artiﬁcially created using MATLAB (MathWorks, Natick, MA) code that increased the HU of the wood structure to the range of 50–800, mimicking soft tissue to bone. This was the study used in the testing of the Varian iX with ExacTrac. Each of the 12 targets was therefore imaged 35 times on each linac. The average offset between the treatment ﬁeld and DRR was calculated along with the maximum and the standard deviation. The 95% conﬁdence limit on the offset was estimated by adding the average with twice the standard deviation. These results were then plotted as a function of the distance of the target from the isocenter. 4 \| DISCUSSION Target Distance from isocenter (cm) Average offset (mm) Maximum offset (mm) Std Dev (mm) Avg + 2SD (mm) (A) iX/ExacTrac platform T0 0 0.5 1.1 0.2 1.0 T4 3.1 0.5 1.0 0.2 0.9 T3 3.3 0.5 1.0 0.2 1.0 T5 6.7 0.5 1.0 0.3 1.0 T2 7.2 0.4 0.8 0.2 0.9 T6 9.9 0.6 1.1 0.3 1.1 T1 10.2 0.4 0.9 0.2 0.9 T11 10.5 1.1 1.8 0.4 2.0 T10 10.9 1.2 2.0 0.5 2.2 T8 11.6 0.9 1.6 0.3 1.6 T9 13.8 0.8 1.7 0.4 1.6 T7 13.8 1.1 1.8 0.4 1.8 (B) TrueBeam/CBCT platform T0 0 0.3 0.7 0.2 0.6 T4 3.1 0.5 1.2 0.3 1.1 T3 3.3 0.5 0.9 0.2 0.9 T5 6.7 0.4 0.8 0.2 0.7 T2 7.2 0.5 0.8 0.2 0.9 T6 9.9 0.7 1.1 0.2 1.1 T1 10.2 0.7 1.1 0.2 1.1 T11 10.5 0.5 0.8 0.2 0.9 T10 10.9 0.4 0.8 0.2 0.8 T8 11.6 0.3 0.7 0.2 0.6 T9 13.8 0.7 1.2 0.2 1.1 T7 13.8 0.6 1.3 0.2 1.1 (b) F I G . 3. The average and maximum offsets (in mm) between the planned and imaged targets along with the average plus two standard deviations as a function of distance from the isocenter (in cm): (a) for the iX with ExacTrac and (b) for the TrueBeam with CBCT. F I G . 4. The 95% conﬁdence limits, approximated by the average offset plus two standard deviations, for the two platforms plotted on a single graph. F I G . 4. The 95% conﬁdence limits, approximated by the average offset plus two standard deviations, for the two platforms plotted on a single graph. smaller than the region of interest in the CBCT. While that ﬁeld of view is generally adequate, in this case six to seven of the targets (and all the associated phantom structure) more distant from the isocenter were not visualized. To some extent that is an artifact of this nonanthropomorphic phantom design. The CBCT system visual- ized the entire phantom which was then subjected to the automatic registration. so that physicists can perform end-to-end tests for multitarget SRS with realistic phantoms. The observed offset between the target on the planned DRR and the MV image represents the geometric error in beam delivery that would have occurred for that target and ﬁeld without regard for the direction of the offset. 4 \| DISCUSSION Target Distance from isocenter (cm) Average offset (mm) Maximum offset (mm) Std Dev (mm) Avg + 2SD (mm) (A) iX/ExacTrac platform T0 0 0.5 1.1 0.2 1.0 T4 3.1 0.5 1.0 0.2 0.9 T3 3.3 0.5 1.0 0.2 1.0 T5 6.7 0.5 1.0 0.3 1.0 T2 7.2 0.4 0.8 0.2 0.9 T6 9.9 0.6 1.1 0.3 1.1 T1 10.2 0.4 0.9 0.2 0.9 T11 10.5 1.1 1.8 0.4 2.0 T10 10.9 1.2 2.0 0.5 2.2 T8 11.6 0.9 1.6 0.3 1.6 T9 13.8 0.8 1.7 0.4 1.6 T7 13.8 1.1 1.8 0.4 1.8 (B) TrueBeam/CBCT platform T0 0 0.3 0.7 0.2 0.6 T4 3.1 0.5 1.2 0.3 1.1 T3 3.3 0.5 0.9 0.2 0.9 T5 6.7 0.4 0.8 0.2 0.7 T2 7.2 0.5 0.8 0.2 0.9 T6 9.9 0.7 1.1 0.2 1.1 T1 10.2 0.7 1.1 0.2 1.1 T11 10.5 0.5 0.8 0.2 0.9 T10 10.9 0.4 0.8 0.2 0.8 T8 11.6 0.3 0.7 0.2 0.6 T9 13.8 0.7 1.2 0.2 1.1 T7 13.8 0.6 1.3 0.2 1.1 (a) (b) F I G . 3. The average and maximum offsets (in mm) between the planned and imaged targets along with the average plus two standard deviations as a function of distance from the isocenter (in cm): (a) for the iX with ExacTrac and (b) for the TrueBeam with CBCT. F I G . 4. The 95% conﬁdence limits, approximated by the average offset plus two standard deviations, for the two platforms plotted on a single graph. smaller than the region of interest in the CBCT. While that ﬁeld of view is generally adequate, in this case six to seven of the targets (and all the associated phantom structure) more distant from the isocenter were not visualized. To some extent that is an artifact of this nonanthropomorphic phantom design. The CBCT system visual- so that physicists can perform end-to-end tests for multitarget SRS with realistic phantoms. The observed offset between the target on the planned DRR T A B L E 2 Offsets between the planned and imaged target positions for the two platforms. For each target, the distance from the isocenter (in cm) and the average and maximum offsets between the planned and imaged targets (in mm) with the standard deviation in the 35 measurements for each target. The 95% conﬁdence limit is approximated by the last column, which sums the average offset with twice the standard deviation. 4 \| DISCUSSION In the study, the offsets seen for the ExacTrac system at 10 cm from the isocenter and beyond were more than those seen for the CBCT- based alignment system. This may be attributed to two factors. First, the ExacTrac tolerances that were applied, 0.7 mm and 0.7 degrees, inherently permit some variation that will be magniﬁed at distance. Second, the ﬁeld of view of the ExacTrac images (13 9 13 cm2) is \| 41 EZZELL EZZELL 41 (a) (b) F I G . 3. The average and maximum offsets (in mm) between the planned and imaged targets along with the average plus two standard deviations as a function of distance from the isocenter (in cm): (a) for the iX with ExacTrac and (b) for the TrueBeam with CBCT. \| (a) (b) F I G . 3. The average and maximum offsets (in mm) between the planned and imaged targets along with the average plus two standard deviations as a function of distance from the isocenter (in cm): (a) for the iX with ExacTrac and (b) for the TrueBeam with CBCT. \| smaller than the region of interest in the CBCT. While that ﬁeld of T A B L E 2 Offsets between the planned and imaged target positions for the two platforms. For each target, the distance from the isocenter (in cm) and the average and maximum offsets between the planned and imaged targets (in mm) with the standard deviation in the 35 measurements for each target. The 95% conﬁdence limit is approximated by the last column, which sums the average offset with twice the standard deviation. (A) for the iX with ExacTrac and (B) for the TrueBeam with CBCT. 4 \| DISCUSSION (A) for the iX with ExacTrac and (B) for the TrueBeam with CBCT. Target Distance from isocenter (cm) Average offset (mm) Maximum offset (mm) Std Dev (mm) Avg + 2SD (mm) (A) iX/ExacTrac platform T0 0 0.5 1.1 0.2 1.0 T4 3.1 0.5 1.0 0.2 0.9 T3 3.3 0.5 1.0 0.2 1.0 T5 6.7 0.5 1.0 0.3 1.0 T2 7.2 0.4 0.8 0.2 0.9 T6 9.9 0.6 1.1 0.3 1.1 T1 10.2 0.4 0.9 0.2 0.9 T11 10.5 1.1 1.8 0.4 2.0 T10 10.9 1.2 2.0 0.5 2.2 T8 11.6 0.9 1.6 0.3 1.6 T9 13.8 0.8 1.7 0.4 1.6 T7 13.8 1.1 1.8 0.4 1.8 (B) TrueBeam/CBCT platform T0 0 0.3 0.7 0.2 0.6 T4 3.1 0.5 1.2 0.3 1.1 T3 3.3 0.5 0.9 0.2 0.9 T5 6.7 0.4 0.8 0.2 0.7 T2 7.2 0.5 0.8 0.2 0.9 T6 9.9 0.7 1.1 0.2 1.1 T1 10.2 0.7 1.1 0.2 1.1 T11 10.5 0.5 0.8 0.2 0.9 T10 10.9 0.4 0.8 0.2 0.8 T8 11.6 0.3 0.7 0.2 0.6 T9 13.8 0.7 1.2 0.2 1.1 T7 13.8 0.6 1.3 0.2 1.1 (a) (b) F I G . 3. The average and maximum offsets (in mm) between the planned and imaged targets along with the average plus two standard deviations as a function of distance from the isocenter (in cm): (a) for the iX with ExacTrac and (b) for the TrueBeam with CBCT. F I G . 4. The 95% conﬁdence limits, approximated by the average offset plus two standard deviations, for the two platforms plotted on a single graph. (a) T A B L E 2 Offsets between the planned and imaged target positions for the two platforms. For each target, the distance from the isocenter (in cm) and the average and maximum offsets between the planned and imaged targets (in mm) with the standard deviation in the 35 measurements for each target. The 95% conﬁdence limit is approximated by the last column, which sums the average offset with twice the standard deviation. (A) for the iX with ExacTrac and (B) for the TrueBeam with CBCT. (B) for the TrueBeam with CBCT. 5 \| CONCLUSION 5 Stanhope et al.9 analyzed sequential CBCT images for 22 patients who had SRS treatments to two targets with isocenters with the purpose of measuring the rotational difference in the skull between the two scans. By aligning the second scan to the ﬁrst, they measured how much the patient had moved within the mask and therefore estimated the uncertainty associated with patient motion after an initial alignment, which they termed “intraoperational” uncertainty. They found that 0.1 mm/cm of target-isocenter separa- tion would account for 95% if this uncertainty. This assumed that the initial correction completely removed any initial setup error. What practical recommendations follow from these results? Most publications about single-isocenter, multitarget treatments advocate centering the isocenter among the targets. This will generally keep the distance from any part of a target to the isocenter to about 8 cm. At such distances, it would be prudent to increase the PTV margin by 1 mm. If one chose to place the isocenter on a target near the brainstem or chiasm while also treating targets near the superior aspect of the brain, then the distance from isocenter would be larger and an additional margin might be advisable, depending on the size of the lesions and the eloquence of the surrounding tissue. Adding margin is not necessarily benign. Kirkpatrick et al.14 in a randomized trial involving 49 patients with 80 metastases, found more radionecrosis in those patients having a 3-mm margin compared to a 1-mm margin. Roper et al.10 analyzed 50 SRS cases, each having two targets treated with a single-isocenter VMAT technique. They simulated rotational errors of 0.5i°, 1.0°, and 2.0° about all axes to determine how the dose coverage of the PTVs was affected. Distances from the plan isocenter to the PTV centroid varied from 0.6 cm to 7.3 cm. They found that the dose to 95% of the PTV (D95) fell by 1.5%/cm with a rotational error of 1.0° and by 4.3%/cm with a rota- tional error of 2.0°. This study did not recommend how PTV margins might be increased to account for uncertainties that increase with distance from isocenter. A fundamental outcome of this study is that it conﬁrms that either platform in our institution is suitable for single-isocenter, mul- titarget SRS. REFERENCES 1. Nichol A, Ma R, Hsu F, et al. Volumetric radiosurgery for 1 to 10 brain metastases: a multicenter, single-arm, phase 2 study. Int J Radiat Oncol Biol Phys. 2016; 94:312–321. 1. Nichol A, Ma R, Hsu F, et al. Volumetric radiosurgery for 1 to 10 brain metastases: a multicenter, single-arm, phase 2 study. Int J Radiat Oncol Biol Phys. 2016; 94:312–321. 2. Clark GM, Popple RA, Prendergast BM, et al. Plan quality and treat- ment planning technique for single isocenter cranial radiosurgery with volumetric modulated arc therapy. Pract Radiat Oncol. 2012;2:306–313. 2. Clark GM, Popple RA, Prendergast BM, et al. Plan quality and treat- ment planning technique for single isocenter cranial radiosurgery with volumetric modulated arc therapy. Pract Radiat Oncol. 2012;2:306–313. Winey and Bussiere12 studied geometric uncertainties in single- isocenter, multitarget fractionated treatments. They used retrospec- tive data from 45 patients aligned with their orthogonal kV system with a ﬁducial-based 2D/3D algorithm to determine the translational and rotational accuracy and uncertainty, both interfractional and intrafractional, in each of the six degrees of freedom. They then determined the maximum error value if all of the errors aligned for 1 and 2 sigma uncertainty levels, noting that in practice only 3.6% and 0.3% of all cases exceeded these two values, respectively. For the most direct comparison to this study, one can inspect their (Fig. 2) for the case that six degree of freedom corrections are made, and use the curve for 1 sigma, which they found to correspond to 96.4% of the treatments. Reading from the graph, at isocenter the error is approximately 0.8 mm, at 3 cm it is 1.0 mm, and at 7.5 cm is 1.5 mm for the immobilization system used in their study and 1.5 mm, 2.0 mm, and 3.1 mm for the ExacTrac systems described by van Santvoort et al.13 3. Clark GM, Popple RA, Young PE, Fiveash JB. Feasibility of single-iso- center volumetric modulated arc radiosurgery for treatment of multi- ple brain metastases. Int J Radiat Oncol Biol Phys. 2010;76:296–302. 3. Clark GM, Popple RA, Young PE, Fiveash JB. Feasibility of single-iso- center volumetric modulated arc radiosurgery for treatment of multi- ple brain metastases. Int J Radiat Oncol Biol Phys. 2010;76:296–302. 4. Wolff HA, Wagner DM, Christiansen H, Hess CF, Vorwerk H. Single fraction radiosurgery using RapidArc for treatment of intracranial tar- gets. Radiat Oncol. 2010;5:77. 5. 4 \| DISCUSSION This study did not attempt to quantify the effect on dose coverage for a full treatment employing arcs at these couch angles, but it is intuitively clear that such errors will compromise target coverage. Other studies9,10 have shown that the decrease in target coverage expressed as the dose covering 95% of the target (D95) is more signiﬁcant for small targets. Another potential weakness of this nonanthropomorphic phan- tom is that the grain structure on the ExacTrac radiographs approxi- mate parallel lines, as seen in Fig. 2. This is very different from the appearance provided by a human skull, so it is possible that the ExacTrac algorithms would be more accurate with a realistic phan- tom. It is hoped that phantom vendors will soon provide such tools EZZELL CONFLICT OF INTEREST None. ACKNOWLEDGMENTS Thomas Boltz, Ph.D., provided the MATLAB code used in this study. 5 \| CONCLUSION Wen et al.11 evaluated the systematic accuracy of the Varian Edge TM linac-based SRS platform with a variety of tests involving a 15 9 15 9 15 cm3 phantom with 5-mm diameter ceramic BBs at the isocenter and embedded into the outer shell. In the test most comparable to this study, they aligned the phantom to a reference CT with CBCT using automatic registration and then imaged the phantom at couch 0 and at the four cardinal angles with MV beams. They determined the targeting accuracy for the target at the isocen- ter and for six others from ranging from 5.7 to 9.2 cm from the isocenter. They found the accuracy for the target at the isocenter to be 0.54 0.24 mm and for the others to be 0.51 0.24 mm at 5.7 cm away from the isocenter, 0.62 0.43 mm at 6.5 cm away, and 0.63 0.35 mm at 9.2 mm away. These results are similar to those of this study, but it did not include couch kicks and used a phantom with more symmetry and simplicity than was employed here. 9. Stanhope C, Chang Z, Wang Z, et al. Physics considerations for single-isocenter, volumetric modulated arc radiosurgery for treat- ment of multiple intracranial targets. Pract Radiat Oncol. 2016;6:207–213. 10. Roper J, Chanyavanich V, Betzel G, Switchenko J, Dhabaan A. Sin- gle-isocenter multiple-target stereotactic radiosurgery: risk of com- promised coverage. Int J Radiat Biol Oncol Phys. 2015;93:540–546. 11. Wen N, Snyder KC, Scheib SG, et al. Evaluation of the systematic accuracy of a frameless, multiple image modality guided, linear accel- erator based stereotactic radiosurgery system. Med Phys. 2016;43:2527–2537. REFERENCES Mayo CS, Ding L, Addesa A, Kadish S, Fitzgerald TJ, Moser R. Initial experience with volumetric IMRT (RapidArc) for intracranial stereo- tactic radiosurgery. Int J Radiat Oncol Biol Phys. 2010;78:1457–1466. 6. Salkeld AL, Unicomb K, Hayden AJ, Van Tilburg K, Yau S, Tiver K. Dosimetric comparison of volumetric modulated arc therapy and lin- ear accelerator-based radiosurgery for the treatment of one to four brain metastases. J Med Imaging Radiat Oncol. 2014;58:722–728. 7. Huang Y, Chin K, Robbins JR, et al. Radiosurgery of multiple brain metastases with single-isocenter dynamic conformal arcs (SIDCA). Radiat Oncol. 2014;112:128–132. 8. Thomas EM, Popple RA, Wu X, et al. Comparison of plan quality and delivery time between volumetric arc therapy (RapidArc) and gamma knife radiosurgery for multiple cranial metastases. Neurosurg. 2014;75:409–417; discussion 417–418. EZZELL EZZELL 43 12. Winey B, Bussiere M. Geometric and dosimetric uncertainties in intracranial stereotatctic treatments for multiple nonisocentric lesions. J Appl Clin Med Phys. 2014;15:122–132. 13. van Santvoort J, Wiggenraad R, Bos P. Positioning accuracy in stereotactic radiotherapy using a mask system with added vacuum mouth piece and stereoscopic X-ray positioning. Int J Radiat Oncol Biol Phys. 2008;72:261–267. 14. Kirkpatrick JP, Wang Z, Sampson JH, et al. Deﬁning the optimal planning target volume in image-guided stereotactic radiosurgery of brain metastases: results of a randomized trial. Int J Radiat Oncol Biol Phys. 2015;91:100–108.
https://openalex.org/W4240989744	https://zenodo.org/records/1865190/files/article.pdf	German	null	Eier	European food research & technology	1,902	public-domain	2,008	210 210 [Zeitchr. f. Uutm~u~hm~ ~_d. Nahr.- u. O~almomi~L [Zeitchr. f. Uutm~u~hm~ ~_d. Nahr.- u. O~almomi~L Referate. ~ Eier. Nachdem nun die Xanthinstoffe nicht nur im Fleischextrakte Mlelm vor- kommen, sondern auch in vegetabilischen Extrakten enthalten sein kSnnem, ist die fiir die Untersuchung und Beurtheilung derartiger Extrakte nicht unwidtti~ Frage zu beantworten, ob in den beiden Arten yon Extrakten auch stetl die= selben Xanthinstoffe enthulten sind und ferner ob ihr gegenseitiges Meul~r- hiiitniss dasselbe ist. Die diesbeziiglichen Untersuchungen habe ich bereits in Angriff genommen. Reterate. Eier. Eier. Uhlenhut: ~'euer Beitrag zum specifischen Nachweis yon Eiereiwei~ auf biologischem Wege. ~ Deutsch. medlein. Wochenschr. 1900, ~ 734 bis 735. Verf. suchte festzustellen, ob in dem Serum mit :Eiereiweiss behandelter Thiere sich speeifi.~che AntikSrper entwickeln und ob sich etwa auf diese Weise die Ei- weissstoffe verschiedener Vogeleier unterscheiden lassen. Zu diesem Zweck wurde Hfihnereiweiss, mit physiologischer KochsaizlSsung verdiinnt, Kaninchen in die Bauch- hShle gespritzt. Wenige Tropfen des Serums dieser Thiere rufen in verdfmnten HfihnereiweisslSsungen Trfibungen hervor, und wurde bei einer Verdiinnung 1 : 100000 noch eine deudiche Reaktion erhalten, w~ihrend die chemischen Agentien schon bei ~erdiinnungen des Eiweisses 1:1000 versagen. ]Normales Kaninchenserum zeigte die Reaktion gegeniiber Hiithnereiweiss nicht. Bei tier Untersuehuug verschiedener EiweissprRpara~ des Handels ergab sich, dass das Serum yon in oben angegebener Weise mit Eiweiss behandelten Kaninchen nur in den Pr~paraten Triibungen hervor- fief, welche aus Eieralbumin hergestellt waren. Abet auch bei TaubeneiweisslSsungen trat eine deutlich positive Reaktion ein; ebenso trfibte das Serum yon Kaninchen, welchen wiederholt intraperitoneal Taubeneiweiss eingespritzt wurde, LSsungen yon ltiihnereiweiss. Von besonderem Interesse ist ferner, dass auch die intrastomachale Einverleibung yon Hfihnereiweiss (mittelst Schlundsonde) bei Kaninchen im Serum Stoffe bildet, welche beim Zusatz yon Hiihner- und TaubeneiweisslSsungen Trfilmngen bezw. Niederschl~ge hervorrufen. g Verf. hatte ferner gefunden, dass das Serum eines mit lliihnerblut behandelten Kaninchens in einer sehr stark verdiinnten HiihnerblutlSsung schnell eine doutlich auftretende Triibung hervorruft, w~hrend sich dasselbe Serum gegen LSsungeu yon Pferde-, Esel-, Rinder-, Hammel- uud Taubenblut indifferent zeigte. Verf. hat sieh in Folge dessen die Aufgabe gestellt, mit Hilfe dieser biologischen Methode die ver- schiedenen Blutarten zu differenciren. Bemerkenswerth ist noch, dass das Serum der mit Eiereiweiss behandelten Ka- ninchen dutch einstiindige Erhitzung auf 600 seine ReaktionsfiLhigkeit nieht ein- biisste. A. J~uck. gkeit nieht ein A. J~uck. 5. Jahrgaag. 1. ~arz 19o.2. 211 Referate. -- Eier. Leo Langstein: Die Kohlenhydratgruppe des kryst~llisirten Ovalbu- rains. -- Zeitschr. physiol. Chem. 1900, 31, 49--57. Leo Langstein: Die Kohlenhydratgruppe des kryst~llisirten Ovalbu- rains. -- Zeitschr. physiol. Chem. 1900, 31, 49--57. Leo Langstein: Die Kohlenhydratgruppe des kryst~llisirten Ovalbu- rains. -- Zeitschr. physiol. Chem. 1900, 31, 49--57. p y , , In der Frage nach dem Kohlenhydratkomplex des Ovalbumins ist eine Einigung bisher noch nicht erreicht. Einerseits wird genannte Spaltung des Ovalbmnins geleugnet, andererseits herrscht keine Uebereinstimmung in der Charakterisirung des abgespaltenen Komplexes. Verf. Reterate. stellte sich 100 g krystallisirten Ovalbumins (aus 4 1 Eierklar) nach dem Verfahren yon Hop ki n s und P i n k u s dar. Eine Verunreinigung durch Ovomu- cold wurde dadurch vermieden, dass das Ovalbumin aus mit Ammonsulfat halbges~t- tigter L5sung durch Salzs~urezusatz wiederholt umkrystallisirt wurde. Dutch Vorver- suche mit 5 g der sorgfMtigst gereinigten, mit Alkohol koagulirten, getmckneten und gepulverten Substanz, welehe nach Quellung in Alkali mit 100 ccm 3~ Salz- s~iure am Rfickflusskfihler auf dent Sandbade erhitzt wurde, ergab sich, dass nach 4 Stunden die grSsste Kohlenhydratmenge (7O]o) mit Fehling'scher LSsung nachge- wiesen werden konnte. In Folge dessen wurde zur Charakterisirung des abspaltbaren Kohlenhydratcs die tIauptma~se des gereinigten Ovalbumins in ~hnlicher Weise be- handelt, l~ach der Inversion wurde das Filtrat neutralisirt und ohne die gel5sten EiweisskSrper zu entfernen benzoylirt. Iqach der Neutralisation der Benzoylirungs- flfissigkeit wurde das Benzoylprodukt abgesaugt, mit verdfinntem Ammoniak und Wasser gewaschen, getrocknet und in Alkohol gelSst. Aus der filtrirten LSsung schieden sich im Verlaufe von drei Tagen seidengl~.nzende Nadeln und amorphe Krfi- me]n aus; die mit kaltem absolutem Alkohol von dem amorphen K5rper getrennten :Nadeln zeigten einen Schmelzpunkt yon 202--203 ~ und einen Stickstoffgehalt yon 1,962o/o. Es liegt demnach anscheinend Pentabenzoylglykosamin vor. Aus den vorerw~hnten alkoholischen Mutterlaugen wurde nunmehr der Zucker selbst abzuspalten versucht. Zu dem Zwecke wurde mit grossen Mengen destillirten Wassers das Benzoylprodukt als weisse, bald kSrnig werdende Floeken abgeschieden. Dasselbe wurde 24 Stunden bei 100 o mit Salzs~ure (Spec. Gew. 1,1) im zugesehmolzenen Rohr behandelt. Nach dem Erkalten wurde filtrirt, mit Aether ausgeschfittelt und die Salzs~ure bei 600 aly- geraucht. Aus dem so erhaltenen braunen Syrup krystallisirten neben Koehsalz und Gyps (?)Sphenoide, sp~tel: rhombische Platten. Durch Behandlung des Syrups mit al)- solutcm Alkohol und Umkrystallisiren der erhaltenen Substanz aus salzs~iurehaltigem Wasser wurden die letztgenannten Krystalle rein erhalten. Dieselben zeigten zwar nicht die yon L e d d er ho s e ffir Glykosamin beschriebenen charakteristischen Formen, jedoch ]iessen die analytischen Befunde keinen Zweifel fiber die Identit~t mit salz- saurem Glykosamin bestehen (33,51~ Kohlenstoff; 6,53~ Wasserstoff; 6,38O/o Stickstoff). Aus den l:Yntersuchungen geht demnach unzweifelhaft hervor, dass Glykos- amin am Aufbau des krystallisirten Ovalbumins zu etwa 10--11~ betheiligt ist. y g Bemerkenswerth ist es ferner, dass es Verf. weder dutch Spaltung yon krystaUi- sirtem Ovalbumin mit koncentrirter Salzs~ure noch mit koncentrirter Salzs~.ure und Zinnchloriir ge]ang, aus Eiweiss Kohlenhydrat zu erhalten. Reterate. Es erklart sich dies da- durch, dass das durch starke Basen aus dem Eiweiss abgespaltene Ammoniak die Reaktion in andercm Sinne verlaufen lasst. Die Untersnchungen sollen auch auf krystallisirtes Sermnalbumin ausgedehnt werden. A. Juekenaek. Die Untersnchungen sollen auch auf krystallisirtes Sermnalbumin ausgedehnt A J k k 14" 2l 2 ]-Zeitschr. f. U n S u n g [d. Nahr.- ~ Oenumm\|tt*l. R e fe r a t e. -- Eier. Loges und Pingel: Ueber ,,Eisen-Eier". -- Sichs. landw. Zeitschr. 1900, 22, 409--411. Loges und Pingel: Ueber ,,Eisen-Eier". -- Sichs. landw. Zeitschr. 1900, 22, 409--411. Verff. haben das Verfahren yon Carl Aufsberg (Diese Zeitschrift 1901, 4, 309) zur ]~rzielung eisenreicher Eier mittelst be~nderen ei~nhaltigen Futters naehge- prfift. Zu dem Zwecke wurde der eine Theil yon Hfihnern gleichen Alters und 8iron- rues drei ~,Vochen in der von A u f s b e r g angegebenen Weise geffittert, wiltrend der andere Theil das gleiche Futter ohnc Zusatz des Eisenprfiparates erhielt. Zur Untersuchung gelangten je 10 Eier. Das Ergebniss war folgendes: Ohne Eisenftttterung Mit Eisenftitterung Eiweiss Eigelb Schale Eiinhalt Eiweiss Eigelb Schale ]FAimJUl t Eisenoxyd 0,002400 0,00880/0 0,02720/0 0,00470'o 0,0040~ 0,00950/0 0,02720/0 0,064~~ Asche 0,699 , 1,500 , -- 0,992 , 0,699 . 1,430 , -- 0,963 , Eisenoxyd ~ . . . . 0,474 -- -- -- 0,612 inder Asche J ~ 9 Demnach entsprechen (lie in beiden Ver.~uehsreihen erhaltenen Befunde snn&ksrud r . r " dem Durchschnitte tier bisherigen Untersuchun,~se gebmsse, zudem enthalten die ,Ei- sen"-Eier nur 0,0012 ~ Eisenoxyd mehr als die gew5hnliehen Eier. Ganz abgesshen davon, dass dieser Unterschied innerhalb der unvermeidllchen Fehlergrenzen liegt, kann, wenn er als wirklich bestehend angenommen wird, yon einer di~tetisch wichtigen An- reicherung des Ei~ns in den Eiern keine Rede sein, da im Verh/ilt,niss zu den sonst Verwendung findenden Ei~nprfiparaten t/iglicb 88--117 Ei~neier von einer Person zu nehmen sein wfirden, welehe nebenbei 17--23 M. kosten. A. Juckenack. einer Person z A. Juckenack. P. Hoffmann: Ueber den Eisengehalt des Hfihnereies, sowie Versuehe fiber Anreicherung des Eisens im Ei nach Ffitterung ]nitH~.mogallol und Ferroh/imol. -- Zeitschr. analyt. Chemie 1901, 40, 450--459. Nach den bisher fiber den Eisengehalt ,..,ewShnlieher Hfihnereier in der Litteratur gemachten Angaben schwankt derselbe zwisehen 10,7 und 29,88 mg Eisenoxyd in 100 g Eidotter. Verf. land im Durchschnitt von 10 Bestimmungen in 100 g Ei- dotter 12,065 mg, in einem ganzen Ei ohne Schale 1,81 mg Eisenoxyd. Socin hat nun schon vor Jahren (Zeitschr. Reterate. physiol. Chemie 1891, 15, 112) festgestellt, dass die organische Eisenverbindung des Hfihnereies, (la~, Hamatogen, resorbirbar ist. Bel der Bedeutung des Eisens ffir den KSrper wiirde es nun nieht unwichtig sein, wenn auf kfinstlichem Wege (let Hfi, matogengehalt erhSht und die MSglichkeit einer grSsseren Eisenzufuhr in dieser Form geboten wfirdc. Es erhielten daher l&ngere Zeit hindurch 2 Hfihner neben der nieht besonders geregelten :Nahrung thglich je 10 g Hfi.mogallol- pastillen, ent.~prechend 16,24 mg Eisenoxyd. Wie die Vemuche ergaben, hatte sich nun in der That der Eisenoxydgehalt der Dotter yon 12.06~ beim Beginn der Ffitterung auf 15,26~ beim Schluss derselben erhSht. Darauf wurden dieselben Hfihner mit Ferroh/~mol, einem von Prof. Kobert dumh Fi~llung des Rinderblut- hiimoglobins mit Metallsalzen dargestellten Erzeugniss geffittert, jedoch konnte nur bei einem Theil der Eier ein Ansteigen tier Eisenmenge festgestellt werden, wfi~hrend im Durchschnitt yon 14 Dotter-Analvsen der Eisengehalt sich auf 10,74~ belief, somit nicht einmal die vom Verf. in gewShnlichen Eiern gefundene Menge errvichte. Diese Schwankungen sind wahrscheinlich auf ~ussere Verhfiltnisse, z. B. auf das Futter, zuriickzuffihren, da (lie niedri~'swn Zahlen zu einer Zeit erhalten wurden, als den Hfihnern hauptsiichlieh Griinfutter gereieh! wurde. Aus den Versuchen ergiebt sieh 5. Jahrgaug. ] 1. Marz 1902.] R e f e r a t e. -- Butter, Speisefette und Oele. 213 also, dass das eisenRrmere H~mogallol gfinstiger ffir eine Anreicherung des Eisens im Ei zu verwenden ist als alas Ferroh~tmol. Nach Ansicht des Verf.'s ist es nicht wahr- scheinlich, dass die Men~ des Eisens im Ei noch wesentlich starker erhSht werden kann, als es in den vorliegenden Versuchen der Fall war. Durch mikroskopische Untersuehung konnte niimlich nachgewiesen werden, dass die zum Eierstock fiihren- den Lymphwege yon Eisen strotzten. Wenn dennoch nicht mehr yore Ei au~genommen wurde, so muss die Natur Vorkehrungen besitzen, das iiberschfissig dem Eierstock zugefiihrte Eisen nicht ins Ei gelangen zu lassen. Schliesslich wurde noch ein Huhn mit Cuproh~molpillen gefiittert. Das Thier, das sich andauernd wohl befand, erhielt im Ganzen innerhalb 32 Tagen 235 PiUen, trotzdem fand sieh niemals Kupfer im Ei; beim Verfiittern yon Kupferverbindungen geht somit kein Kupfer in das Ei fiber. Max ~)Biller. Max ~)Biller. H. Kreis: Eisengehalt der Eier. -- Jahresbericht des kantonalen chemhchen Laberatoriums Basel 1900, S. 15. H. Kreis: Eisengehalt der Eier. -- Jahresbericht des kantonalen chemhchen Laberatoriums Basel 1900, S. 15. Verf. erhielt den Auftrag, drei Hiihnereier auf einen dutch eigenartige Ffitte, rung erzielten angel)lichen Mehrgehalt an :Eisen zu prfifen. Reterate. Die Untersuchung ergab indessen, dass diese Eier noch etwas weniger :Eisen enthielten, als ein zum Vergleich hcrangezogenes gewShnliches El. g g g Der Eisenoxydgehalt betmg n~mlich 0,0038, 0,0040 und 0,0042 ~ vom Ei- inhalt~ w~hrend im gew5hnliehen Ei 0,0046 o/~ gefunden wurden. ~. Mai. ~. Mai. Patente. Ernst Uteseher in Hamburg: Verfahren zur Herstellung eines Eierkonservi- rungsmittels und Anwendung desselben zur Konservirung yon Eiern. D.R.P. 119575 yore 11. M~z 1900. -- Patentbl. 1901, 22, 657. Das neue Konservirungsmittel wird hergestellt, indem man ein Elsenoxydulsa]z, Calcium- hydroxyd und Paraffin(ll innig mit einander vermischt. Die Anwendung des Konservirungs- mittels geschieh~ zweckm~issig in der Weise, dass man die Eier in die wasserige Mischung dieses Pr~iparates legt und ab und zu beim Einlegen bezw. Einsenken neuer Eier yon dem Konservirungsmittel, welches noch mit Talcum, Paraflinpulver oder einem anderen indifferenten Stoff vermischt werden kann, in die Fltissigkeit einstreut. E. Utescher in Hamburg: Verfahren zur Konservirung yon Eiern. D.R.P. 122388 yore 6. Oktober 1900. -- Patentbl. 1901, 22, 1185. y Die Eierschalen werden nor dem Einlegen in eine Konservirungsfliissigkeit, z. B. eine Calciumhydroxydltlsung, mit ParaffinS1 benetzt, was zweckmitssig in der Weise geschieht, dass man die Eier durch eine auf der Konservirungsflilssigkeit schwimmende Schicht ParatfinSl fallen l~tsst. A. Oelker. y Die Eierschalen werden nor dem Einlegen in eine Konservirungsfliissigkeit, z. B. eine Calciumhydroxydltlsung, mit ParaffinS1 benetzt, was zweckmitssig in der Weise geschieht, dass man die Eier durch eine auf der Konservirungsflilssigkeit schwimmende Schicht ParatfinSl fallen l~tsst. A. Oelker. A. Oelker. Butter, Speisefette lind 0ele. D. Holde und M. Stange: GemischteGlyceride in natfirlichen Fetten. -- Ber. Deutsch. Chem. Ges. 1901, 34, 2402--2408. Bis vor wenigen Jahren nahm man allgemein an, d&ss die natfirlichen Pflanzen- und Thierfette im Wesentlichen aus Gemisehen einfacher Triglyceride der Typen Ole'in CaHs(ClsHsa02) a und Stearin, C3H.~,(CIsHssO2) s bestehen. Blith undRobertson dagegen stellten aus der Butter durch fraktionirte Krystallisation mittelst Alkohol-Aether ein festes Bis vor wenigen Jahren nahm man allgemein an, d&ss die natfirlichen Pflanzen- und Thierfette im Wesentlichen aus Gemisehen einfacher Triglyceride der Typen Ole'in CaHs(ClsHsa02) a und Stearin, C3H.~,(CIsHssO2) s bestehen. Blith undRobertson dagegen stellten aus der Butter durch fraktionirte Krystallisation mittelst Alkohol-Aether ein festes
https://openalex.org/W4306780923	https://periodicos.utfpr.edu.br/rbta/article/download/15218/9378	Portuguese	null	Caracterização físico-química e sensorial de vinhos espumantes elaborados com uvas da espécie Vitis rotundifólia	Revista brasileira de tecnologia agroindustrial	2,022	cc-by	7,893	Edson Luiz de Souza edsonluizdesouza@gmail.com orcid.org/0000-0001-8238-1529 Universidade do Oeste de Santa Catarina, Videira, Santa Catarina, Brasil https://periodicos.utfpr.edu.br/rbta Angelica Bender bender.angelica.fruti@gmail.com orcid.org/0000-0002-1141-6090 Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil. PALAVRAS-CHAVE: Muscadínea. Qualidade. Aceitação. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022 RESUMO As uvas Vitis rotundifolia possuem aroma e sabor bastante característicos e tem despertado interesse dos produtores pois apresentam grande potencial para produção orgânica devido à resistência à maioria das pragas e doenças da videira, além dos atributos medicinais, nutricionais e terapêuticos devido aos níveis de antioxidantes presentes nas bagas, demonstrando uma alternativa para diversificação da cadeia produtiva da uva e do vinho. O objetivo deste estudo foi avaliar as características físico-químicas e sensoriais de espumantes produzidos a partir de uvas Vitis rotundifolia utilizando o método tradicional com adição de diferentes formulações de licor de expedição. O experimento foi realizado na Epagri-Videira com uvas de vinhedo implantado em 2002. As variedades avaliadas foram ‘Regale’ e ‘Noble’, de acordo com os tratamentos: T1- Testemunha (próprio espumante); T2- 20g. L-1 de açúcar; T3- 20 g.L-1 de açúcar + suco de amora preta; T4- 20 g.L-1 de açúcar + suco da própria variedade (‘Regale’/ ‘Noble’); T5- 20 g.L-1 de açúcar + 0,2 g.L-1 de ácido cítrico, quanto a composição físico-química e sensorial. Os maiores valores de acidez total foram nos vinhos espumantes do T5, para ambas as variedades. Os espumantes de ‘Noble’ apresentaram maior teor alcoólico em relação a ‘Regale’. Nos aspectos físico-químicos, os espumantes elaborados com uvas V. rotundifolia apresentaram diferentes teores de açúcares totais culminando em diferentes classificações. Sensorialmente os espumantes de ‘Regale’ se destacaram em relação aos de ‘Noble’ para a persistência de espuma. Em relação à avaliação global os espumantes de ‘Noble’ obtiveram maior aceitação por parte dos avaliadores, independente da composição do licor de expedição. Os espumantes obtidos a partir de ‘Noble’ receberam melhor avaliação global. André Luiz Külkamp de Souza andresouza@epagri.sc.gov.br orcid.org/0000-0003-4889-6291 Empresa de Pesquisa e Extensão Rural de Santa Catarina, Videira, Santa Catarina, Brasil. André Luiz Külkamp de Souza andresouza@epagri.sc.gov.br orcid.org/0000-0003-4889-6291 Empresa de Pesquisa e Extensão Rural de Santa Catarina, Videira, Santa Catarina, Brasil. Vinicius Caliari caliari@epagri.sc.gov.br orcid.org/0000-0003-2206-0293 Empresa de Pesquisa e Extensão Rural de Santa Catarina, Videira, Santa Catarina, Brasil. Vinicius Caliari caliari@epagri.sc.gov.br orcid.org/0000-0003-2206-0293 Empresa de Pesquisa e Extensão Rural de Santa Catarina, Videira, Santa Catarina, Brasil. Edson Luiz de Souza edsonluizdesouza@gmail.com orcid.org/0000-0001-8238-1529 Universidade do Oeste de Santa Catarina, Videira, Santa Catarina, Brasil PALAVRAS-CHAVE: Muscadínea. Qualidade. Aceitação. Página \| 3954 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022 Página \| 3955 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. INTRODUÇÃO A produção mundial de vinhos espumantes apresentou um aumento de 40% na última década (MARTÍNEZ-GARCÍA et al., 2017). No Brasil a produção de vinhos espumantes aumentou rapidamente nos últimos anos, houve ampliação na área de cultivo de uvas Vitis vinifera, para elaboração de vinhos finos, com destaque para os vinhos espumantes (RUIZ-MORENO et al., 2017). O forte aumento na demanda pela categoria dos vinhos espumantes pode ser explicado pelas mudanças nas preferências e gostos dos consumidores (OIV, 2020). O termo vinho espumante refere-se exclusivamente a vinhos que passaram por uma fermentação alcoólica em um recipiente fechado e consequente incorporação de gás carbônico decorrente desse procedimento (BRASIL, 2018). A elaboração de vinho espumante tradicional consiste basicamente de duas etapas, a elaboração do vinho base, ou seja, um vinho tranquilo com acidez pronunciada que, quando finalizado recebe adição de açúcar, nutrientes e leveduras (licor de tirage) para sofrer novo processo fermentativo em recipiente fechado (MARTÍNEZ- GARCÍA et al., 2017), que pode ser em garrafas seladas (método tradicional) ou em grandes recipientes (método Charmat) (CALIARI et al., 2015; CULBERT et al., 2016; UBEDA et al., 2016). A quantidade de açúcar final do espumante pode ser corrigida pelo licor de expedição, que consiste basicamente do próprio espumante, algum vinho ou brandy com quantidade específica de sacarose para determinar o tipo de produto (SIMONAGGIO e LEHN, 2014) e assim a classificação dos espumantes varia entre nature (até 3g L-1), extra brut (> 3g L-1 a 8 g L-1), brut (> 8 g L-1 a 15 g L-1), seco (> 15 g L-1 a 20 g L-1), meio seco (> 20 g L-1 a 60 g L-1) e doce (> 60 g L-1) (BRASIL, 2014). Mediante o aumento na produção de vinhos espumantes, várias pesquisas vêm sendo realizadas no intuito de melhorar a qualidade da bebida, e avaliar diferentes cultivares que apresentem aptidão para a elaboração (CAMPOS et al., 2016). As uvas Vitis rotundifolia também conhecidas como uvas muscadíneas possuem aroma e sabor bastante característicos (PARK e OH, 2015). Embora, no Brasil são ainda pouco conhecidas, nos Estados Unidos apresentam importância econômica, onde são comercializadas como fruta fresca, sucos, geleias e vinhos (FONSAH e AWONDO, 2016). Esta espécie tem despertado interesse pois R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. INTRODUÇÃO apresenta grande potencial para produção orgânica devido à grande resistência à maioria das pragas e doenças da videira (WEI et al. 2017), bem como, aos atributos medicinais, nutricionais e terapêuticos devido aos altos níveis de antioxidantes presentes nas bagas (FONSAH e AWONDO, 2016). Diante das características apresentadas, as uvas muscadíneas apresentam-se como uma alternativa para diversificação da cadeia produtiva da uva e do vinho. Sendo assim, o objetivo deste estudo foi avaliar as características físico-químicas e sensoriais de espumantes produzidos a partir de uvas V. rotundifolia utilizando o método tradicional de elaboração com adição de diferentes formulações de licor de expedição. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3956 MATERIAL E MÉTODOS Os espumantes foram elaborados a partir de uvas muscadíneas colhidas manualmente no mês de março de 2015, na área experimental da Epagri - Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina, Estação Experimental de Videira (Videira, SC/Brasil), localizada sob as coordenadas 27º02’27,59’’ S, 51º08’04,73’’ W e, altitude de 830 metros acima do nível do mar. O clima da região de acordo com Köppen é classificado como mesotérmico úmido e verão ameno (Cfb). As médias históricas (1960-2016) de precipitação, temperatura média e umidade relativa nos meses que englobam a brotação e colheita (setembro a março) são de 1.240 mm, 19,8°C e 73,4% UR, respectivamente (EPAGRI/CIRAM). As uvas empregadas na elaboração dos espumantes foram da espécie V. rotundifolia. Foram elaborados vinhos varietais das variedades tintas ‘Regale’ e ‘Noble’. O vinhedo foi implantado em 2002 no sistema de condução em latada sem o uso de porta-enxerto (pé franco) em espaçamento de 3,0 x 2,0 m, entre linhas e entre plantas, respectivamente. As amostras de espumantes foram elaboradas na vinícola experimental da Epagri-Videira-SC, seguindo duas etapas: 1ª etapa: elaboração do vinho base, as uvas V. rotundifolia foram previamente desengaçadas e esmagadas mecanicamente, com adição de enzimas pectolíticas R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. (Everzym XPL), posteriormente o líquido juntamente com a parte sólida foi conduzido para recipiente de aço inoxidável permanecendo por 24 horas em maceração. Após o período de contato líquido/casca, a massa sólida foi levada para prensa, promovendo a separação do líquido e bagaço. Os mostos foram acrescidos com sacarose para elevação do potencial alcoólico, ativante de fermentação e leveduras Saccharomyces cerevisiae PB2019 (Fermol Blanc-AEB - Bréscia, Itália). A fermentação alcoólica ocorreu em tanques de aço inoxidável e não foram submetidos à fermentação maloláctica. Os vinhos base foram estabilizados a frio e foi adicionado dióxido de enxofre antes do engarrafamento (Vinoaromax e AEB Spa e Brescia e Itália) e apresentaram teores de álcool de 7,55% v/v e 7,93% v/v e de açúcares residuais de 2,31 g L-1 e 2,38 g L-1 para ‘Regale’ e ‘Noble’, respectivamente. Estas etapas foram realizadas de forma individual para cada variedade de uva empregada no experimento. 2ª etapa: tomada de espuma, o vinho base foi acondicionado em garrafas champanheiras, adicionados de licor de tireage contendo 24 g L-1 de sacarose para fornecer 6,0 atm de pressão, leveduras S. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3957 MATERIAL E MÉTODOS cerevisiae PB2002 (Fermol Reims Champagne AEB Spa - Bréscia, Itália) e solução de bentonite 0,7 mL.L-1 (AEB Spa - Brescia, Itália), posteriormente selados com tampas tipo corona. Os vinhos foram armazenados a temperatura próxima de 17 °C até a segunda fermentação estar completa. Depois de 12 meses em garrafa sob borras a 10 °C, os vinhos espumantes passaram pelos processos de remoagem em pupitres de madeira e posteriormente pelo degorgement, onde o bico da garrafa foi congelado a -30°C e a tampa seladora removida, onde as sujidades (borras) foram expelidas naturalmente pela pressão interna da garrafa. O volume foi ajustado com diferentes composições de licores de expedição T1- Testemunha (próprio espumante); T2 -20 g L-1 de açúcar (sacarose); T3- 20 g.L-1 de açúcar (sacarose) + suco de amora preta; T4- 20 g L-1 de açúcar (sacarose) + suco da variedade empregada na elaboração do espumante (Regale ou Noble); T5- 20 g L-1 de açúcar (sacarose) + 0,2 g L-1 de ácido cítrico. A adição destes foi realizada nos espumantes prontos para beber. Página \| 3957 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Os tratamentos foram definidos como: R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3958 Os tratamentos foram definidos como: O experimento contou com delineamento experimental inteiramente casualizado, arranjado em esquema bifatorial (2x5), sendo o fator A as variedades (‘Regale’ e ‘Noble’) e o fator B as composições do licor de expedição (T1, T2, T3, T4 e T5), com três repetições. Foram utilizadas três garrafas de vidro com capacidade de 500 mL cada para cada tratamento, sendo escolhida aleatoriamente uma garrafa para as análises físico-químicas, colorimétricas e de compostos bioativos. Cada garrafa representou uma repetição. O restante das amostras foi destinado à análise sensorial. As análises físico-químicas de acidez total (AT), acidez volátil, teor de sólidos solúveis (SS), relação SS/AT e pH foram realizadas seguindo Instrução Normativa n° 24 de 08/09/2005 (BRASIL, 2005). O teor de sólidos solúveis foi determinado em refratômetro digital de bancada com compensação automática de temperatura (QUIMIS®) e o resultado expresso em °Brix. O pH foi determinado em pHmetro Meter AD1030® e as determinações de acidez total foram realizadas por titulação da amostra, com solução padronizada de NaOH 0,1N, adotando-se, como ponto final, o pH = 8,2, e o resultado expresso em mEq.L-1. A acidez volátil foi obtida pela destilação da amostra em equipamento Cazenave-Ferré®, sendo recolhido 100 mL do destilado acrescido de fenolftaleína e titulado com NaOH 0,1N adotando-se, como ponto final a presença da coloração rosa, expresso em mEq L-1. O teor alcoólico foi medido em balança hidrostática após a destilação em destilador enológico eletrônico Gibertini®. Para os açúcares redutores (g.L-1) e açúcares totais (redutores + não redutores) utilizou-se o método DNS descrito por Maldonade et al. (2016). A densidade foi determinada com um densímetro de vidro Aton Paar®, expressa em g.cm3. Para avaliar a coloração dos espumantes foi empregado o uso do espectrofotômetro da Konica Minolta®, modelo CM-5, pesquisando as coordenadas L, a e b. Sendo que o L representa a luminosidade da amostra e os valores de a* e b* foram empregados nos cálculos da saturação (croma) e tonalidade (°HUE) da cor, obtidas pelas seguintes fórmulas, conforme recomendações de McGuire (1992): C= [(a)2+(b)2]1/4 Hº = tan−1 (b/a) quando a > 0; b* ≥ 0 Hº = 180 + tan−1 (b/a) quando a* < 0 Hº = tan−1 (b/a) quando a* > 0; b* ≥ 0 Hº = 180 + tan−1 (b/a) quando a* < 0 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3959 RESULTADOS E DISCUSSÃO As variáveis físico-químicas de acidez total, pH, sólidos solúveis, teor alcoólico, açúcares redutores, açúcares totais e densidade apresentaram interação entre os fatores avaliados (variedades x licor de expedição) estão listados na Tabela 1. Tabela 1- Variáveis de acidez total, pH, sólidos solúveis, teor alcoólico, açúcares redutores, açúcares totais e densidade de vinhos espumantes elaborados com uvas V. rotundifolia ‘Regale’ e ‘Noble’ Variáveis Composição do Licor de Expedição Acidez Total (meq L-1) T1 T2 T3 T4 T5 Regale 80,87 Ca* 79,30 Ca** 86,34 Ba 86,91 Ba 99,57 Aa Noble 75,95 Cb 70,37 Db 80,53 Bb 77,41 Cb 90,52 Ab pH T1 T2 T3 T4 T5 Regale 2,75 Db 2,84 Cb 3,11 Aa 3,00 Bb 2,87 Cb Noble 3,04 Ba 3,13 Aa 3,11 Aa 3,05 Ba 2,92 Ca Sólidos solúveis (°Brix) T1 T2 T3 T4 T5 Regale 4,23 Db 4,68 Cb 5,28 Bb 5,68 Ab 5,4 Bb Noble 4,60 Da 5,65 Ba 5,95 Aa 6,18 Aa 5,93 Aa Teor Alcoólico (%v/v) T1 T2 T3 T4 T5 Regale 9,08 Ab 9,03 Ab 8,88 ABb 8,73 BCb 8,60 Cb Noble 9,95 Aa 9,35 Ca 9,68 Ba 9,50 Bca 9,33 Ca Açúcares Redutores (g L-1) T1 T2 T3 T4 T5 Regale 2,15 Da 10,52 Cb 18,46 Bb 25,48 Aa 19,33 Bb Noble 2,50 Da 21,90 Ba 21,36 Ba 23,47 ABa 25,13 Aa Açúcares Totais (g L-1) T1 T2 T3 T4 T5 Regale 2,78 Da 11,42 Cb 20,03 Bb 26,72 Ab 20,89 Bb Noble 3,00 Da 25,45 Ba 26,30 Ba 31,56 Aa 26,40 Ba Densidade (g.cm3) T1 T2 T3 T4 T5 Regale 995 Da 996 Cb 999 Ba 1002 Aa 999 Ba Noble 994 Db 999 Ba 1000 Ba 1001 Aa 1000 Ba Acidez Volátil (meq L-1) T1 T2 T3 T4 T5 Regale 7,54a# 6,87 B## 6,80 B 7,42 B 7,04 AB 6,69 B Noble 6,34b Mesma letra maiúscula na linha não difere entre si para os tratamentos dentro de variedades (P<0,05). Mesma letra minúscula na coluna não difere entre si para variedades dentro de composição do licor de expedição (P<0,05). #Mesma letra minúscula na coluna não difere entre variedades (P<0,05). ## Mesma letra maiúscula na linha não difere entre composição do licor de expedição (P<0,05). Os tratamentos foram definidos como: O conteúdo de polifenóis totais dos vinhos espumantes foi determinado por espectrofotometria de acordo com o método colorimétrico de Folin-Ciocalteu (SINGLETON e ROSSI, 1965) e expressos em mg de ácido gálico (GAE) L-1. As medições de absorbância foram realizadas em espectrofotômetro Ray Leigh modelo UV-2601. O teor total de antocianinas monoméricas foi determinado através do método diferencial de pH (GIUSTI e WROLSTAD, 2001) usando soluções tampão de cloreto de potássio (pH 1,0) e acetato de sódio (pH4.5), os resultados foram expressos em mg L-1 de cianidina-3-glicosídio. A capacidade antioxidante foi determinada por DPPH de acordo com metodologia descrita por Kim et al. (2002), e os resultados expressos em μM TEAC mL-1. A avaliação sensorial dos espumantes foi realizada em laboratório de análise sensorial localizado na Epagri-Videira/SC através da análise quantitativa (NBR 12994) (Associação Brasileira De Normas Técnicas, 2014). A intensidade dos atributos foi avaliada em escala não estruturada de 9 pontos, ancorada nos extremos com as palavras pouca intensidade e muita intensidade para cor âmbar, quantidade e persistência de espuma, tamanho de bolhas, intensidade e desprendimento de bolhas, sabor amargo, doce, ácido e adstringente, retrogosto e sensação alcoólica, volume, corpo e equilíbrio e 0-desgostei muitíssimo e 9- gostei muitíssimo para avaliação global, quanto ao aroma foi solicitado aos julgadores que descrevessem individualmente a percepção de cada um. A compreensão dos atributos e descritores pelos provadores foi verificada e avaliada durante a seleção e treinamento da equipe de degustadores. O treinamento foi realizado durante 06 meses precedentes ao início das avaliações. Foram efetuados testes de gostos (doce, ácido e amargo) com soluções teste e posteriormente familiarização com as amostras e espumantes comerciais. Foi selecionado um grupo de 10 pessoas que assinaram o Termo de Consentimento Livre e Esclarecido aprovado pelo Comitê de Ética da Universidade Federal de Pelotas, sob protocolo CAAE 92226218.8.0000.5317. Os avaliadores receberam as amostras (4 ± 1 °C) em taças de vidro codificadas com três dígitos aleatórios, juntamente com as fichas de avaliação. Os dados foram submetidos à análise de variância (ANOVA) e quando detectados efeitos de tratamento, procedeu-se o teste de comparação de médias pelo Teste Tukey a 5% de probabilidade de erro. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3960 RESULTADOS E DISCUSSÃO Tratamentos: T1- Testemunha (próprio espumante); T2- 20 g L-1 de açúcar; T3- 20 g L-1 de açúcar + suco de amora preta; T4- 20 g L-1 de açúcar + suco da variedade empregada na elaboração do espumante (Regale ou Noble); T5- 20 g L-1 de açúcar + 0,2 g L-1 de ácido cítrico. Fonte: Elaborado pelo autor (2022). R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Os maiores valores de acidez total foram verificados nos vinhos espumantes oriundos do tratamento com adição de açúcar e ácido cítrico (T5), para ambas as variedades, diferindo dos demais tratamentos, o que pode ser justificada pela adição de ácido cítrico na composição do licor de expedição (Tabela 1). Quanto às variedades, os espumantes elaborados com ‘Regale’ apresentaram-se mais ácidos do que os de ‘Noble’ em todos os tratamentos testados, resultado que vai ao encontro dos observados no mosto antes da fermentação, onde o valor para Regale apresentou-se mais elevado. Valores elevados de acidez total garantem as características de frescor da bebida, e os níveis de acidez nos vinhos espumantes estão relacionados ao grau de maturação da uva, a cultivar e a forma de extração do mosto (MENEGUZZO, 2010; MENEGUZZO et al., 2018). A legislação brasileira determina limites de 40 a 130 meq L-1 para acidez total em vinhos espumantes (BRASIL, 2018). Os valores encontrados nos espumantes analisados compreenderam os valores de 70,37 meq L-1 a 99,57 meq L-1, respeitando os limites estabelecidos em lei. De acordo com Meneguzzo (2010), os espumantes brasileiros apresentam valores elevados de acidez, ou seja, superiores a 70 meq L-1, afirmação que corrobora com os resultados encontrados para os espumantes de uvas muscadíneas em estudo. Os valores de pH dos espumantes que receberam o licor de expedição com açúcar e suco de amora (T3) não apresentaram diferenças significativas entre as variedades. Nos demais tratamentos, os espumantes de ‘Noble’ apresentaram os maiores valores, oscilando de 2,92 a 3,13, enquanto os espumantes de ‘Regale’ variaram de 2,87 a 3,11 (Tabela 2). Maior parte das amostras ficaram fora da margem descrita por Meneguzzo (2010) para o pH dos espumantes brasileiros, onde os autores alegam que os valores variam de 3,10 a 3,40. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3961 RESULTADOS E DISCUSSÃO No entanto, níveis muito elevados de pH podem desestabilizar o vinho tanto biologicamente como do ponto de vista físico-químico, uma vez que o torna mais propenso à oxidação e à proliferação microbiana e compromete, portanto, a sua vida útil. O teor alcoólico dos espumantes brasileiros deve ser compreendido entre os valores de 10 a 13 % v/v (BRASIL, 2018). Os resultados dos espumantes de ‘Noble’ diferiram em relação aos de ‘Regale’ devido aos valores mais elevados, no entanto, em ambos os casos foram abaixo do preconizado, variando de 9,33 % v/v a 9,95% R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. v/v para ‘Noble’ e 8,60 % v/v a 9,08 % v/v para ‘Regale’. O grau alcoólico do espumante representa a soma da graduação do vinho base, mais aquela formada durante a espumatização, que é de aproximadamente 1,4% v/v (RIZZON et al., 2000). Meneguzzo et al. (2018) avaliaram vinhos espumantes da Serra Gaúcha, os quais apresentaram baixo teor alcoólico, 10,40 % v/v para ‘Riesling Itálico’ a 11,4 % v/v para ‘Chardonnay’. Para açúcares redutores dos espumantes produzidos com a variedade ‘Noble’ diferiram estatisticamente daqueles elaborados com ‘Regale’ dentro dos tratamentos T2, T3 e T5 devido as maiores concentrações. Quanto aos açúcares totais, foi observada diferença apenas no tratamento com o próprio espumante (T1). Os espumantes de ‘Noble’ e ‘Regale’ apresentaram maior teor de açúcares redutores e açúcares totais nos tratamentos T4 e T5, bem como os menores no T1. Os açúcares totais representam a soma dos açúcares redutores remanescentes do mosto da uva que permaneceram no vinho base e não redutores compostos pela quantidade de sacarose tanto no vinho base, como no licor de tiragem que não foram exauridos na primeira e segunda fermentação mais a quantidade adicionada no licor de expedição por ocasião do “dégorgement”. As diferenças entre os espumantes que receberam a mesma composição de licor de expedição pode ser justificada pelo método de elaboração, uma vez que no sistema tradicional “champenoise” a tomada de espuma ocorre individualmente em cada garrafa, sendo assim, o consumo dos açúcares pelas leveduras pode ser distinto, bem como, a porcentagem de líquido expelido no momento do “dégorgement” para eliminação das borras e que vai dar espaço para o licor de expedição é diferente de uma garrafa para outra, o que pode resultar em uma composição diferente no produto final. Página \| 3962 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. RESULTADOS E DISCUSSÃO A densidade do vinho está relacionada principalmente ao seu teor alcoólico e de açúcares residuais (OLIVEIRA et al., 2011), fator condizente aos observados no presente estudo, uma vez que as amostras com maior teor de açúcares totais resultaram em maiores densidades. Da mesma forma, o teor de sólidos solúveis é influenciado pelos açúcares, uma vez que estes são responsáveis por mais de 90% dos sólidos solúveis da uva e derivados, o que justifica o fato dos espumantes da variedade ‘Noble’ terem apresentado teores mais elevados quando comparados R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. com aqueles elaborados com ‘Regale’ dentro dos cinco tratamentos. O tratamento que consistiu na adição de açúcar mais suco da mesma variedade empregada como matéria prima para o espumante (T4) obteve os maiores valores para essa mesma variável (Tabela 1). Em uma média geral os espumantes de ‘Regale’ apresentaram uma acidez volátil um pouco mais elevada em relação aos espumantes de ‘Noble’, chegando a 7,54 meq L-1 (Tabela 1), permanecendo dentro do especificado em lei, onde o limite está estipulado em 20 meq L-1. Ribéreau-Gayon et al. (2003) e Kunz et al. (2010) explicam que a acidez volátil dos vinhos espumantes é fundamental para a qualidade da bebida desde que seja inferior ao limite tolerável sensorialmente de 10 meq L-1 a 12 meq L-1. Os resultados verificados no presente estudo se mantiveram abaixo do limite indicado por estes autores. Bem como, inferiores aos encontrados por Meneguzzo et al. (2018), que avaliaram a acidez volátil de espumantes das cultivares ‘Riesling Itálico’, ‘Chardonnay’ e ‘Pinot Noir’ produzidos na Serra Gaúcha, com valores que oscilaram de 10 a 13 meq L-1. A luminosidade, coordenadas a e b, tonalidade e intensidade de cor, bem como, a atividade antioxidante apresentaram interação entre os fatores avaliados (variedades x licor de expedição) e estão listadas na Tabela 2. Os espumantes de ‘Noble’ apresentaram valores mais elevados para luminosidade nos diferentes tratamentos diferindo de ‘Regale’. Os espumantes do T3 obtiveram os menores valores para L, indicando produtos mais escuros, para ambas as variedades (Tabela 2). A coordenada a varia entre +a vermelho e o –a verde, a coordenada b varia do +b amarelo e –b azul e, a leitura destas coordenadas fornecem a tonalidade e a intensidade da cor (CHITARRA e CHITARRA, 2005). R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3963 RESULTADOS E DISCUSSÃO De maneira geral os espumantes de ‘Regale’ apresentaram valores de a mais elevados, enquanto que os de ‘Noble’ os valores de b foram maiores nos tratamentos T1, T2, T4 e T5, indicando que a variedade ‘Regale’ gerou uma bebida com nuances mais avermelhados, e ‘Noble’ a tendência foram espumantes mais amarelados. Situação observada também na tonalidade (HUE), às médias encontraram-se mais afastadas do eixo 0° (vermelho), tendendo à direção do eixo 90° (amarelo), os valores para ‘Noble’ ficaram na faixa do amarelo, e aqueles oriundos de ‘Regale’, a faixa de cor obtida compreende do amarelo/laranja. Os R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. espumantes de ‘Noble’ apresentaram maior intensidade de cor nos tratamentos T1, T2 e T5 em relação aos produzidos com ‘Regale’. Os espumantes do T3 para as duas variedades obtiveram valores de a, b e intensidade de cor maiores e tonalidade menor em relação aos demais tratamentos, o que pode ter ocorrido devido a influência do suco de amora. espumantes de ‘Noble’ apresentaram maior intensidade de cor nos tratamentos T1, T2 e T5 em relação aos produzidos com ‘Regale’. Os espumantes do T3 para as duas variedades obtiveram valores de a, b e intensidade de cor maiores e tonalidade menor em relação aos demais tratamentos, o que pode ter ocorrido devido a influência do suco de amora. Tabela 2- Variáveis de luminosidade, a, b, tonalidade de cor, intensidade de cor, DPPH e polifenóis totais de vinhos espumantes elaborados com uvas V. rotundifolia ‘Regale’ e ‘Noble’ Tabela 2- Variáveis de luminosidade, a, b, tonalidade de cor, intensidade de cor, DPPH e polifenóis totais de vinhos espumantes elaborados com uvas V. rotundifolia ‘Regale’ e ‘Noble’ e polifenóis totais de vinhos espumantes elaborados com uvas V. Página \| 3964 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. RESULTADOS E DISCUSSÃO rotundifolia Regale e ‘Noble’ Variáveis Composição do Licor de Expedição Luminosidade L T1 T2 T3 T4 T5 Regale 91,34 Bb 92,56 Ab** 90,67 Cb 91,38 Bb 92,73 Ab Noble 92,52 Ba 93,19 Aa 91,66 Ca 92,60 Ba 92,99 Aa a T1 T2 T3 T4 T5 Regale 2,56 Ca 2,14 Da 3,74 Aa 2,96 Ba 2,08 Da Noble 1,34 Bb 1,08 Cb 2,60 Ab 1,23 BCb 1,17 BCb b T1 T2 T3 T4 T5 Regale 8,77 Cb 7,85 Db 9,86 Ab 9,35 Ba 7,59 Eb Noble 9,68 Ba 8,48 Ca 10,01 Aa 9,50 Ba 8,58 Ca Tonalidade de cor (°HUE) T1 T2 T3 T4 T5 Regale 73,77 Ab 74,75 Ab 69,24 Cb 72,43 Bb 74,68 Ab Noble 82,14 Aa 82,77 Aa 75,52 Ba 82,65 Aa 82,24 Aa Intensidade de cor (Croma) T1 T2 T3 T4 T5 Regale 9,13 Cb 8,13 Db 10,55 Aa 9,81 Ba 7,86 Eb Noble 9,77 Ba 8,55 Ca 10,39 Aa 9,58 Bb 8,66 Ca DPPH (mmol L-1) T1 T2 T3 T4 T5 Regale 0,99 Ca 0,92 Da 1,14 Aa 1,08 Ba 0,95 Da Noble 0,88 Bb 0,83 Cb 0,96 Bb 1,04 Ab 0,95 Ba Polifenóis Totais (g L-1) T1 T2 T3 T4 T5 Regale 38,89a# 35,24 C## 33,49 D 40,58 A 38,57 B 33,82 D Noble 35,79b Mesma letra maiúscula na linha não difere entre si para os tratamentos dentro de variedades (P<0.05). Mesma letra minúscula na coluna não difere entre si para variedades dentro de composição do licor de expedição (P<0.05). #Mesma letra minúscula na coluna não difere entre variedades (P<0.05). ## Mesma letra maiúscula na linha não difere entre composição do licor de expedição (P<0.05). Tratamentos: T1- Testemunha (próprio espumante); T2-20 g L-1 de açúcar; T3- 20 g L-1 de açúcar + suco de amora preta;T4- 20 g L-1 de açúcar + suco da variedade empregada na elaboração do espumante (Regale ou Noble); T5- 20 g L-1 de açúcar + 0,2 g L-1 de ácido cítrico. Fonte: Elaborado pelo autor (2022) R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Os espumantes de ‘Regale’ apresentaram maior atividade antioxidante, diferindo de ‘Noble’, exceto no T4, que o maior valor foi obtido para espumantes de ‘Noble’ (Tabela 2). Quanto aos tratamentos a maior média de atividade antioxidante para os espumantes de ‘Regale’ foi verificada no T3 e para ‘Noble’ no T4. Página \| 3965 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. RESULTADOS E DISCUSSÃO Quanto aos polifenóis totais, os espumantes do T3 apresentaram maior teor em relação aos demais tratamentos (Tabela 2), provavelmente devido à adição do suco de amora, uma vez que estes sucos são ricos em compostos fenólicos. Os compostos fenólicos são de grande importância nos vinhos pois influenciam nas características sensoriais, contribuindo para cor, sabor e adstringência, além das suas potenciais propriedades bioativas, responsáveis pela atividade antioxidante da bebida (GRIS et al., 2010; CALIARI et al., 2015; IZQUIERDO-LLOPART e SAURINA, 2019). O conteúdo de polifenóis totais e atividade antioxidante podem ser influenciados por diversos fatores, tais como a região, processo de produção, e especialmente a variedade de uva empregada (POZO- BAYÓN et al., 2009; STEFENON, et al., 2014). Caliari et al. (2015) avaliaram vinhos espumantes da variedade ‘Moscato Giallo’ elaborados pelos métodos tradicional, Charmat e Asti e, os valores verificados pelos autores variaram de 0,78 mmol L-1 a 0,84 mmol L-1 para atividade antioxidante (DPPH). Em estudo realizado por Stefenon et al. (2014) com vinhos espumantes varietal ‘Chardonnay’ e cortes entre ‘Chardonnay’, ‘Riesling Italico’ e ‘Pinot Noir’ produzidos pelos métodos tradicional e charmat, mantidos sobre borras até 12 meses, os maiores valores para polifenóis totais foram observados nos espumantes oriundos dos cortes, alegando que o maior teor de polifenóis é devido a uva tinta ‘Pinot Noir’, oscilando de 0,451 .g L-1 a 0,498 g L-1, inferiores aos resultados dos vinhos espumantes de uvas V. rotundifolia estudados. Izquierdo-Llopart e Saurina (2019) explicam que os vinhos elaborados com uvas tintas são mais ricos em polifenóis do que os vinhos rosés ou brancos devido ao simples fato de que esses compostos são extraídos da casca da uva durante o processo de maceração. Os resultados da análise sensorial, foram determinados pela média das notas atribuídas, pelos degustadores, aos vinhos espumantes de V. Rotundifolia. Para as variáveis sensoriais não ocorreram interações entre os fatores de tratamento R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. (variedades x licor de expedição), sendo avaliado apenas o efeito simples. Os aspectos visuais e gustativos dos espumantes que apresentaram diferença significativa estão listados na Tabela 3. Tabela 3- Aspectos visuais (cor âmbar, persistência de espuma, persistência de bolhas), aspectos gustativos (volume em boca e corpo) e avaliação global de vinhos espumantes elaborados com uvas V. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. RESULTADOS E DISCUSSÃO rotundifolia ‘Regale’ e ‘Noble’ Tabela 3- Aspectos visuais (cor âmbar, persistência de espuma, persistência de bolhas), aspectos gustativos (volume em boca e corpo) e avaliação global de vinhos espumantes elaborados com uvas V. rotundifolia ‘Regale’ e ‘Noble’ Aspectos visuais Aspectos Gustativos Variedades Cor Âmbar Persistência espuma Persistência Bolhas Volume em boca Corpo Avaliação Global Regale 7,20ns 5,53a 6,24ns 3,99b 4,20b 3,99b Noble 6,72 3,58b 5,60 5,13a 5,20a 5,13a Comp. Licor Cor Âmbar Persistência espuma Persistência de Bolhas Volume em boca Corpo Avaliação Global T1 6,80ab** 4,73ns 5,40ab 4,10ns 4,50ns 4,10ns T2 6,00b 4,12 6,50ab 4,30 4,60 4,30 T3 8,10a 4,75 5,90ab 5,20 5,04 5,20 T4 7,20ab 4,76 5,02b 4,40 4,50 4,40 T5 6,73b 5,00 6,90a 4,90 4,80 4,90 Mesma letra minúscula na coluna não difere entre variedades (P<0.05). Mesma letra maiúscula na coluna não difere entre composição do licor de expedição (P<0.05). nsNão significativo Teste Tukey a 5% de probabilidade de erro. Tratamentos: T1- Testemunha (próprio espumante); T2- 20 g L-1 de açúcar; T3- 20 g L-1 de açúcar + suco de amora preta; T4- 20 g L-1 de açúcar + suco da variedade empregada na elaboração do espumante (Regale ou Noble); T5- 20 g L-1 de açúcar + 0,2 g L-1 de ácido cítrico. Fonte: Elaborado pelo autor (2022) Mesma letra minúscula na coluna não difere entre variedades (P<0.05). *Mesma letra maiúscula na coluna não difere entre composição do licor de expedição (P<0.05). nsNão significativo Teste Tukey a 5% de probabilidade de erro. Tratamentos: T1- Testemunha (próprio espumante); T2- 20 g L-1 de açúcar; T3- 20 g L-1 de açúcar + suco de amora preta; T4- 20 g L-1 de açúcar + suco da variedade empregada na elaboração do espumante (Regale ou Noble); T5- 20 g L-1 de açúcar + 0,2 g L-1 de ácido cítrico. Mesma letra minúscula na coluna não difere entre variedades (P<0.05). **Mesma letra maiúscula na coluna não difere entre composição do licor de expedição (P<0.05). nsNão significativo Teste Tukey a 5% de probabilidade de erro. Tratamentos: T1- Testemunha (próprio espumante); T2- 20 g L-1 de açúcar; T3- 20 g L-1 de açúcar + suco de amora preta; T4- 20 g L-1 de açúcar + suco da variedade empregada na elaboração do espumante (Regale ou Noble); T5- 20 g L-1 de açúcar + 0,2 g L-1 de ácido cítrico. Página \| 3966 Fonte: Elaborado pelo autor (2022) Fonte: Elaborado pelo autor (2022) Em relação ao aspecto visual os vinhos espumantes foram avaliados quanto à cor, quantidade e intensidade de espuma e bolhas e desprendimento de bolhas (perlage). As variáveis de quantidade e persistência de espuma, bem como, tamanho e desprendimento de bolhas não apresentaram diferença significativa para os fatores de tratamento. Em relação à coloração dos espumantes e a persistência das bolhas, não houve efeito significativo das variedades, mas sim das diferentes formulações do licor de expedição. O tratamento que teve a adição de suco de amora na composição (T3), recebeu maiores notas para a coloração âmbar R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. diferindo dos tratamentos T2 e T5. Para a persistência das bolhas, o T5 obteve maior média, destacando-se em relação ao T4. Os espumantes de ‘Regale’ se destacaram em relação aos de ‘Noble’ para a persistência de espuma. A presença do dióxido de carbono no vinho espumante torna a análise sensorial mais complexa em relação à de vinhos tranquilos, uma vez que a efervescência pode ser usada como um indicativo de qualidade da bebida devido ao prazer visual e gustativo que desperta nos consumidores, além de contribuir com a fineza aromática detectada tanto no olfato como no gosto, as borbulhas devem ser finas e numerosas, persistindo até o momento de ingerir o espumante (RIZZON et al, 2000; MENEGUZZO, 2010). De acordo com a percepção dos avaliadores as amostras analisadas apresentaram boa quantidade de espuma, no entanto, com pouca persistência, já as bolhas foram consideradas pequenas e persistentes, aspectos positivos em vinhos espumantes de maneira geral. Quanto aos aspectos olfativos, foi solicitado aos avaliadores apenas que descrevessem os aromas percebidos. Foram descritos os aromas de tabaco, pão torrado, melado, baunilha, chocolate, moscato, e de frutas como abacaxi, pêssego, maçã e guaraná, frutas secas e passas. Essas observações obtidas pelo grupo de avaliadores nos vinhos espumantes em estudo vão ao encontro das citações encontradas na literatura para os espumantes brasileiros, onde alguns autores relatam que esses possuem aroma floral e frutado, sendo a cultivar ‘Chardonnay’ a principal percursora de aromas de frutas e flores brancas, frutas cítricas com ‘Riesling Itálico’ e flores violetas devido a cultivar ‘Pinot Noir’, os aromas de pão e pão torrado são bastante apreciados e estão relacionados com os processos fermentativos (MENEGUZZO, 2010; GANSS et al., 2011; DUTEURTRE, 2014; GABBARDO e CELOTTI, 2015). R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3967 Fonte: Elaborado pelo autor (2022) Na avaliação gustativa o fator licor de expedição não apresentou diferença significativa dentro das variáveis estudadas, para o fator variedades observou-se destaque dos espumantes de ‘Regale’ para a variável retrogosto, enquanto para volume em boca e corpo os espumantes de ‘Noble’ obtiveram maiores médias. As variáveis gustativas são influenciadas pela variedade e qualidade da uva, bem como pelas técnicas de elaboração, o conjunto destas variáveis contribuem para a estrutura do vinho espumante, e aqueles que apresentam maior equilíbrio entre R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. elas, resultam em bebidas com mais corpo e melhor aceitação pelos consumidores. Observa-se nos resultados gustativos que as médias de notas foram medianas a baixas dentro da escala proposta, atingindo valor máximo de 5,2 para o volume em boca. Apesar de sutil é possível verificar que ocorreu um aumento na percepção de doçura por parte dos avaliadores para os tratamentos que receberam licor de expedição contendo açúcar, esse pequeno aumento também é perceptível para a variável equilíbrio. Em relação à avaliação global (Tabela 4), os espumantes de ‘Noble’ obtiveram maior aceitação por parte dos avaliadores com média das notas superior a 5, independente da composição do licor de expedição, para este fator de tratamento apesar não ter sido detectada diferença estatística significativa é possível observar que o tratamento com suco de amora obteve maior média em relação aos demais. A adição do suco de amora elevou a intensidade da cor âmbar dos espumantes avaliados, fato observado tanto nas análises colorimétricas como na sensorial. A cor é o primeiro contato do avaliador sendo um dos atributos mais importantes, pois está diretamente relacionado à aparência do produto, o que permite especular possíveis defeitos existentes na bebida, apresentando ainda forte influência sobre a percepção de outros atributos sensoriais, como o aroma e o gosto doce (MORROT et al., 2001; OLIVEIRA et al., 2011). R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3968 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3969 CONCLUSÕES Os espumantes elaborados com as uvas V. rotundifolia ‘Regale’ e ‘Noble’ apresentaram características físico-químicas condizentes com a legislação nacional de vinhos espumantes, com exceção do teor alcoólico que ficou abaixo do preconizado em lei. As diferentes formulações do licor de expedição influenciaram o teor de açúcares totais dos espumantes. A adição de suco de amora preta no licor de expedição contribui na maior intensidade de cor dos espumantes, verificada tanto nas análises colorimétricas quanto sensorial. R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Os espumantes elaborados com a variedade ‘Regale’ apresentaram maior persistência de espuma independente da composição do licor de expedição, e os espumantes de ‘Noble’ receberam melhor avaliação quanto ao volume em boca e corpo, e melhores pontuações para a avaliação global. Página \| 3969 R. bras. Tecnol. Agroindustr., Francisco Beltrão, v. 16, n. 2, p. 3954-3974, jul./dez. 2022. Página \| 3970 KEYWORDS: Muscadine. Quality. Acceptance ABSTRACT The grapes Vitis rotundifolia have a very characteristic aroma and flavor and have aroused the interest of producers because they have great potential for organic production due to their resistance to most pests and diseases of the vine, in addition to medicinal, nutritional, and therapeutic attributes due to the levels of antioxidants present in the grapes. berries, demonstrating an alternative for the diversification of the grape and wine production chain. The objective of this study was to evaluate the physicochemical and sensorial characteristics of sparkling wines produced from Vitis rotundifolia grapes using the traditional method with the addition of different expedition liqueur formulations. The experiment was carried out at Epagri-Videira with grapes from a vineyard established in 2002. The varieties evaluated were 'Regale' and 'Noble', according to the treatments: T1- Witness (own sparkling wine); T2- 20 g L-1 of sugar; T3- 20 g L-1 of sugar + blackberry juice; T4- 20 g L-1 of sugar + juice of the variety itself ('Regale'/'Noble'); T5- 20 g L-1 of sugar + 0.2 g L-1 of citric acid, in terms of physical-chemical and sensorial composition. The highest values of total acidity were found in sparkling wines from T5, for both varieties. 'Noble' sparkling wines had higher alcohol content than 'Regale'. In the physicochemical aspects, the sparkling wines made with V. rotundifolia grapes showed different levels of total sugars, culminating in different classifications. Sensorially, the sparkling wines from 'Regale' stood out in relation to those from 'Noble' for foam persistence. Regarding the global evaluation, the 'Noble' sparkling wines obtained greater acceptance by the evaluators, regardless of the composition of the expedition liqueur. The sparkling wines obtained from 'Noble' received the best global evaluation. KEYWORDS: Muscadine. Quality. Acceptance Página \| 3970 REFERÊNCIAS BRASIL. MINISTÉRIO DA AGRICULTURA, PECUÁRIA E ABASTECIMENTO. Instrução normativa n° 24, de 8 de setembro de 2005. Aprova o Manual Operacional de Bebidas e Vinagre. Diário Oficial [da] República Federativa do Brasil, Brasília, DF, 20 set., 2005. BRASIL. MINISTÉRIO DA AGRICULTURA, PECUÁRIA E ABASTECIMENTO. Decreto nº 8.198, de 20 de fevereiro de 2014. Dispõe sobre a produção, circulação e comercialização do vinho e derivados da uva e do vinho. Diário Oficial [da] República Federativa do Brasil, Brasília, DF, 09 mar. 2018. BRASIL. MINISTÉRIO DA AGRICULTURA, PECUÁRIA E ABASTECIMENTO. Instrução normativa nº 14, de 8 de fevereiro de 2018. Página \| 3971 ABSTRACT Complementação dos Padrões de Identidade e Qualidade do Vinho e Derivados da Uva e do Vinho. 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Disponível em: <https://periodicos.utfpr.edu.br/rbta>. Acesso em: XXX. Correspondência: Angelica Bender Programa de Pós-Graduação em Agronomia. Av. Eliseu Maciel, sn. Campus UFPel- C.Postal 354 - CEP 96160-000,Capão do Leão, Rio Grande do Sul, Brasil Formatado por: Mari Eduarda Cristina Brancher Garlet Processo de Editoração: Prof.a Dr.a Alessandra Machado Lunkes Direito autoral: Este artigo está licenciado sob os termos da Licença Creative Commons-Atribuição 4.0 Internacional. Página \| 3974
https://openalex.org/W2763668199	https://scindeks-clanci.ceon.rs/data/pdf/0352-5252/2017/0352-52521775703P.pdf	English	null	The width of the attached gingiva and its variability in people with healthy periodontal status	Acta stomatologica Naissi/Acta Stomatologica Naissi	2,017	cc-by-sa	8,777	1UNIVERSITY OF NIŠ, FACULTY OF MEDICINE, CLINIC OF DENTISTRY, DEPARTMENT OF PERIODONTOLOGY AND ORAL MEDICINE, NIŠ, SERBIA 1UNIVERSITY OF NIŠ, FACULTY OF MEDICINE, CLINIC OF DENTISTRY, DEPARTMENT OF PERIODONTOLOGY AND ORAL MEDICINE, NIŠ, SERBIA 2 PRIVATE DENTAL PRACTICES “MIRKOVIC”, NIŠ, SERBIA PRIVATE DENTAL PRACTICES “MIRKOVIC”, NIŠ, SERBIA Abstract g g f p p y p Results: The greatest width of the attached gingiva was found in subjects aged 15-30 years, primarily in the female population. The mean value of the width of the attached gingiva varied depending on the area in the mouth: the maximum width of the attached gingiva was noted in the area of the upper central incisors, while the lowest recorded value was in the area of the first molars, both in the upper and lower jaw. Conclusion: The width of the attached gingiva varies according to the age and sex of the person, and according to the site in the oral cavity. Rezultati: Kod ispitanika starosti od 15-30 godina, nađena je najveća širina pripojne gingive, kao i kod osoba ženskog pola u odnosu na mušku grupu ispitanika. Srednje vrednosti širine pripojne gingive varirale su u zavisnosti od područja usne Results: The greatest width of the attached gingiva was found in subjects aged 15-30 years, primarily in the female population. The mean value of the width of the attached gingiva varied depending on the area in the mouth: the maximum width of the attached gingiva was noted in the area of the upper central incisors, while the lowest recorded value was in the area of the first molars, both in the upper and lower jaw. p pojne gingive va i ale su u avisnosti od pod učja usne duplje: najveća širina pripojne gingive zabeležena je u predelu gornjih centralnih sekutića, dok je najmanja širina zabeležena u predelu prvih molara gornje i donje vilice. Zaključak: Širina pripojne gingive varira u odnosu na starost i pol osobe, kao i od mesta u usnoj duplji. duplje: najveća širina pripojne gingive zabeležena je u predelu gornjih centralnih sekutića, dok je najmanja širina zabeležena u predelu prvih molara gornje i donje vilice. Š Zaključak: Širina pripojne gingive varira u odnosu na starost i pol osobe, kao i od mesta u usnoj duplji. Conclusion: The width of the attached gingiva varies according to the age and sex of the person, and according to the site in the oral cavity. Ključne riječi: parodont, pripojna gingiva, zdravlje Key words: periodontium, attached gingiva, health Address for correspondence: Ana Pejčić DDS, MSc, PhD Nade Tomić 3/5 18000 Niš, Serbia Phone:381/642572178 E-mail:dranapejcic@hotmail.com © 2017 Faculty of Medicine in Niš. Clinic of Dentistry in Niš. All rights reserved / © 2017. Medicinski fakultet Niš. Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 str./p.1703 -1717 Primljen/ Recived on : 20.02.2017. Revidiran/ Revised on : 13.03.2017. Prihvaćen/ Accepted on : 27.03.2017. ŠIRINA PRIPOJNE GINGIVE I NJENA VARIJABILNOST KOD OSOBA SA ZDRAVIM PARODONTALNIM STATUSOM THE WIDTH OF THE ATTACHED GINGIVA AND ITS VARIABILTY IN PEOPLE WITH HEALTHY PERIODONTAL STATUS Ana S. Pejčić 1, Radmila R. Obradović 1, Dimitrije S. Mirkovic2 1UNIVERZITET U NIŠU, MEDICINSKI FAKULTET, KLINIKA ZA STOMATOLOGIJU, ODELJENJE ZA PARODONTOLOGIJU I ORALNU MEDICINU, NIŠ, SRBIJA 2 PRIVATNA STOMATOLOŠKA ORDINACIJA “MIRKOVIC”, NIŠ, SRBIJA 1UNIVERSITY OF NIŠ, FACULTY OF MEDICINE, CLINIC OF DENTISTRY, DEPARTMENT OF PERIODONTOLOGY AND ORAL MEDICINE, NIŠ, SERBIA ORGINALNI RAD KLINIČKA STUDIJA ORIGINAL ARTICLE CLINICAL STUDY doi:10.5937/asn1775703P Primljen/ Recived on : 20.02.2017. Revidiran/ Revised on : 13.03.2017. Prihvaćen/ Accepted on : 27.03.2017. Acta Stomatologica Naissi Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 str./p.1703 -1717 Abstract Background: Gingiva is part of the mucous membrane that covers the alveolar ridges of the jaw and surrounds the necks of the teeth. Anatomical features of the gingiva are very important in planning the treatment of periodontal disease. The attached gingiva is important for maintaining a healthy periodontal condition. This study measured the normal value width of the attached gingiva in periodontal healthy subjects. Uvod: Gingiva je deo sluzokože koja pokriva avleolarne grebene vilica i okružuje vratove zuba. Anatomske karakteristike gingive su veoma važne u planiranju lečenja oboljenja parodonta. Pripojna gingiva je važna za održavanje parodonta u zdravom stanju. Ovom studijom merene su normalne vrednosti širine pripojne gingive kod parodontalno zdravih osoba. Cilj rada bio je procena širinepripojne gingive različitim metodama. Š The aim was estimating the width of the attached gingiva by warious methods. Materijal i metode: Širina pripojne gingive merena je korišćenjem parodontalne sonde kod parodontalno zdravih osoba. U zavisnosti od starosti pacijenata, formirane su četiri grupe (I - ≤ 14 god; II - 15-30 god; III - 31-45 god i IV - 46-60 Material and Methods: The width of the attached gingiva was measured using a periodontal probe in periodontally healthy subjects. The measurement was performed in the Department of Periodontology and Oral Medicine Clinic of Dentistry, Faculty of Medicine University of Nis. Descriptive statistical analysis was performed to determine the mean values of the attached gingiva, which will represent the normal value width of the attached gingiva for people with healthy periodontium. Materijal i metode: Širina pripojne gingive merena je korišćenjem parodontalne sonde kod parodontalno zdravih osoba. U zavisnosti od starosti pacijenata, formirane su četiri grupe (I - ≤ 14 god; II - 15-30 god; III - 31-45 god i IV - 46-60 god.). Deskriptivna statistička analiza urađena je za određivanje srednjih vrednosti pripojne gingive , koje će predstavljati normalne vrednosti širine pripojne gingive za osobe sa zdravim parodontom. god.). Deskriptivna statistička analiza urađena je za određivanje srednjih vrednosti pripojne gingive , koje će predstavljati normalne vrednosti širine pripojne gingive za osobe sa zdravim parodontom. određivanje srednjih vrednosti pripojne gingive , koje će predstavljati normalne vrednosti širine pripojne gingive za osobe sa zdravim parodontom. određivanje srednjih vrednosti pripojne gingive , koje će predstavljati normalne vrednosti širine pripojne gingive za osobe sa zdravim parodontom. Introduction Pripojna gingiva je jedan od najvažnijih anatomskih i funkcionalnih delova potpornog aparata zuba. Iako u literaturi nema dovoljno podataka o ulozi pripojne gingive u održa- vanju parodontalnog zdravlja, odsustvo ili mala širina pripojne gingive može dovesti do bržeg širenja zapaljenja kod osoba koje ne održavaju dobru oralnu higijenu 1. The attached gingiva is one of the most important anatomical and functional parts of the tooth supporting apparatus. Although there is insufficient literary data on the role of the attached gingiva in maintaining periodontal health, the absence or small width of the attached gingiva may lead to faster spread of inflammation in people who do not maintain good oral hygiene 1. j g j Zdrava pripojna gingiva je svetlo- ružičaste boje sa površinom koja je tačkasta. Pripojna gingiva, zajedno sa palatinalnom sluzokožom, spada u mastikatorni tip sluzo- kože. Iz tih razloga, prekrivena je epitelom koji keratinizira. Deo pripojne gingive pripojen je za cement zuba, dok je veći deo pripojen za alveolarnu kost. Š Healthy attached gingiva is of light pink color with an area that is dotted. Attached gingiva, together with the palatal mucosa is one of the masticatory tips of mucous membranes. For these reasons, it is covered by keratinized epithelium. Part of the attached gingiva was annexed to the cement teeth, while the larger part was annexed to the alveolar bone. Širina pripojne gingive predstavlja rastojanje od donje ivice slobodne gingive u nivou dna gingivalnog sulkusa do muko- gingivalne linije u vestibulumu 1. Uprkos raznim mišljenjima u vezi sa adekvatnom količinom keratinizovanog tkiva za održavanje zdravlja parodonta, mukogingivalna linija služi kao važno kliničko obeležje u proceni parodonta 2. Mukogingivalna linija je diskretna linija koja predstavlja granicu između pokretne i nepokretne sluzokože prilikom pasivnog kretanja usana i obraza 3. Metode za lociranje mukogingivalne linije su vizuelna metoda (VM), funkcionalna metoda (FM) i Šilerova metoda (ŠM) 4. The width of the attached gingiva is the distance from the bottom edge of the free gingiva in the bottom of the gingival sulcus to the gingival line in the vestibulum 1. Despite various opinions regarding the appropriate amount of keratinized tissue to maintain periodontal health, mucogingival line serves as an important landmark in the clinical assessment of periodontium 2. Mucogingival line is a discrete line representing the boundary between mobile and immobile mucous membranes in the passive movement of the lips and cheeks 3. Abstract Klinika za stomatologiju Niš. Sva prava zadržana. 1703 Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 Introduction Methods for locating gingival lines are visual method (VM), functional (FM) and Shiler’method (ŠM) 4. Vizuelna metoda se zasniva na razlici u boji između gingive i alveolarne sluzokože 5. U funkcionalnoj metodi se mukogingivalna linija ocenjuje kao granica između pokretnog i nepokretnog tkiva gde se mobilnost tkiva, utvrđuje korišćenjem parodontalne sonde, koja je pozicionirana laganim pritiskom hori- zontalno u predvorju usana prema ivici gin- give 3. Mukogingivalna linija može se proceniti i vizuelno posle bojenja muko- gingivalnog kompleksa Lugolovim rastvorom (jodna proba). Alveolarna sluzokoža razlikuje se od keratinizovane gingive u sadržaju glikogena, kisele fosfataze i nespecifične esteraze i povećanoj količini elastičnih vlakana, što rezultira u pozitivnoj jodnoj reakciji 6-9. ( ) ( ) Visual method is based on the difference in color between the gingival and alveolarmucous membrane 5. In the functional method, the mucogingival line is estimated as a border between the movable and immovable tissue where the mobility of tissue is determined using a periodontal probe that is positioned horizontally by pressing gently in the lobby of the lips to the edge of the gin- giva 3. Mucogingival line can be assessed visually after staining the mucogingival complex with Lugol solution (iodine test). Alveolar mucosa differs from the gingival curettage in glycogen, acid phosphatase, and the non-specific esterase, and an increased amount of elastic fibers, resulting in a positive reaction on iodine6-9. The attached gingiva, which is keratinized, does not contain glycogen so that the surface layer provides a negative iodine reaction. Thus, the Lugol's solution stains only the alveolar mucosa and clearly demarcates the gingival line. j Pripojna gingiva koja je keratinizovana ne sadrži glikogen, tako da površinski sloj daje negativnu jodnu reakciju. Tako, Lugolov rastvor prebojava samo alveolarnu sluzokožu i jasno razgraničava mukogingivalnu liniju. 1704 Pejčić i sar. - ŠIRINA PRIPOJNE GINGIVE Širina pripojne gingive se znatno razlikuje i kreće se od jedan do devet mili- metara. Širina se razlikuje u predelu pojedinih zuba, šira je u gornjoj vilici nego u donjoj i uža je kod mlečnih zuba 10. The width of the attached gingiva varies considerably, ranging from one to nine millimeters. The width varies in the area of individual tooth; it is wider in the upper jaw than in the lower one and is narrower near the milk teeth 10. j Povećanje širine pripojne gingive ima veliku ulogu u parodontalnoj plastičnoj hirurgiji. Material and Methods U ovom istraživanju učestvovalo je ukupno 120 osoba odabranih iz Službe za parodontologiju i oralnu medicinu Klinike za stomatologiju Medicinskog fakulteta u Nišu. Za odabir učesnika korišćena je nepro- porcionalna slojevita nasumična metoda. Uz ovu metodu, primenjivali su se i kriterijumi uključivanja i isključivanja u istraživanje. Pacijenti starosti do 60 godina, sa dobrim opštim zdravljem, zdravim gingivalnim tkivom (nema gubitka pripoja) i oni koji nisu imali neku vrstu parodontalnog tretmana u poslednjih 6 meseci, bili su uključeni u studiju. Iz studije su isključene trudnice i dojilje, zatim pacijenti sa sistemskim bolestima i oni koji su uzimali lekove koji mogu imati uticaj na stanje gingive. This study involved a total of 120 people selected from the Department of Periodontology and Oral Medicine, Dental Clinic School of Medicine in Nis. For the selection of participants, a disproportionate stratified random method was used. Along with this method, the criteria for inclusion and exclusion were also applied. Patients up to the age of 60 years with good general health, healthy gingival tissue (no loss of attachment) and those who did not have some kind of periodontal treatment in the last six months were included in the study. Pregnant and lactating women, patients with systemic diseases, and those taking drugs that may affect the condition of the gingiva were excluded from the study. Pacijenti su obavešteni o protokolu istraživanja i oni koji su pristali na ispitivanje, potpisali su pristanak. U zavisnosti od starosti pacijenata, formirane su sledeće grupe y Patients were informed about the study protocol, and those who agreed with the examination terms submitted the written informed consent. Depending on the age of the patient, the following groups were formed: I ≤ 14 godina III 31-45 godina II 15-30 godina IV 46-60 godina. I ≤ 14 years III 31-45 years II 15-30 years IV 46-60 years. I ≤ 14 years III 31-45 years II 15-30 years IV 46-60 years. Širina pripojne gingive vizuelnom metodom merena je uz primenu parodontalne sonde Michigen 0 i stomatološkog ogledalceta pri veštačkoj svetlosti stomatološke stolice. The width of the attached gingiva was measured with the use of periodontal probes Michigan 0 and dental mirror artificial light in the dental chair. Drugi način izvođenja vizuelne metode merenja širine pripojne gingive bila je primena rastvora joda (Lugol rastvor) koji je pripremljen razblaživanjem 2 g kalijum jodida i 1 g kristala joda u 60 ml destilovane vode 11. Introduction Malo je studija koje su ispitivale širinu pripojne gingive kod parodontalno zdravog stanovništva. Merenje širine pripojne gingive će pomoći u proceni rizika za pojavu i pogoršanje parodontalnih oboljenja kod parodontalno zdrave populacije. Increasing the width of the attached gingiva has a great role in periodontal plastic surgery. There are very few studies that have examined the width of the attached gingiva in periodontal healthy population. Measuring the width of the attached gingiva will assist in assessing the risk of the emergence and worsening of periodontal disease in a periodontally healthy population. Ciljevi ove studije bili su da se proceni širina pripojne gingive u punom zubnom nizu u ustima i razlike između vizuelne metode, odrеđivane uz pomoć parodontalne sonde i vizuelne metode, određivane nakon Šilerove probe. The objectives of this study were to estimate the width of the attached gingiva in full dental arch in the mouth and to assess the differences between visual methods determined with the help of periodontal probes and visual methods determined after Schiller's probe. Material and Methods Another way of measuring the width of the attached gingiva was the application of iodine solution (Lugol's Solution), which was prepared by diluting 2 g of potassium iodide 1705 Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 and 1 g of a crystal of iodine in 60 ml of distilled water 11. Merenje širine pripojne gingive vršilo se na centralnoj poziciji bukalne površine svih centralnih sekutića, očnjaka, prvih premolara i prvih molara, na ukupno 16 zuba. Ceo postupak je sproveden od strane jednog ispitivača. Measuring the width of the attached gingiva was performed in the central position of the buccal surface of the central incisors, canines, first premolars and first molars, i.e. a total of 16 teeth. The entire procedure was carried out by one examiner. p Da bi se procenila širina pripojne gingive, mukogingivalna linija je označena na sledeći način: y To assess the width of the attached gingiva, mucogingival line is indicated as follows: • vizuelnom metodom – 1. način (Slika 1) • vizuelnom metodom nakon Šilerove probe – vrši se premazivanje pamučnom paletom blagim pritiskom tkiva gingive i alveolarne sluzokože do naglog razgraničenja između keratinizovane gingive i alveolarne mukoze (Slika 2). Š • visual method - Method 1 (Figure 1) • visual method - Method 1 (Figure 1) • visual method after a Schiller's probe – staining is performed using a coating cotton palette, applying a slight pressure over the gingival and alveolar mucous membrane to a sharp demarcation between the subgingival curettage and alveolar mucosa (Figure 2). Širina keratinizovane gingive je merena kao udaljenost od gingivalne ivice do mukogingivalne linije. Dubina sulkusa je merena kao udaljenost od gingivalne ivice do donje ivice slobodne gingive (dna gingi- valnog sulkusa). Sa ovim vrednostima, širina pripojne gingive bila je izračunavana kao razlika dubine sulkusa i širine keratinizovanog tkiva. ( g ) The width of subgingival curettage is measured as the distance from the gingival edge to the mucogingival line. Sulcus depth was measured as the distance from the gingival edge to the bottom edge of the free gingiva (bottom of the gingival sulcus). With these values, the width of attached gingiva was calculated as the difference sulcus depth and width keratinized tissue. Slika 1. Vizuelna metoda Figure 1. Visual method Slika 2 Vizuelna metoda nakon Šilerove probe Figure 2. Visual method after a Shiler’s method Slika 2 Vizuelna metoda nakon Šilerove probe Figure 2. Results Ukupno 120 ispitanika je učestvovalo u ovom istraživanju, od kojih je 74 (61,67 %) bilo ženskog pola. Ž A total of 120 respondents participated in the survey, of which 74 (61.67%) were female. Ženske ispitanice su imale veću prosečnu širinu pripojne gingive (3,65 mm) od muških, koji su imali prosek od 2,42 mm (Tabela 1). Female respondents had a greater average width of the attached gingiva (3.65 mm) than men in whom the average width of width of the attached gingiva was 2.42 mm (Table 1). Maksilarni centralni sekutići imali su najveću širinu pripojne gingive sa prosekom od 3,57 mm, dok su mandibularni prvi molari imali najmanju širinu pripojne gingive (2,35 mm). Centralni sekutići u mandibuli imali su širinu 2,48 mm, maksilarni premolari 2,99 mm, mandibularni premolari 2,67 mm, dok su maksilarni prvi molari bili sa širinom od 2,58 mm (Tabela 2). ( ) The maxillary central incisors had a maximum width of the attached gingiva with an average of 3.57 mm, while the mandi- bular first molars had a minimal width of the attached gingiva (2.35 mm). The width of the central incisors in the mandible was 2.48 mm, 2.99 mm of the maxillary premolars, 2.67 mm of the mandibular premolars, while the width of the maxillary first molars was 2.58 mm (Table 2). Procena širina pripojne gingive u različitim starosnim grupama vizuelnom metodom na 1. način otkrila je da se širina gingive povećava sa starenjem, gde je prosečna širina na maksilarnim zubima u prvoj starosnoj grupi (≤14 godina) bila 1,73 mm, sa povećanjem od 2,25 do 2,89 u drugoj grupi (15-30 godina) i trećoj starosnoj grupi (31-45 godina), dok je u starosnoj grupi od 46-60 godina bila 3,25 mm. Visual assessment of the attached gingiva width in different age groups using the method 1 revealed that the width of the gingiva increases with age, where the average width of the maxillary teeth in the first age group (≤14 years of age) was 1.73 mm, with an increase of 2.25 to 2.89 in the second group (15-30 years) and the third age group (31-45 years), while in the age group of 46-60 years it was 3.25 mm. Material and Methods Visual method after a Shiler’s method Slika 1. Vizuelna metoda Figure 1. Visual method Slika 2 Vizuelna metoda nakon Šilerove probe Figure 2. Visual method after a Shiler’s method Slika 1. Vizuelna metoda Figure 1. Visual method 1706 Pejčić et al. THE WIDTH OF THE ATTACHED GINGIVA Statistička obrada Svi prikupljeni podaci su obrađeni u programskom paketu SPSS 15.0. Rađena je srednja vrednost i standardna devijacija dobijenih rezultata. Podaci pokazuju homo- genost i razlike između analiziranih grupa korišćenjem ANOVA testa . Razlika između grupa je analizirana korišćenjem post-hoc testa. Kriterijum za statističku značajnost je prihvaćen na nivou verovatnoće p < 0,05. All collected data was analyzed by SPSS 15.0. The mean value and standard deviation of the results were determined. The data show homogeneity and differences between the groups analyzed using ANOVA test. The difference between groups was analyzed using post-hoc test. The criteria for statistical significance was accepted at the probability level of p < 0.05. Results g , Slično tome, kod donjih zuba u prvoj starosnoj grupi (≤14 godina), prosečna širina bila je 1,35 mm, sa povećanjem na 2,13 mm u drugoj i 2,10 mm u trećoj starosnoj grupi (15-45 godina) i 2,45 mm u starosnoj grupi od 45-60 godina (Tabela 3). Procena širine pripojne gingive u različitim starosnim grupama vizuelnom metodom nakon Šilerove probe pokazala je u proseku širinu pripojne gingive od 2,11 mm u prvoj starosnoj grupi (≤14 godina) do 3,25 mm u starosnoj grupi od 45-60 godina starosti za gornju vilicu. Kod donjih zuba, srednja vrednost širine pripojne gingive kod prve starosne grupe (≤14 godina) bila je 1,64 mm, dok je u najstarijoj starosnoj grupi od 45-60 godina bila 2,48 mm (Tabela 2). y Similarly, in the lower teeth in the first age group (≤14 years) the average width was 1.35 mm, increasing to 2.13 mm in the second and 10.2 mm in the third age group (15-45 years) and 2.45 mm in the age group of 45-60 years (Table 3). y ( ) Assessment of the width of the attached gingiva in different age groups by visual method after Schiller's probe showed that the average width of the attached gingiva was 2.11 mm in the first age group (≤14 years) i.e. 3.25 mm in the age group of 45-60 years of age for the upper jaw. In the lower teeth, the mean width of the attached gingiva in the first age group (≤14 years of age) was 1.64 mm, while in the oldest age group of 45-60 years it was 2.48 mm (Table 2). 1707 Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 There was no statistically significant difference in the width of the attached gingiva in both jaws in the age group (≤14 years), regardless of the method used for the assessment (Table 5). Ne postoji statistički značajna razlika u širini pripojne gingive obe vilice u starosnoj grupi (≤14 godina) bez obzira na metod koji se koristi za procenu (Tabela 5). j p ( ) Poređenje dva različita metoda razli- čitih zuba u starosnoj grupi od 15-30 godina nije otkrilo nikakve značajne razlike, iako je najšira zona pripojne gingive pronađena kod sekutića primenom obe metode (metoda 1 - 4,07, metoda 2 – 3,85) i najmanja zona širine u premolarnom regionu (metoda 1 – 1,92, metoda 2 – 2,07). Ovakve varijacije su slične i kod donjih zuba (Tabela 6). Results Evaluation of the width of the attached gingiva in different age groups, using a visual method (method 1) SV– srednja vrednost; SD – standardna devijacija; F – vrednost signifakntnosti; p – statistički značajna razlika SV– mean; SD – standard deviation; F – the value of significance; p – statistically significant difference vrsta zuba type of teeth SV ± SD (mm) gornji centralni sekutići upper central incisors 3.57 ± 1.761 gornji očnjaci upper canines 3.28 ± 1.527 gornji prvi premolari upper first premolars 2.99 ± 1.929 gornji prvi molari upper first molars 2.58 ± 1.581 donji centralni sekutići lower central incisors 2.48 ± 1.370 donji očnjaci lower canines 2.18 ± 1.211 donji prvi premolari lower first premolars 2.67 ± 1.730 donji prvi molari lower first molars 2.35 ± 1.273 godine age SV ± SD (mm) F; p-vredn. F; p-value Post-hoc test gornji zubi upper teeth ≤ 14 1.73 ± 0.77 F - 10.629 p < 0.001 II > I 15-30 2.25 ± 0.42 IV > I 31-45 2.89 ± 0.40 IV > II 46-60 3.25 ± 0.25 donji zubi lower teeth ≤ 14 1,35 ± 0.30 F - 6.398 p < 0.001 II > I 15-30 2.13 ± 0.42 III > I 31-45 2.10 ± 0.36 IV > I 46-60 2.45 ± 0.50 Tabela 2. Širina pripojne gingive kod različitih zuba u vilicama Table 2. The width of the attached gingiva in different teeth in the jaw SV d j d t SD t d d d ij ij vrsta zuba type of teeth SV ± SD (mm) gornji centralni sekutići upper central incisors 3.57 ± 1.761 gornji očnjaci upper canines 3.28 ± 1.527 gornji prvi premolari upper first premolars 2.99 ± 1.929 gornji prvi molari upper first molars 2.58 ± 1.581 donji centralni sekutići lower central incisors 2.48 ± 1.370 donji očnjaci lower canines 2.18 ± 1.211 donji prvi premolari lower first premolars 2.67 ± 1.730 donji prvi molari lower first molars 2.35 ± 1.273 Tabela 2. Širina pripojne gingive kod različitih zuba u vilicama Table 2. The width of the attached gingiva in different teeth in the jaw Tabela 3. Procena širine pripojne gingive u različitim starosnim grupama vizuelnom metodom (način 1) Table 3. Results ( ) Comparison of two different methods of different teeth in the age group of 15-30 years did not show any significant differences, though the widest zone of the attached gingiva was found for incisors by the application of both methods (method 1 - 4.07, method 2 - 3.85),whereas the minimum zone width was seen in the premolar region (method 1 - 1.92, method 2 - 2.07). Such vari- ations are similar in the lower teeth as well (Table 6). j Slične vrednosti maksimalne širine kod gornjih sekutića i kod donjih premolara uočeni su i u drugim starosnim grupama, odnosno u grupi od 31 _ 45 godina i grupi od 46 _ 60 godina, tako da nije uočena statistički značajna razlika između dve metode (Tabele 7 i 8). Similar values for the maximum width of the upper incisors and the lower premolars were observed in other age groups, i.e. in groups of 31 - 45 years and the group of 46-60 years, so there was not a significant difference between the two methods ( Table 7 and 8). Tabela 1. Širina pripojne gingive kod muškaraca i žena Table 1. The width of the attached gingiva in men and women SV – srednja vrednost; SD – standardna devijacija SV – mean; SD – standard deviation pol sex broj (procenat) number (percentage) SV ± SD (mm) ženski female 74 (61.67%) 3.654 ± 1.269 muški male 46 (38.33%) 2.426 ± 1.536 Tabela 1. Širina pripojne gingive kod muškaraca i žena Table 1. The width of the attached gingiva in men and women SV – srednja vrednost; SD – standardna devijacija SV – mean; SD – standard deviation SV – srednja vrednost; SD – standardna devijacija SV – mean; SD – standard deviation 1708 Pejčić i sar. - ŠIRINA PRIPOJNE GINGIVE j Tabela 2. Širina pripojne gingive kod različitih zuba u vilicama Table 2. The width of the attached gingiva in different teeth in the jaw SV – srednja vrednost; SD – standardna devijacija SV – mean; SD – standard deviation Tabela 3. Procena širine pripojne gingive u različitim starosnim grupama vizuelnom metodom (način 1) Table 3. SV– mean; SD – standard deviation; F – the value of significance; p – statistically significant difference Results Evaluation of the width of the attached gingiva in different age groups, using a visual method (method 1) SV– srednja vrednost; SD – standardna devijacija; F – vrednost signifakntnosti; p – statistički značajna razlika SV– mean; SD – standard deviation; F – the value of significance; p – statistically significant difference godine age SV ± SD (mm) F; p-vredn. F; p-value Post-hoc test gornji zubi upper teeth ≤ 14 1.73 ± 0.77 F - 10.629 p < 0.001 II > I 15-30 2.25 ± 0.42 IV > I 31-45 2.89 ± 0.40 IV > II 46-60 3.25 ± 0.25 donji zubi lower teeth ≤ 14 1,35 ± 0.30 F - 6.398 p < 0.001 II > I 15-30 2.13 ± 0.42 III > I 31-45 2.10 ± 0.36 IV > I 46-60 2.45 ± 0.50 Tabela 3. Procena širine pripojne gingive u različitim starosnim grupama vizuelnom metodom (način 1) able 3. Evaluation of the width of the attached gingiva in different age groups, using a visual method (method 1) Tabela 3. Procena širine pripojne gingive u različitim starosnim grupama vizuelnom metodom (način 1) SV– mean; SD – standard deviation; F – the value of significance; p – statistically significant difference 1709 Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 Tabela 4. Procena širine pripojne gingive u različitim starosnim grupama vizuelnom metodom nakon Šilerove probe (način 2) Table 4. Visual assessment of the width of the attached gingiva in different age groups after a Schiller’s probe (method 2) SV– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference Tabela 5. Poređenje dva različita metoda u starosnoj grupi ≤ 14 godina Table 5. Comparison of two different methods in the age group ≤ 14 years zubi teeth vizuelna metoda visual method SV ± SD (mm) F; p-vredn. F; p-value gornji upper način 1 method 1 1.89 (0.42) F – 0.78 način 2 method 2 2.05 (0.43) n.s. donji lower način 1 method 1 1.50 (0.43) F – 0.84 način 2 method 2 1.57 (0.35) n.s. SV– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika; n.s. – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. Results – it is not statistically significant godine age SV ± SD (mm) F; p-vredn. F; p-value Post-hoc test gornji zubi upper teeth ≤ 14 2.11 ± 0.42 F - 8.558 p < 0.001 III > I 15-30 2.21 ± 0.52 IV > I 31-45 2.74 ± 0.49 IV > II 46-60 3.25 ± 0.63 donji zubi lower teeth ≤ 14 1.64 ± 0.38 F - 9.082 p < 0.001 II > I 15-30 2.08 ± 0.29 III > I 31-45 2.34 ± 0.38 IV > I 46-60 2.48 ± 0.41 Tabela 4. Procena širine pripojne gingive u različitim starosnim grupama vizuelnom metodom nakon Šilerove probe (način 2) Table 4. Visual assessment of the width of the attached gingiva in different age groups after a Schiller’s probe (method 2) godine age SV ± SD (mm) F; p-vredn. F; p-value Post-hoc test gornji zubi upper teeth ≤ 14 2.11 ± 0.42 F - 8.558 p < 0.001 III > I 15-30 2.21 ± 0.52 IV > I 31-45 2.74 ± 0.49 IV > II 46-60 3.25 ± 0.63 donji zubi lower teeth ≤ 14 1.64 ± 0.38 F - 9.082 p < 0.001 II > I 15-30 2.08 ± 0.29 III > I 31-45 2.34 ± 0.38 IV > I 46-60 2.48 ± 0.41 Tabela 4. Procena širine pripojne gingive u različitim starosnim grupama vizuelnom metodom nakon Šilerove probe (način 2) Table 4. Visual assessment of the width of the attached gingiva in different age groups after a Schiller’s probe (method 2) Tabela 5. Poređenje dva različita metoda u starosnoj grupi ≤ 14 godina Table 5. Comparison of two different methods in the age group ≤ 14 years zubi teeth vizuelna metoda visual method SV ± SD (mm) F; p-vredn. F; p-value gornji upper način 1 method 1 1.89 (0.42) F – 0.78 način 2 method 2 2.05 (0.43) n.s. donji lower način 1 method 1 1.50 (0.43) F – 0.84 način 2 method 2 1.57 (0.35) n.s. SV– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika; n.s. – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant Tabela 5. Poređenje dva različita metoda u starosnoj grupi ≤ 14 godina Table 5. Results Comparison of two different methods in the age group ≤ 14 years zubi teeth vizuelna metoda visual method SV ± SD (mm) F; p-vredn. F; p-value gornji upper način 1 method 1 1.89 (0.42) F – 0.78 način 2 method 2 2.05 (0.43) n.s. donji lower način 1 method 1 1.50 (0.43) F – 0.84 način 2 method 2 1.57 (0.35) n.s. Tabela 5. Poređenje dva različita metoda u starosnoj grupi ≤ 14 godina Table 5. Comparison of two different methods in the age group ≤ 14 years V– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika; n.s. – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant 1710 Pejčić et al. THE WIDTH OF THE ATTACHED GINGIVA Tabela 6. Poređenje dve različite metode u starosnoj grupi od 15 do 30 godina starosti Table 6. Comparison of two different methods in the age group of 15-30 years zubi teeth vilica jaw Vizuelna metoda Visual method SV ± SD (mm) F; p-vredn. F; p-value sekutići incisors gornja upper način 1 method 1 4.07 ± 0,58 F – 0.82 način 2 method 2 3.85 ± 0.84 n.s. donja lower način 1 method 1 3.22 ± 0.73 F – 0.62 način 2 method 2 3.12 ± 0.76 n.s. očnjaci canines gornja upper način 1 method 1 3.80 ± 0,71 F – 0.72 način 2 method 2 3.75 ± 0.98 n.s. donja lower način 1 method 1 2.45 ± 0,48 F – 0.90 način 2 method 2 2.65 ± 0.42 n.s. premolari premolars gornja upper način 1 method 1 1.92 ± 0.65 F – 0.90 način 2 method 2 2.07 ± 0.75 n.s. donja lower način 1 method 1 1.50 ± 0.59 F – 0.84 način 2 method 2 1.67 ± 0.38 n.s. molari molars gornja upper način 1 method 1 2.67 ± 0.47 F - 0,97 način 2 method 2 2.80 ± 0.57 n.s. donja lower način 1 method 1 1.65 ± 0.65 F – 0.84 način 2 method 2 1.92 ± 0.71 n.s. SV– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statističk značajna razlika; n.s. Results – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant V– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika; n.s. – nije statistički značajna razlika SV– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika; n.s. – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant 1711 1711 Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 Tabela 7. Poređenje dve različite metode u starosnoj grupi od 31 do 45 godina starosti Table 7. Comparison of two different methods in the age group of 31-45 years SV– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statističk značajna razlika; n.s. – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant zubi teeth vilica jaw vizuelna metoda visual method SV ± SD (mm) F; p-vredn. F; p-value sekutići incisors gornja upper način 1 method 1 3.30 ± 1.08 F – 0.23 način 2 method 2 3.62 ± 0.71 n.s. donja lower način 1 method 1 3.22 ± 0.98 F – 0.87 način 2 method 2 3.05 ± 0.72 n.s. očnjaci canines gornja upper način 1 method 1 2.80 ± 1.25 F – 0.15 način 2 method 2 2.95 ± 0.95 n.s. donja lower način 1 method 1 1.80 ± 1.06 F – 0.86 način 2 method 2 1.70 ± 0.94 n.s. premolari premolars gornja upper način 1 method 1 2.07 ± 0.65 F – 0.54 način 2 method 2 2.32 ± 0.73 n.s. donja lower način 1 method 1 1.52 ± 0.47 F – 0.24 način 2 method 2 1.47 ± 0.31 n.s. molari molars gornja upper način 1 method 1 2.22 ± 0.74 F – 0.75 način 2 method 2 2.42 ± 0.88 n.s. donja lower način 1 method 1 1.97 ± 0.31 F – 0.68 način 2 method 2 2.10 ± 0.48 n.s. V– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika; n.s. SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant Results – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant 1712 Pejčić i sar. - ŠIRINA PRIPOJNE GINGIVE Tabela 8. Poređenje dve različite metode u starosnoj grupi od 46 do 60 godina starosti Table 8. Comparison of two different methods in the age group of 46-60 years zubi teeth vilica jaw vizuelna metoda visual method SV ± SD (mm) F; p-vredn. F; p-vrednost sekutići incisors gornja upper način 1 method 1 3.65 ± 1.08 F – 0.14 način 2 method 2 3.30 ± 0.71 n.s. donja lower način 1 method 1 3.05 ± 0.98 F – 0.23 način 2 method 2 3.22 ± 0.72 n.s. očnjaci canines gornja upper način 1 method 1 2.95 ± 1.25 F – 0.21 način 2 method 2 2.80 ± 0.95 n.s. donja lower način 1 method 1 1.70 ± 1.06 F – 0.84 način 2 method 2 1.80 ± 0.94 n.s. premolari premolars gornja upper način 1 method 1 2.32 ± 0.65 F – 0.52 način 2 method 2 2.07 ± 0.73 n.s. donja lower način 1 method 1 1.47 ± 0.47 F – 0.33 način 2 method 2 1.52 ± 0.31 n.s. molari molars gornja upper način 1 method 1 2.42 ± 0.74 F – 0.80 način 2 method 2 2.22 ± 0.88 n.s. donja lower način 1 method 1 2.10 ± 0.31 F – 0.06 način 2 method 2 1.97 ± 0.48 n.s. SV– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statističk značajna razlika; n.s. – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant Tabela 8. Poređenje dve različite metode u starosnoj grupi od 46 do 60 godina starost Table 8. Comparison of two different methods in the age group of 46-60 years V– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika; n.s. – nije statistički značajna razlika SV– srednja vrednost; SD – standardna devijacija; F – vrednost značajnosti; p – statistički značajna razlika; n.s. – nije statistički značajna razlika SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. Discussion The assessment of the width of the attached gingiva is vital to assess the risk for the occurrence of periodontal disease. For the assessment of the width of the attached gingiva, mucogingival line serves as an important anatomical landmark, which can be delineated by various methods. As suggested by Fasske and Morgenroth, the exact location of this line can be seen after staining the mucosa with different solutions (such as Lugol iodine solution) which allow accurate determination of the line on which the keratinization ends 12. For the purpose of elimination of the difference in probing, all measurements were performed by a single examiner. Procena širine pripojne gingive je od vitalnog značaja za procenu rizika parodon- cijuma za pojavu oboljenja. U proceni širine pripojne gingive mukogingivalna linija služi kao važan anatomski orijentir, koja se može razgraničiti različitim metodama. Kako sugeriše Fasske i Morgenroth, tačna lokacija ove linije može se videti posle bojenja sluzokože različitim rastvorima ( kao što je Lugolov jodni rastvor), koji omogućavaju određivanje tačne linije na kojoj se keratinizacija završava 12. U cilju eliminacije razlika u sondiranju, sva merenja su vršena od strane jednog ispitivača. There is a large number of studies that have examined the width of the attached gingiva. The most read studies related to the examination of the width of the attached gingiva are those authored by Bowers in 1963 13 and 1976 and Ainamo in 1976 14. In this study, the measurement of the attached gingiva was performed using a periodontal probe similar to Tenenbaum 15, and using a solution of iodine (Schiller's test) as Talari16, Ainamo 17 and Saario 18,19 did. Bowers 13 found that the width of the attached gingiva varies in different areas of the oral cavity, as it was found in this study. Ainamo 14 found the maximum width of the attached gingiva in the area of the maxillary incisors. Similar results were found in this study. Minimum width in this study was in the part of the first molar in the mandible, while Ainamo 14 found that the minimum width was in the region of the premolars in the mandible. Ainamo 14 and Vincent 20 found that the width of the attached gingiva increases with age, which is consistent with the results of this research. Results – it is not statistically significant SV – mean; SD – standard deviation; F – the value of significance; p – statistically significant difference: n.s. – it is not statistically significant 1713 Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 Discussion These results show that the maximum width of the attached gingiva was in the second age group (15-30 yrs.), while in the middle age group of 31-45 years the width of the attached gingiva was the smallest (Table 3). j g p Postoji veliki broj studija koje su ispitivale širinu pripojne gingive. Najčitanije studije koje su ispitivale širinu pripojne gingive su studije Boversa iz 1963.13 i 1976. godine i Ainima iz 1976. godine 14.U ovom ispitivanju merenje pripojne gingive vršilo se pomoću periodontalne sonde slično kao Tenenbaum 15, kao i korišćenjem rastvora joda (Šilerova jodna proba) kao Talari 16, Ainamo 17 i Saario 18,19. Bovers 13 je pronašao da širina pripojne gingive varira na različitim mestima usne duplje, kao što je nađeno i u ovom ispitivanju. Ainamo 14 je pronašao najveću širinu pripojne gingive u predelu maksilarnih inciziva slično rezultatima u našoj studiji. Najmanja širina u našoj studiji je bila u predelu prvog molara u mandibuli, dok je Ainamo 14 pronašao da je najmanja širina u predelu premolara u mandibuli. Ainamo 14 i Vincent 20 su utvrdili da se širina pripojne gingive povećava sa uzrastom , što je u saglasnosti sa rezultatima iz ovog istraživanja, koji pokazuju da je najveća širina pripojne gingive bila u drugoj starosnoj grupi (15-30 god.), dok je u srednjoj starosnoj grupi od 31- 45 godina širina pripojne gingive bila najmanja (tabela 3). P ši i i j i i Procena širine pripojne gingive u različitim starosnim grupama vizuelnom meto- dom otkrila je da se širina pripojne gingive povećava sa godinama, što je u saglasnosti sa autorima poput Ainama i Talarija 16 i Vinsent sa sar.20. Širina pripojne gingive varira u različitim oblastima u usnoj duplji i iznosi od 1-9 mm, 21 1-4 mm, 22 0-5 mm 23. The assessment of the width of the attached gingiva in different age groups using a visual method revealed that the width of the attached gingiva increases with age which is in accordance with the results obtained by Ainamo and Talari16 and Vincent et al. 20 The width of the attached gingiva varies in different areas of the oral cavity, ranging from 1-9 mm, 21 1-4 mm, 22, 0-5 mm23. hi d h f di U ovoj studiji, opseg srednje širine pripojne gingive varira od 1 mm do 4 mm. Discussion Slične varijacije se vide u Boverovoj studiji, gde je najšira zona pripojne gingive pronađena In this study, the extent of medium width of the attached gingiva can vary from 1 mm to 4 mm. Similar variations are seen in 1714 Pejčić et al. THE WIDTH OF THE ATTACHED GINGIVA u predelu sekutića , a najmanja u predelu premolara, bez obzira na metod koji se koristi u proceni 21. the Bover’s study, where the widest zone of the attached gingiva was found in the area of incisors, and the lowest one in the region of the premolars, regardless of the method used in the assessment 21. Kategorizacija različitih tipova zuba urađena je samo u poslednje tri starosne grupe, dok učesnici prve starosne grupe ≤ 14 godina, imaju različitu konfiguraciju prisutnih zuba zbog prisustva mešovite denticije. U slučaju mešovite denticije ne može se komentarisati zbog različite prirode prisutnih zuba. The categorization of different types of teeth was done only in the last three age groups, while the participants of the first age group ≤14 years had a different configuration of the present teeth due to the presence of mixed dentition. The width of the attached gingiva cannot be compared in cases of mixed dentition because of the different nature of the present teeth. Rezultati ove studije su pokazali da nije bilo značajne razlike u širini pripojne gingive dobijene merenjem vizuelnom metodom ili korišćenjem vizuelne metode nakon izvedene Šilerove probe. Ovi rezultati su u skladu sa istraživanjem Guglielmoni et al. 24, dok Bernimoulin et al. 25 prijavljuje da izvođenje funkcionalne metode daje najveću širinu keratinizirane gingive. Osim toga, vizuelna metoda, nakon bojenja gingivalnog tkiva, dovodi do najmanjeg neslaganja vrednosti širine pripojne gingive u poređenju sa drugim metodama određivanja širine pripojne gingive. p The results of this study showed that there was no significant difference in the width of the attached gingiva obtained by measuring using the visual method or using visual methods implemented after Schiller probe. These results are consistent with the research of Guglielmone et al., 24 while Bernimoulin et al. 25 reported that performing the functional method gives a maximum width of keratinized gingiva. In addition, the visual method after staining the gingival tissue leads to the smallest disagreements in the values of the attached gingiva width compared to other methods of determining the width of the attached gingiva. Discussion p p j g g Najšira je u predelu sekutića, zatim se sužava u predelu premolara, da bi se opet nešto širila u predelu molara. U pogledu širine pripojne gingive postoji simetrija sa leve i desne strane lica, dok razlika između polova ne postoji. Širina pripojne gingive je važna i za lekara i za pacijenta, jer utiče na prognozu oboljenja. Ako je pripojna gingiva uzana, prognoza oboljenja parodonta će biti nepovoljnija. Kod osoba koje imaju uzanu pripojnu gingivu, proces destrukcije koji nastaje u toku bolesti, brže dospeva do mukogingivalne linije. Tada dolazi do izražaja vuča preko pokretne sluzokože i daljeg napredovanja patološkog procesa. Vuča koja deluje odvaja ivicu gingive od zuba i omogućava lakše stvaranje i zadržavanje dentalnog plaka, sa svim neželjenim posledicama. Ovaj problem postaje izrazitiji ako se na to nadovežu i anomalije u mekom tkivu. g g It is the widest in the area of incisors, and then it narrows in the premolar area to expand again slightly in the area of the molars. In terms of the width of the attached gingiva, there is a symmetry with the left and right sides of the face, while the difference between the sexes does not exist. The width of the attached gingiva is important for physicians and patients because it affects the prognosis of the disease. If the attached gingiva is narrow, the periodontal disease prognosis will be worse. Considering people with narrow attached gingiva, the process of destruction that occurs in the course of the disease reaches faster a mucogingival line. Then, there is a marked traction over the movable mucous membrane and further progression of the pathological process. Traction separates the edge of the gingiva from the teeth and facilitates the creation and retention of dental plaque, with all the adverse consequences. This problem becomes more noticeable if this is combined with the anomalies in the soft tissue. 1715 Acta Stomatologica Naissi Jun/June 2017, Vol. 33, br./num. 75 LITERATURA / REFERENCES 1. Newman MG, Takei HH, Klokkevold PR, Carranza FA Eds Carranza's Clinical Periodontology, 10th (eds.), 2006. 13. Bowers GM. A study of the width of the attached gingiva. J Periodontology, 1963; 34: 210-213. 14. Ainamo J, Talari A. The increase with age of the width of attached gingiva. J Periodontal Res 1976; 11: 182- 188. 2. Wennström JL, Zucchelli G, Pini GP. Mucogingival therapy‑ periodontal plastic surgery. In: Lindhe J, Lang NP, Karring T, editors. Clinical Periodontology and Implant Dentistry. 5th ed. Oxford: Blackwell Munksgaard Publishers; 2008. p. 956‑ 7. 15. Tenenbaum H, Tenenbaum M. A clinical study of the width of the attached gingiva in the deciduous, transitional and permanent dentitions. J Clin Periodontol 1986; 13(4):270-275. 3. Hilming F, Jervoe P. Surgical extension of vestibular depth. On the results in various regions of the mouth in periodontal patients. Tandlaegebladet 1970; 74: 329‑ 343. 16. Talari A, Ainamo J. Orthopantomographic assessment of the width of attached gingiva. J Periodontal Res 1976; 11(4): 177-181. 4. Guglielmoni P, Promsudthi A, Tatakis DN, Trombelli L. Intra‑ and inter‑ examiner reproducibility in keratinized tissue width assessment with 3 methods for mucogingival junction determination. J Periodontol 2001; 72: 134‑ 139. 17. Ainamo A, Ainamo J. The width of attached gingiva on supraerupted teeth. J Periodontal Res 1978; 13(3): 194- 198. 18. Saario M, Ainamo A, Mattila K, Suomalainen K, Ainamo J. The width of radiologically-defined attached gingiva over deciduous teeth. J Clin Periodontology 1995; 22(12): 895-898. 5. Orban B. Clinical and histologic study of the surface characteristics of the gingiva. Oral Surg Oral Med Oral Pathol 1948; 1: 827‑ 841. 19. Saario M, Ainamo A, Mattila K, Ainamo J. The width of radiologicallydefined attached gingiva over permanent teeth in children. J Clin Periodontology 1994; 21(10): 666-669. 6. Weinmann JP, Meyer J, Mardfin D, Weiss M. Occurrence and role of glycogen in the epithelium of the alveolar mucosa and of the attached gingiva. Am J Anat 1959; 104: 381‑ 402. 20. Vincent JW, Machen JB, Levin MP. Assessment of attached gingiva using the tension test and clinical measurements J Periodontology 1976; 47(7): 412-414. 7. Lozdan J, Squier CA. The histology of the muco‑ gingival junction. J Periodontal Res 1969; 4: 83‑ 93. gy 21. Bowers GM. A study of the width of attached gingiva. J Periodontol 1963; 34: 201‑ 209. 8. Kapur KK, Chauncey HH, Shapiro S, Shklar G. Conclusion Procena širine pripojne gingive u punom zubnom nizu otkriva različite širine u različitim predelima usne duplje u predelu maksilarnih inciziva je zabeležena najveća širina, dok je najmanja širina u predelu donjih prvih molara. Starost pacijenata utiče na širinu pripojne gingive, dok je kod ženskih osoba zabeležena veća širina pripojne gingive. Assessment of the width of the attached gingiva in full dental arch reveals different widths in different areas of the oral cavity. The maximum width is in the area of the maxillary incisors, while the smallest width is in the area of the lower first molar. Patients’ age affects the width of the attached gingiva, while the females had greater width of the attached gingiva. Further research is needed regarding the periodontally healthy people from different parts of Serbia in order to determine whether the geographical position may affect the different values of width of the attached gingiva. It is also necessary to perform this research on a larger number of participants to obtain a definite scope that could be defined as adequate or not, making it easier to treat periodontal disease. p p j g g Potrebna su dalja istraživanja kod parodontalno zdravih osoba sa različitih teritorija Srbije kako bi se utvrdilo da li i geografski položaj možda utiče na različite vrednosti širine pripojne gingive. Takođe je potrebno da se ova istraživanja izvrše na većem broju uzoraka, kako bi se dobio definitivan opseg koji bi se mogao definisati kao adekvatan ili ne, što olakšava lečenje oboljenja parodonta. 1716 Pejčić i sar. - ŠIRINA PRIPOJNE GINGIVE LITERATURA / REFERENCES A comparative study of the enzyme histochemistry of human edentulous alveolar mucosa and gingival mucosa. Periodontics 1963; 1: 137‑ 147. 22. Jacob P, Zade RM. Width of attached gingiva in an Indian population: A descriptive study. Bangladesh J Med Sci 2009; 8: 1‑ 4. 9. Tencate AR. The distribution of acid phosphatase, non‑ specific esterase and lipid in oral epithelia in man and the macaque monkey. Arch Oral Biol 1963; 8: 747‑ 753. 23. Subbaiah R, Manohar B. Assessment of the width of attached gingiva in different regions of the mouth in an Indian subpopulation. J Indian Dent Assoc 2012; 6: 96‑ 98. 10. Kojović D, Pejčić A, Obradović R, Marjanović D. Parodontologija, Galaksija Niš, 2015. 24. Guglielmoni P, Promsudthi A, Tatakis DN, Trombelli L. Intra‑ and inter‑ examiner reproducibility in keratinized tissue width assessment with 3 methods for mucogingival junction determination. J Periodontol 2001; 72: 134‑ 139. g j j 11. Sheehan DC, Hrapchak BB. Staining and labelling‑ laboratory manuals. In: Sheehan DC, Hrapchak BB, editors. Theory and Practice of Histotechnology. 2nd ed. St. Louis: CV Mosby Publishers; 1980. p. 219. 25. Bernimoulin JP, Son S, Regolati B. Biometric comparison of three methods for determining the mucogingival junction. Helv Odontol Acta 1971; 15: 118‑ 120. ; p 12. Fasske E, Morgenroth K. Comparitive stomatoscopic and histochemical studies of the marginal gingiva in man. Parodontologie 1958; 12: 151‑ 160. 1717
https://openalex.org/W2766526664	https://europepmc.org/articles/pmc5656072?pdf=render	English	null	Congenital Zika syndrome and neuroimaging findings: what do we know so far?	Radiologia brasileira/Radiologia Brasileira	2,017	cc-by	8,286	Study conducted in the Radiology Department of the Instituto Estadual do Cére- bro Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. 1. Masters Student, MD, Neuroradiologist at the Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. 2. Full Member of the Colégio Brasileiro de Radiologia e Diagnóstico por Imagem (CBR), MD, Neuroradiologist at the Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. 3. PhD, MD, Neuroradiologist, Head of the Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. 4. PhD, MD, Neuroradiologist at the Instituto Estadual do Cérebro Paulo Nie- meyer, Rio de Janeiro, RJ, Brazil. 5. Full Professor at the Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil. Mailing address: Dr. Bruno Niemeyer de Freitas Ribeiro. Instituto Estadual do Cé- rebro Paulo Niemeyer – Departamento de Radiologia. Rua do Rezende, 156, Centro. Rio de Janeiro, RJ, Brazil, 22231-092. E-mail: bruno.niemeyer@hotmail.com. Received May 9, 2017. Accepted after revision July 28, 2017. Congenital Zika syndrome and neuroimaging findings: what do we know so far? O objetivo deste artigo é revisar os achados de tomografia computadorizada e ressonância magnética da síndrome congênita pelo vírus Zika. Unitermos: Ressonância magnética; Tomografia computadorizada; Vírus Zika; Síndrome congênita pelo vírus Zika; Infecção congê- nita. Congenital Zika syndrome and neuroimaging findings: what do we know so far? Bruno Niemeyer de Freitas Ribeiro1, Bernardo Carvalho Muniz2, Emerson Leandro Gasparetto3, Nina Ventura4, Edson Marchiori5 Niemeyer B, Muniz BC, Gasparetto EL, Ventura N, Marchiori E. Congenital Zika syndrome and neuroimaging findings: what do we know so far? Radiol Bras. 2017 Set/Out;50(5):314–322. Edson Marchiori5 Niemeyer B, Muniz BC, Gasparetto EL, Ventura N, Marchiori E. Congenital Zika syndrome and neuroimaging findings: what d Bras. 2017 Set/Out;50(5):314–322. Abstract Although infection with the Zika virus was first recognized in 1942, it received little attention until 2007, when a true pandemic spread throughout Africa, Asia, and the Americas. Since then, numerous forms of central nervous system involvement have been described, mainly malformations related to congenital infection. Although the neuroimaging findings in congenital Zika syndrome are not pathognomonic, many are quite suggestive of the diagnosis, and radiologists should be prepared to interpret such findings accordingly. The objective of this article is to review the computed tomography and magnetic resonance imaging findings in con- genital Zika syndrome. Keywords: Magnetic resonance imaging; Computed tomography; Zika virus; Congenital Zika syndrome; Congenital infection. Magnetic resonance imaging; Computed tomography; Zika virus; Congenital Zika syndrome; Congenital infection. Resumo A infecção pelo vírus Zika, apesar de conhecida desde 1942, apresentou destaque somente a partir de 2007, quando uma verda- deira pandemia se espalhou pela África, Ásia e Américas. Durante este período, numerosas formas de acometimento do sistema nervoso central têm sido descritas, principalmente as malformações relacionadas a infecção congênita. Apesar de os achados de neuroimagem na síndrome congênita pelo vírus Zika não serem patognomônicos, muitos são bastante sugestivos, devendo o ra- diologista estar preparado para saber interpretar e sugerir o diagnóstico. O objetivo deste artigo é revisar os achados de tomografia computadorizada e ressonância magnética da síndrome congênita pelo vírus Zika. Unitermos: Ressonância magnética; Tomografia computadorizada; Vírus Zika; Síndrome congênita pelo vírus Zika; Infecção congê- nita. Resumo A infecção pelo vírus Zika, apesar de conhecida desde 1942, apresentou destaque somente a partir de 2007, quando uma verda- deira pandemia se espalhou pela África, Ásia e Américas. Durante este período, numerosas formas de acometimento do sistema nervoso central têm sido descritas, principalmente as malformações relacionadas a infecção congênita. Apesar de os achados de neuroimagem na síndrome congênita pelo vírus Zika não serem patognomônicos, muitos são bastante sugestivos, devendo o ra- diologista estar preparado para saber interpretar e sugerir o diagnóstico. et al. / Congenital Zik Review Article et al. / Congenital Zik Review Article 0100-3984 © Colégio Brasileiro de Radiologia e Diagnóstico por Imagem Received May 9, 2017. Accepted after revision July 28, 2017. Mailing address: Dr. Bruno Niemeyer de Freitas Ribeiro. Instituto Estadual do Cé- rebro Paulo Niemeyer – Departamento de Radiologia. Rua do Rezende, 156, Centro. Rio de Janeiro, RJ, Brazil, 22231-092. E-mail: bruno.niemeyer@hotmail.com. 1. Masters Student, MD, Neuroradiologist at the Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. 3. PhD, MD, Neuroradiologist, Head of the Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. 2. Full Member of the Colégio Brasileiro de Radiologia e Diagnóstico por Imagem (CBR), MD, Neuroradiologist at the Instituto Estadual do Cérebro Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. Study conducted in the Radiology Department of the Instituto Estadual do Cére- bro Paulo Niemeyer, Rio de Janeiro, RJ, Brazil. 4. PhD, MD, Neuroradiologist at the Instituto Estadual do Cérebro Paulo Nie- meyer, Rio de Janeiro, RJ, Brazil. 5. Full Professor at the Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil. INTRODUCTION humans was not reported until 1952(1–8). Typically, it oc- curs in tropical and subtropical areas of the world, mainly in Africa and Asia, the two major lineages (Asian and Afri- can) originating from a common ancestor(1–8). Like other arboviruses, ZIKV presents many barriers to the accumu- lation of mutations, as a consequence of double replica- tion in mammalian and invertebrate hosts, and the rate of fixation of its mutations is therefore relatively slow(5). Flaviviruses are among the most important emerging viruses known to man, being transmitted by mosquitoes and ticks. The Zika virus (ZIKV) pandemic is the most recent of the arthropod-borne viral diseases, following on the heels of dengue, West Nile virus, and chikungunya, for which outbreaks were recorded in 1990, 1999, and 2013, respectively(1). ZIKV is an arbovirus of the family Flaviviridae, composed of ribonucleic acid, which was discovered in Uganda in 1947, although the first case in Beginning in 2007, ZIKV, which had until then been confined to a narrow equatorial zone in Africa and Asia, began to become more widespread, affecting Micronesia; in the 2013–2014 period, there was a ZIKV epidemic in French Polynesia and New Caledonia. Since then, there has been a progressive expansion of the virus, cases of ZIKV infection having been reported in South America, Central America, and the Caribbean Islands(1,4), the dis- placement of people and the presence of vectors being important factors for its dissemination. The first autochthonous ZIKV transmission in Brazil occurred in May 2015(4,8), with probable dissemination from the Pacific region, given that phylogenetic studies have shown high similarity (99.7% for nucleotides and 99.9% for amino acids) with the virus circulating among islands in the Pacific Ocean(2). Brazil was the Latin American country most affected by ZIKV, approximately Radiol Bras. 2017 Set/Out;50(5):314–322 Radiol Bras. 2017 Set/Out;50(5):314–322 314 0100-3984 © Colégio Brasileiro de Radiologia e Diagnóstico por Imagem http://dx.doi.org/10.1590/0100-3984.2017.0098 Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings 1,500,000 cases being reported in 2015 and 2016(3). CLINICAL ASPECTS OF ZIKV INFECTION In 75–80% of cases, ZIKV infection is asymptom- atic(3). In symptomatic cases, after an incubation period that typically lasts for 3–12 days, the most common mani- festation is a self-limiting profile—low-grade fever (37.8– 38.5°C); headache; muscle aches; pain in the small joints of the hands and feet; non-purulent conjunctivitis; ocular pain; prostration; and pruritic maculopapular rash—simi- lar to that of dengue, albeit milder and typically evolving to regression of the symptoms in 2–7 days(1,3,4). Hema- tological and biochemical parameters are usually nor- mal(1,3). After the first infection, the person develops im- munity and will not develop the disease if again exposed to the virus(2–5). Although the neuroimaging findings in congenital Zika syndrome are not pathognomonic, many are quite suggestive of the diagnosis, and radiologists should be prepared to recognize those findings, interpret them, and propose the diagnosis. The objective of this article is to review the CT and MRI findings in congenital Zika syn- drome. To that end, we searched the PubMed database using the following search terms: “Zika virus”; “congeni- tal Zika virus infection”; “zika neuroimaging”; “zika mag- netic resonance imaging”; “zika computed tomography”; and “zika ultrasound”. We analyzed articles indexed up through May 2017. Although severe and fatal forms of ZIKV are quite rare, cases of Guillain-Barré syndrome, meningoencepha- litis, and acute disseminated encephalomyelitis have been reported(1,2,8,9,11), such cases having been attributed to the high degree of tropism of ZIKV for the CNS(8). INTRODUCTION Global warming and climate change, together with the El Niño phenomenon, might have contributed to accelerat- ing the spread of the virus and its vector, additional fac- tors including the low socioeconomic status and lack of awareness of the population, as evidenced by the fact that the epidemic was more intense in impoverished areas of the northern and northeastern regions of the country, espe- cially in the states of Pernambuco, Bahia, and Paraíba(3). and transmission via urine, have been described(1,2,4,8). Because most patients infected with ZIKV are asymptom- atic, there is an extremely high risk of blood donors act- ing as a source of transmission in endemic areas(8). Other suspected routes of ZIKA transmission include monkey bite, organ transplantation, and hemodialysis(3). In kidney transplantation, the risk of ZIKV infection should be con- sidered if the donor is a resident of or is returning from an endemic area, given ZIKV can be detected in the urine of people infected for more than 30 days(3). Despite the fact that the viral particle has been isolated in breast milk, there is as yet no evidence of transmission through breast- feeding, although that route of transmission has been de- scribed for other flaviviruses(3,5,10). During epidemics, various forms of central nervous system (CNS) involvement associated with ZIKV infec- tion have been reported, such as meningoencephalitis, Guillain-Barré syndrome, and acute disseminated en- cephalomyelitis(2,3,5,7,9). Simultaneously, numerous cases of CNS malformations potentially related to congenital ZIKV infection, characterizing congenital Zika syndrome, have gained prominence in the scientific community, hav- ing been widely documented by computed tomography (CT) and magnetic resonance imaging (MRI). ZIKV TRANSMISSION The transmission of ZIKV occurs mainly by the bite of mosquitoes of the genus Aedes, which are common in the tropics and are also recognized vectors of dengue fever, yellow fever, and chikungunya. Although the main vector of ZIKV transmission is A. aegypti, other Aedes spe- cies, such as A. albopictus, A. africanus, A. luteocephalus, A. vittatus, A. furcifer, A. hensilli and A. apicoargenteus, can transmit the virus, as can mosquitoes of the genera Anopheles, Eretmapodites, Culex, and Mansonia(1,3,5,8). DIAGNOSIS Fauci et al.(12) reported that the diagnosis of ZIKV infection can be made mainly on the basis of the clini- cal findings. However, the authors stated that this is only applicable in areas in which there is a current ZIKV epi- demic and other diseases with a similar clinical presenta- tion, such as dengue and chikungunya, are not endemic. The diagnosis of ZIKV can be confirmed through am- plification of the viral genome by reverse transcriptase- polymerase chain reaction (RT-PCR) in samples of blood, saliva, urine, cerebrospinal fluid and amniotic fluid, al- though the RT-PCR procedure is expensive and subject to contamination. In addition, due to the kinetics of ZIKV viremia, the timing of the investigation depends on the material collected(1,3,8): in blood and saliva, RT-PCR can be performed only up until the seventh day after the onset of symptoms; in urine, the virus can be detected for more than 30 days after the onset of symptoms; and, in male patients, the virus can be detected in semen within the first 3–8 weeks after the onset of symptoms. Serological Aedes aegypti is widely distributed in the Americas, usually lives in close proximity to people and their homes, lays its eggs in still waters, as well as in water that collects in buckets, flowerpots, empty pipes, and other containers, and bites mainly during the daytime(3,4,7,8). The infection cycle begins when Aedes species ingest ZIKV-infected blood, which initiates a process of viral rep- lication in the epithelial cells of the midgut and migration of the virus to the salivary glands of the mosquito; after 5–10 days, the mosquito is able to transmit the virus to healthy individuals(3,4). Other forms of transmission, such as blood transfusion, sexual (oral, anal, or vaginal) trans- mission, vertical (transplacental or perinatal) transmission, 315 Radiol Bras. 2017 Set/Out;50(5):314–322 Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings during pregnancy(13,15); and the identification of a causal relationship, which can be achieved through the use of epidemiological studies, as in the case of valproic acid, which was identified as a teratogen after a case-control study demonstrated a 20-fold increase in the incidence of spina bifida when valproic acid was administered during the first trimester of pregnancy(13,16). In 1994, Thomas Shepard, a pioneer in the field of teratology, proposed seven criteria, including those previously cited, to confirm tera- togenicity(17). TREATMENT Because ZIKV infection is usually self-limiting, the treatment consists of increased fluid intake and rest. Pharmacological treatment should be restricted to symp- tom relief(3). There are as yet no vaccines or antiviral therapies for ZIKV infection. Therefore, preventive measures, focusing on vector eradication—such as reducing the number of water reservoirs available for oviposition and using insec- ticides—and on mosquito bite avoidance—such as the use of repellents—are fundamental as the best form of controlling the disease(3,8). It is also recommended that to that travelers returning from endemic areas to nonen- demic areas be advised to use repellents for a minimum of 14 days, in order to prevent local mosquitoes from getting the virus(3). Patients with clinical signs of the disease and individuals returning from endemic areas should also be advised to use condoms or abstain from sex, especially if their partner is pregnant(2,3). Some health authorities also recommend that women avoid becoming pregnant dur- ing ZIKV epidemics and that those who are pregnant or intending to become pregnant avoid traveling to endemic areas(3). Pregnant women who are symptomatic should be tested for ZIKV by RT-PCR, as should those in whom there is evidence of microcephaly on ultrasound. In the latter cases, follow-up ultrasound examinations should be performed every 3–4 weeks(2). In the Shepard system, criterion 1 is exposure to a given agent at a critical time during the course of the pregnancy. Microcephaly and other brain abnormalities that have been observed in many infants are consistent with infection occurring in the first trimester or at the be- ginning of the second trimester of pregnancy. An analysis of the time to biochemical confirmation of ZIKV trans- mission in certain Brazilian states and of the increased incidence of microcephaly identified the first trimester of pregnancy as the critical period of ZIKV infection(18). Criterion 2 in the Shepard system is the existence of at least two high-quality epidemiological studies support- ing the association. Data from Brazil and French Poly- nesia demonstrated temporal and geographic associations between ZIKV infection and the subsequent appearance of infants with congenital microcephaly(18–30). In one of those studies, conducted during the outbreak in Brazil, 88 pregnant women with a skin rash were tested for ZIKV RNA. Of the 72 who tested positive, 42 underwent ultra- sound. Among those 42 women, fetal abnormalities were observed in 12 (29%). TREATMENT The 16 women who tested negative did not present abnormalities on the ultrasound examina- tion(21). Although those studies provided important evi- dence of a causal relationship, they presented limitations, as cited by their authors(18,19,21), including a lack of con- trol for confounding factors and a relatively small number of cases. Therefore, criterion 2 is not strictly met. Recently, the vector control group of the World Health Organization discussed the use of genetically modified mosquitoes for the control of A. aegypti. By competing with wild A. aegypti males, the male OX513A mosquito was successful in controlling dengue in Brazil(2). DIAGNOSIS According to the Shepard system, causal- ity is established through a rare exposure-rare defect ap- proach—when criteria 1, 3, and 4 (as defined below) are met—or through an epidemiological approach—when criteria 1, 2, and 3 (as defined below) are met(13). tests for the detection of immunoglobulin M antibodies against ZIKV are usually conducted 4–5 days after the on- set of symptoms, and those antibodies can remain detect- able for an additional 2–3 months, as has been reported for other flaviviruses(2–4,8). CAUSALITY OF MALFORMATIONS AND CONGENITAL ZIKV INFECTION The findings included mi- crocephaly, pachygyria/lissencephaly, and hydrocephalus, together with multifocal dystrophic calcifications in the cortex and subcortical white matter, as well as signs of lo- cal inflammation and placental calcifications. There were no relevant pathological abnormalities in the remaining organs, and RT-PCR of the brain tissue was positive only for ZIKV, no other flaviviruses being identified. The ZIKV strain identified bore a high (99.7%) resemblance to the Asian lineage identified in the outbreak that occurred in French Polynesia in 2013(25). p y In the literature, there is no consensus regarding the definition of microcephaly. Microcephaly is a brain growth disorder in which the head circumference is smaller than that expected for age, gender, and race; the cut-off value can be the 3rd or 5th percentile or 2–3 standard devia- tions below the mean(31–33). It can be caused by numerous conditions, including genetic disorders and infectious dis- eases (rubella, cytomegalovirus infection, toxoplasmosis, herpes virus infection, HIV infection), as well as exposure to alcohol, drugs, or other toxic substances present in the environment. Clinically, patients with microcephaly pres- ent severe neurological impairment, common symptoms being hypertonia, spasticity, and seizures(13,15–17,33,34). The pathogenesis of ZIKV microcephaly is not fully understood, although it is believed to begin with placen- tal infection. In the placental tissue of pregnant women infected with ZIKV, Noronha et al.(29) detected viral pro- teins in Hofbauer cells and some histiocytes in the in- tervillous spaces, suggesting that ZIKV damages the pla- cental barrier, inducing chronic placentitis(29), as has also been documented in rats(28). As gestation progresses, the virus disseminates to the fetal brain, where it preferen- tially infects neuronal progenitor cells, decreasing their viability and impeding their growth, thus inhibiting cell proliferation, cell differentiation, and neuronal apoptosis, with consequent thinning of the cortex and macroscopic signs of microcephaly(3,30). It has also been suggested that the placental inflammatory process can act in synergy with cerebral ZIKV infection in the genesis of brain mal- formations(32). Shepard system criterion 5 is the finding of terato- genicity in animal models. Animal studies of ZIKV have demonstrated not only its neurotropism but also its tera- togenic potential(26–29). Criterion 6 in the Shepard system is that the associa- tion makes biological sense. CAUSALITY OF MALFORMATIONS AND CONGENITAL ZIKV INFECTION Such sense has clearly been demonstrated, given that other viral agents have similar effects, as well as the fact that studies have shown that ZIKV has a high degree of tropism for and a deleterious effect on fetal brain tissue(24,25). Despite the neurotropism of ZIKV, there have been no reports of microcephaly in infected children soon after birth. That is probably attributable to the low infective potential of ZIKV in developed neural cells(3,32,33). The criterion 7 in the Shepard system is used only for exposure to medications and chemicals. Therefore, it does not apply to exposure to infectious agents. In view of the above, according to the Shepard sys- tem, there is strong evidence that ZIKV is a teratogen. CAUSALITY OF MALFORMATIONS AND CONGENITAL ZIKV INFECTION The cautious approach to implicating ZIKV as a cause of birth defects is not surprising, given that the last epi- demic of birth defects caused by an infectious pathogen (the rubella virus) occurred more than 50 years ago(13). In addition, no flaviviruses have been definitively demon- strated to be a cause of birth defects in humans and there have been no reports of adverse pregnancy events during previous ZIKV outbreaks(13,14). Shepard system criterion 3 is a specific defect or syndrome occurring after exposure to a given teratogen. In fact, many fetuses and infants with congenital ZIKV infection have presented a characteristic appearance, in- cluding severe microcephaly, intracranial calcifications and other brain abnormalities, sometimes accompanied by ocular alterations, cutis verticis gyrata (excessive scalp skin), arthrogryposis, and talipes equinovarus (clubfoot), leading some authors to coin the term “congenital Zika syndrome” to describe the presentation(22,23). In addition, cutis verticis gyrata is not a typical finding in other forms of microcephaly. Two methods have been used in order to identify po- tential teratogens: the identification of the combination of a rare exposure and presentation of a rare defect, as was the case for rubella virus, which was identified by an ophthalmologist after noticing a characteristic form of cataract in children born to mothers with rubella infection Criterion 4 in the Shepard system is an association between a rare exposure and the subsequent appearance 316 Radiol Bras. 2017 Set/Out;50(5):314–322 Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Of all the congenital manifestations of ZIKV infec- tion, the one that has had the greatest impact is micro- cephaly, which has been well documented, in Brazil and French Polynesia, and has been associated with the Asian lineage of the virus. During the ZIKV epidemic in Brazil (from March 2015 to February 2016), there was a 20-fold increase in the incidence of microcephaly in comparison with previous years(3). of a rare defect, the rationale of which is that it is unlikely that two rare events will occur in conjunction, especially with the appropriate chronology to suggest causation. Mi- crocephaly is a rare defect estimated to occur in 6 out of 10,000 live births in the United States(13). CAUSALITY OF MALFORMATIONS AND CONGENITAL ZIKV INFECTION Although exposure to ZIKV was not uncommon among women re- siding in Brazil during the outbreak, there were reports of adverse events in fetuses among female travelers who were in endemic areas for only a short time, constituting a rare exposure(24,25). In one report, a European woman presented fever and a skin rash at the end of week 13 of gestation, while in the state of Rio Grande do Norte, in northeastern Brazil. Prior to that, the woman had under- gone a number of ultrasound examinations, none of which had shown any abnormalities. However, ultrasound per- formed at 29 weeks of gestation revealed multiple cerebral malformations. The fetus was aborted, and an autopsy was conducted. All organs were examined, as were the placenta and umbilical cord. The findings included mi- crocephaly, pachygyria/lissencephaly, and hydrocephalus, together with multifocal dystrophic calcifications in the cortex and subcortical white matter, as well as signs of lo- cal inflammation and placental calcifications. There were no relevant pathological abnormalities in the remaining organs, and RT-PCR of the brain tissue was positive only for ZIKV, no other flaviviruses being identified. The ZIKV strain identified bore a high (99.7%) resemblance to the Asian lineage identified in the outbreak that occurred in French Polynesia in 2013(25). of a rare defect, the rationale of which is that it is unlikely that two rare events will occur in conjunction, especially with the appropriate chronology to suggest causation. Mi- crocephaly is a rare defect estimated to occur in 6 out of 10,000 live births in the United States(13). Although exposure to ZIKV was not uncommon among women re- siding in Brazil during the outbreak, there were reports of adverse events in fetuses among female travelers who were in endemic areas for only a short time, constituting a rare exposure(24,25). In one report, a European woman presented fever and a skin rash at the end of week 13 of gestation, while in the state of Rio Grande do Norte, in northeastern Brazil. Prior to that, the woman had under- gone a number of ultrasound examinations, none of which had shown any abnormalities. However, ultrasound per- formed at 29 weeks of gestation revealed multiple cerebral malformations. The fetus was aborted, and an autopsy was conducted. All organs were examined, as were the placenta and umbilical cord. MANIFESTATIONS OF CONGENITAL ZIKV INFECTION involve the cortical-subcortical junction (Figure 3); one possible explanation is that ZIKV infection has a vascular component, given that other processes that mainly affect this region are associated with vascular alterations(34–43). As illustrated in Figure 4, there are other sites that pres- ent calcifications in ZIKV infection(34–40), which are, in descending order of frequency, the following: the basal nuclei and thalamus (in 29–65% of cases); the periven- tricular region (in 14–65%); the cortical region (in 14– 24%); and the infratentorial region (in 4–18%). It is note- worthy that periventricular and cortical involvement are more common in newborns in whom there is a significant loss of brain parenchymal volume; therefore, the precise location of calcifications in such newborns is difficult to determine(34). According to Oliveira-Szejnfeld et al., in- fratentorial calcifications are present in more severe man- ifestations, being accompanied by brainstem malforma- tion, aqueduct stenosis, and secondary hydrocephalus(34). involve the cortical-subcortical junction (Figure 3); one possible explanation is that ZIKV infection has a vascular component, given that other processes that mainly affect this region are associated with vascular alterations(34–43). As illustrated in Figure 4, there are other sites that pres- ent calcifications in ZIKV infection(34–40), which are, in descending order of frequency, the following: the basal nuclei and thalamus (in 29–65% of cases); the periven- tricular region (in 14–65%); the cortical region (in 14– 24%); and the infratentorial region (in 4–18%). It is note- worthy that periventricular and cortical involvement are more common in newborns in whom there is a significant loss of brain parenchymal volume; therefore, the precise location of calcifications in such newborns is difficult to determine(34). According to Oliveira-Szejnfeld et al., in- fratentorial calcifications are present in more severe man- ifestations, being accompanied by brainstem malforma- tion, aqueduct stenosis, and secondary hydrocephalus(34). Figure 1. A 3-month-old patient. Sagittal T1-weighted MRI slice, without con- trast, showing craniofacial disproportion with a microcephalic aspect, together with occipital prominence and cutis verticis gyrata (arrowheads). Note also the confluence of the enlarged dural venous sinuses and the heterogeneous mate- rial (arrow). Abnormalities of cortical development are frequent findings, occurring in 94–100% of cases, commonly pre- senting as agyria-pachygyria (Figure 2), and probably varying according to the stage of cortical development in which the infection occurs(34–43). MANIFESTATIONS OF CONGENITAL ZIKV INFECTION In CT and MRI studies of congenital Zika syndrome, the findings most commonly reported are a reduction in head circumference and a dramatic reduction in brain volume, both of which are indicative of microcephaly and are more common when the infection occurs in the first trimester of pregnancy, with a risk of 1–13%(34–41). Micro- cephaly can be asymmetric. The condition is mild in 25% of cases and moderate-to-severe in 75%(35). The effects of intrauterine ZIKV infection are more severe when they occur in the first or second trimesters of pregnancy, especially in the first trimester, ranging from fetal death to various congenital abnormalities, which in- clude cutis verticis gyrata (of the scalp, neck, or both), low birth weight, polyhydramnios, anasarca, arthrogrypo- sis, and hearing loss, as well as ocular and CNS malfor- mations with craniofacial manifestations(3,25,30–33). An uncommon appearance of the skull, a finding that is common in congenital Zika syndrome and uncommon Radiol Bras. 2017 Set/Out;50(5):314–322 317 Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Figure 2. A 2-month-old patient. Axial T2-weighted MRI slice showing marked simplification of the gyral pattern, with agyria (arrows). Note also the diffuse increase in the extra-axial CSF space (asterisks). in microcephaly of other causes, is characterized by a collapsed aspect of the skull cap, with overlapping su- tures and prominent occipital bones, creating a “shelf” appearance, commonly accompanied by excessive, folded skin on the scalp (Figure 1). That presentation could be due, in part, to the continued growth of the skull and skin while the size of the brain regresses, or even, at some point, collapse of the skull, which previously had larger dimensions due to cerebral ventriculomegaly(34–40). One unusual finding that occurs in some newborns and sug- gests the latter hypothesis is herniation of the orbital fat to the skull, which could be secondary to the abrupt de- formation of the skull, rather than direct infection of the eye, as occurs in other congenital infections(34). Figure 1. A 3-month-old patient. Sagittal T1-weighted MRI slice, without con- trast, showing craniofacial disproportion with a microcephalic aspect, together with occipital prominence and cutis verticis gyrata (arrowheads). Note also the confluence of the enlarged dural venous sinuses and the heterogeneous mate- rial (arrow). Figure 2. A 2-month-old patient. Axial T2-weighted MRI slice showing marked simplification of the gyral pattern, with agyria (arrows). Note also the diffuse increase in the extra-axial CSF space (asterisks). Radiol Bras. 2017 Set/Out;50(5):314–322 MANIFESTATIONS OF CONGENITAL ZIKV INFECTION Ax- ial T1-weighted MRI slice, without con- trast, showing multiple punctate foci of hyperintensity at the cortical-sub- cortical junction of the frontal and pa- rietal lobes, indicative of calcifications (arrows). Note also the cutis verticis gyrata (arrowheads). B: A 6-month-old patient. Axial susceptibility-weighted MRI sequence showing foci of hy- pointensity at the cortical-subcortical junction, affecting both hemispheres of the brain and more conspicuous in the frontal regions, because of the presence of calcifications (arrows). A B B A Figure 3. A: A 2-month-old patient. Ax- ial T1-weighted MRI slice, without con- trast, showing multiple punctate foci of hyperintensity at the cortical-sub- cortical junction of the frontal and pa- rietal lobes, indicative of calcifications (arrows). Note also the cutis verticis gyrata (arrowheads). B: A 6-month-old patient. Axial susceptibility-weighted MRI sequence showing foci of hy- pointensity at the cortical-subcortical junction, affecting both hemispheres of the brain and more conspicuous in the frontal regions, because of the presence of calcifications (arrows). B age(34–43). Other associated abnormalities are hippocam- pal malformation and thickening of the fornix(34–40). Brainstem abnormalities, which have been reported in 21–70% of patients with congenital ZIKV infection, are characterized by a tapered, atrophic encephalic trunk with preferential involvement of the pons (Figure 7), are com- monly associated with more severe conditions and can be related to synergism between the reduction in the number of descending fibers and the direct effects of the virus(34–43). Other abnormalities of the posterior fossa include cerebel- lar hypoplasia, which is usually diffuse and symmetric (Figure 8), occurring in 27–82% of cases, and an enlarged B B A Figure 4. A: A 5-month-old patient. Axial CT scan, without contrast, show- ing foci of calcification in the basal nuclei (arrows) and left thalamus (ar- rowhead). B: A 5-month-old patient. Axial CT scan, without contrast, show- ing calcifications in the dorsolateral regions of the mesencephalic tegmen- tum (arrows), as well as at the cortical- subcortical junction in the temporal lobes (arrowheads). Figure 4. A: A 5-month-old patient. Axial CT scan, without contrast, show- ing foci of calcification in the basal nuclei (arrows) and left thalamus (ar- rowhead). B: A 5-month-old patient. Axial CT scan, without contrast, show- ing calcifications in the dorsolateral regions of the mesencephalic tegmen- tum (arrows), as well as at the cortical- subcortical junction in the temporal lobes (arrowheads). B A age(34–43). Other associated abnormalities are hippocam- pal malformation and thickening of the fornix(34–40). MANIFESTATIONS OF CONGENITAL ZIKV INFECTION The involvement is usu- ally diffuse, predominantly occurring in the frontal, insu- lar, and parietal lobes, with varying degrees of severity; it is frequently accompanied by wide sylvian and interhemi- spheric fissures, as well as by an increase in the extra-axial cerebrospinal fluid (CSF) space, the last being associated with a loss of brain volume and impaired CSF reabsorp- tion(34–43). Gray matter heterotopia is rare(38). There are inherent differences between CT and MRI, the former presenting greater sensitivity in the detection of calcifications even when compared with susceptibility- weighted MRI sequences, whereas MRI presents a greater capacity for characterizing cortical abnormalities and the development of the corpus callosum(34–40). Calcifications are common in congenital Zika syn- drome, occurring in 88–100% of patients; unlike classic infections (toxoplasmosis, rubella, cytomegalovirus, her- pes simplex, HIV, and syphilis), in which calcifications are periventricular and cortical, ZIKV infection tends to Enlargement of the lateral ventricles, as depicted in Figure 5, is common, occurring in 94–100% of cases; in 318 Radiol Bras. 2017 Set/Out;50(5):314–322 Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings most cases, it is moderate-to-pronounced and symmetric; it is accompanied by septations in 10–29% of cases, and occipital horns are commonly seen, sometimes making it difficult to distinguish periventricular cysts(34–43). It should also be borne in mind that the presence of ven- triculomegaly is directly related to a reduction in brain volume and can cause the head circumference to remain age(34–43). Other associated abnormalities are hippocam- pal malformation and thickening of the fornix(34–40). Brainstem abnormalities, which have been reported in 21–70% of patients with congenital ZIKV infection, are characterized by a tapered, atrophic encephalic trunk with preferential involvement of the pons (Figure 7), are com- monly associated with more severe conditions and can be Figure 4. A: A 5-month-old patient. Axial CT scan, without contrast, show- ing foci of calcification in the basal nuclei (arrows) and left thalamus (ar- rowhead). B: A 5-month-old patient. Axial CT scan, without contrast, show- ing calcifications in the dorsolateral regions of the mesencephalic tegmen- tum (arrows), as well as at the cortical- subcortical junction in the temporal lobes (arrowheads). B A Figure 3. A: A 2-month-old patient. MANIFESTATIONS OF CONGENITAL ZIKV INFECTION Confluence of the enlarged dural venous sinuses with heterogeneous material (Fig- ure 1) which can correspond to a thrombus or a hemato- Figure 6. A 3-month-old patient. Sagittal T1-weighted MRI slice, without con- trast, showing diffuse tapering of the corpus callosum (arrows). Note also the craniofacial disproportion with a microcephalic aspect. presenting all of the abnormalities described in the lit- erature and with a symmetric appearance; with postnatal microcephaly, the presentation of which is comparable to that of microcephaly at birth, minus the calcifications outside the cortical-subcortical junction and the agyria; and without microcephaly which presents with calcifica- Figure 7. A 2-month-old patient. Sagittal T1-weighted MRI slice, without con- trast, showing hypoplasia of the pons (arrow), with loss of the usual convexity. Also shown are an enlarged cisterna magna (asterisk), reduced cerebellar vol- ume, and excessive skin in the nuchal region (arrowheads). Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Figure 5. A: A 7-month-old p Magnetic resonance imaging, T section showing diffuse enlarg of the lateral ventricles (asterisk simplification of the gyral patte rowheads). B: An 8-month-old p Axial T2-weighted MRI slice s asymmetric dilation of the po portions of the lateral ventricl terisks), constituting a colpoc configuration. B A B Figure 5. A: A 7-month-old patient. Magnetic resonance imaging, T2, axial section showing diffuse enlargement of the lateral ventricles (asterisks) and simplification of the gyral pattern (ar- rowheads). B: An 8-month-old patient. Axial T2-weighted MRI slice showing asymmetric dilation of the posterior portions of the lateral ventricles (as- terisks), constituting a colpocephaly configuration. B A Figure 7. A 2-month-old patient. Sagittal T1-weighted MRI slice, without con- trast, showing hypoplasia of the pons (arrow), with loss of the usual convexity. Also shown are an enlarged cisterna magna (asterisk), reduced cerebellar vol- ume, and excessive skin in the nuchal region (arrowheads). Figure 6. A 3-month-old patient. Sagittal T1-weighted MRI slice, without con- trast, showing diffuse tapering of the corpus callosum (arrows). Note also the craniofacial disproportion with a microcephalic aspect. Figure 6. A 3-month-old patient. Sagittal T1-weighted MRI slice, without con- trast, showing diffuse tapering of the corpus callosum (arrows). Note also the craniofacial disproportion with a microcephalic aspect. Figure 7. A 2-month-old patient. Sagittal T1-weighted MRI slice, without con- trast, showing hypoplasia of the pons (arrow), with loss of the usual convexity. MANIFESTATIONS OF CONGENITAL ZIKV INFECTION most cases, it is moderate-to-pronounced and symmetric; it is accompanied by septations in 10–29% of cases, and occipital horns are commonly seen, sometimes making it difficult to distinguish periventricular cysts(34–43). It should also be borne in mind that the presence of ven- triculomegaly is directly related to a reduction in brain volume and can cause the head circumference to remain normal or even increase(34–40,43). Brainstem abnormalities, which have been reported in 21–70% of patients with congenital ZIKV infection, are characterized by a tapered, atrophic encephalic trunk with preferential involvement of the pons (Figure 7), are com- monly associated with more severe conditions and can be related to synergism between the reduction in the number of descending fibers and the direct effects of the virus(34–43). Other abnormalities of the posterior fossa include cerebel- lar hypoplasia, which is usually diffuse and symmetric (Figure 8), occurring in 27–82% of cases, and an enlarged Brainstem abnormalities, which have been reported in 21–70% of patients with congenital ZIKV infection, are characterized by a tapered, atrophic encephalic trunk with preferential involvement of the pons (Figure 7), are com- monly associated with more severe conditions and can be related to synergism between the reduction in the number of descending fibers and the direct effects of the virus(34–43). Other abnormalities of the posterior fossa include cerebel- lar hypoplasia, which is usually diffuse and symmetric (Figure 8), occurring in 27–82% of cases, and an enlarged Hypoplasia, dysgenesis, and agenesis of the corpus callosum are often seen in congenital Zika syndrome (Figure 6), reportedly occurring in 75–94% of cases and presenting a direct correlation with parenchymal dam- 319 Radiol Bras. 2017 Set/Out;50(5):314–322 Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Figure 5. A: A 7-month-old patient. Magnetic resonance imaging, T2, axial section showing diffuse enlargement of the lateral ventricles (asterisks) and simplification of the gyral pattern (ar- rowheads). B: An 8-month-old patient. Axial T2-weighted MRI slice showing asymmetric dilation of the posterior portions of the lateral ventricles (as- terisks), constituting a colpocephaly configuration. A B cisterna magna, the frequency of which increases in pro- portion to the severity of the infection, although there is no direct correlation between enlarged cisterna magna and the occurrence of cerebellar hypoplasia(34–43). Alteration of the white matter MRI signal secondary to a delay in myelination is seen in 88–100% of cases of congenital ZIKV infection. MANIFESTATIONS OF CONGENITAL ZIKV INFECTION Also shown are an enlarged cisterna magna (asterisk), reduced cerebellar vol- ume, and excessive skin in the nuchal region (arrowheads). cisterna magna, the frequency of which increases in pro- portion to the severity of the infection, although there is no direct correlation between enlarged cisterna magna and the occurrence of cerebellar hypoplasia(34–43). presenting all of the abnormalities described in the lit- erature and with a symmetric appearance; with postnatal microcephaly, the presentation of which is comparable to that of microcephaly at birth, minus the calcifications outside the cortical-subcortical junction and the agyria; and without microcephaly, which presents with calcifica- tions restricted to the cortical-subcortical junction, areas of pachygyria, a delay in myelination, and slight ventricu- lar enlargement, with an asymmetric appearance. In ad- dition, as proposed by Aragão et al.(41), the presence of presenting all of the abnormalities described in the lit- erature and with a symmetric appearance; with postnatal microcephaly, the presentation of which is comparable to that of microcephaly at birth, minus the calcifications outside the cortical-subcortical junction and the agyria; and without microcephaly, which presents with calcifica- tions restricted to the cortical-subcortical junction, areas of pachygyria, a delay in myelination, and slight ventricu- lar enlargement, with an asymmetric appearance. In ad- dition, as proposed by Aragão et al.(41), the presence of Alteration of the white matter MRI signal secondary to a delay in myelination is seen in 88–100% of cases of congenital ZIKV infection. Confluence of the enlarged dural venous sinuses with heterogeneous material (Fig- ure 1), which can correspond to a thrombus or a hemato- crit effect due to dehydration with hemoconcentration, is seen in 28–53% of cases(34,35,38,41,42). Currently, there are three severity spectra in con- genital Zika syndrome(41): with microcephaly at birth, 320 Radiol Bras. 2017 Set/Out;50(5):314–322 Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Niemeyer B et al. / Congenital Zika syndrome and neuroimaging findings Figure 8. A 2-month-old patient. Axial T2-weighted MRI slice showing diffuse, symmetric cerebellar hypoplasia (arrows), with prominence of the CSF spaces in the posterior fossa. Figure 9. A 14-month-old patient. Sagittal T1-weighted MRI slice showing ta- pering of the dorsal medulla (arrows) in a congenital Zika syndrome patient without arthrogryposis. Also shown are pontine hypoplasia (arrowhead) and an enlarged cisterna magna (asterisk). Figure 8. A 2-month-old patient. MANIFESTATIONS OF CONGENITAL ZIKV INFECTION Axial T2-weighted MRI slice showing diffuse, symmetric cerebellar hypoplasia (arrows), with prominence of the CSF spaces in the posterior fossa. Figure 9. A 14-month-old patient. Sagittal T1-weighted MRI slice showing ta- pering of the dorsal medulla (arrows) in a congenital Zika syndrome patient without arthrogryposis. Also shown are pontine hypoplasia (arrowhead) and an enlarged cisterna magna (asterisk). polymicrogyria mainly in the frontal lobes is seen only in patients without microcephaly or with postnatal micro- cephaly. neuronal migration and of cortical organization between weeks 12 and 16 of gestation, whereas polymicrogyria oc- curs around week 20(42). Recently, spinal and nerve root changes have been described in congenital Zika syndrome, the severity of those changes presenting an apparent correlation with ar- throgryposis, defined as contracture of two or more joints from birth(42). Visual inspection of T2-weighted MRI se- quences in the sagittal and axial planes has revealed a significant reduction in the thickness of the entire spinal cord, accompanied by a significant reduction in the thick- ness of the anterior roots of the medullary cone, in pa- tients with congenital Zika syndrome and arthrogryposis. However, patients who do not present such joint changes have been found to show a reduction in spinal thickness only in the dorsal region and only discrete tapering of the anterior roots of the medullary cone (Figure 9)(42). Clear involvement of the descending anterior medullary tracts, with apparent preservation of ascending posterior tracts, has also been seen, as reported by Mlakar et al.(25). CONCLUSION Fetal infection with ZIKV causes severe CNS de- velopmental abnormalities. Although the neuroimaging findings are not pathognomonic, they can be suggestive of congenital Zika syndrome when the clinical and bio- chemical data are consistent with the diagnosis. The main findings in congenital Zika syndrome are craniofacial disproportion with a microcephalic aspect, accompanied by calcifications (predominantly at the cortical-subcortical junction), malformations of cortical development, ventriculomegaly, and abnormalities in the formation of the corpus callosum. However, attention should be paid to the spectrum of potential presentations of congenital Zika syndrome, and the possibility of ZIKV involvement should not be ruled out when microcepha- ly is not present or when the neuroimaging findings are more subtle. 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https://openalex.org/W4254169278	https://www.researchsquare.com/article/rs-796403/latest.pdf	English	null	Long COVID in hospitalized and non-hospitalized patients in a large cohort in Northwest Spain, a prospective cohort study	medRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	5,113	Long COVID in Hospitalized and Non-Hospitalized Patients in a Large Cohort in Northwest Spain, A Prospective Cohort Study Alexandre Pérez González (  alexandre.perez@iisgaliciasur.es ) Instituto de Investigación Galicia Sur Research Article Keywords: COVID-19, coronavirus, long COVID, persisting symptoms. Posted Date: August 30th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-796403/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: August 30th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/14 Page 1/14 Page 1/14 Abstract Background. Survivors to COVID-19 have described long-term symptoms after acute disease. These signs constitute a heterogeneous group named long COVID or persistent COVID. Objective: The aim of this study is to describe persisting symptoms six months after COVID-19 diagnosis in a prospective cohort in the Northwest Spain Objective: The aim of this study is to describe persisting symptoms six months after COVID-19 diagnosis in a prospective cohort in the Northwest Spain Design. This is a prospective cohort study performed in the COVID-19 Cohort of Galicia Sur Health Institute (COHVID-GS). Design. This is a prospective cohort study performed in the COVID-19 Cohort of Galicia Sur Health Institute (COHVID-GS). Participants: This cohort includes patients in clinical follow-up in a health area of 569,534 inhabitants after SARS-CoV-2/COVID-19 diagnosis. Clinical and epidemiological characteristics were collected during the follow up. Main measures and key results. A total of 284 patients completed 6 months follow-up, 176 (69.4%) required hospitalization and 29 (10.2%) of them needed critical care. At six months, 119 (48.0%) patients described one or more persisting symptoms. The most prevalent were: extra-thoracic symptoms (39.1%), chest symptoms (27%), dyspnoea (20.6%), and fatigue (16.1%). These symptoms were more common in hospitalized patients (52.3% vs 38.2%) and in women (59.0% vs 40.5%). The multivariate analysis identified Chronic Obstructive Pulmonary Disease (COPD), female gender and tobacco consumption as risk factors for long COVID. Conclusions. Persisting symptoms are common after COVID-19 especially in hospitalized patients compared to outpatients (52.3% vs. 38.2%). Based on these findings, special attention and clinical follow- up after acute SARS-CoV-2 infection should be provided for hospitalized patients with previous lung diseases, tobacco consumption, and females. Study design This is a prospective cohort study performed in the COVID-19 Cohort of Galicia Sur Health Institute (COHVID-GS). This is an open cohort associated to a Biobank for sample collection including patients in clinical follow-up at Álvaro Cunqueiro Hospital (Vigo, Spain), a reference hospital for 564,534 inhabitants after SARS-CoV-2/COVID-19 diagnosis (all had a positive PCR for SARS-CoV-2 on nasal swab) (https://www.iisgaliciasur.es/apoyo-a-la-investigacion/cohorte-covid19/). Clinical and epidemiological characteristics were collected at the diagnosis time and during the follow up after 6 months of the diagnosis. The aim of this study is to describe the frequency of long COVID symptoms in hospitalized and non-hospitalized subjects. Introduction COVID-19 pandemic has been a major health challenge around the world, with an unprecedented impact on health systems, society and modern medicine. Acute phase of COVID-19 varies widely, from asymptomatic disease to pneumonia, acute respiratory distress syndrome (ARDS) and multisystem organ failure. Most commonly reported symptoms are fever, cough, dyspnoea, myalgia and anosmia. Several comorbidities have been identified as risk factors for severe disease, such as arterial hypertension, chronic heart disease or diabetes mellitus [1]. In the past few months, many patients reported persisting symptoms after the acute phase of coronavirus disease. These signs are a heterogeneous group, affecting multiple organ systems, including respiratory tract (dyspnoea, chest pain, cough), muscle and joints (arthralgia, myalgia), nervous system (taste or smell alterations, headache, concentration difficulty). There are also many general or unspecified symptoms such as fatigue or hair loss. This group of signs has been named “long COVID” or “persisting Page 2/14 Page 2/14 Page 2/14 COVID”. However, the definition of this syndrome remains unclear and there is not consensus regarding the name and the diagnostic criteria [2]. It is not clear which symptoms could be related to coronavirus and how to define this persistent period after the acute phase [3, 4]. The origin of these symptoms remains unknown. However, it is likely to be a combination of a direct damage caused by SARS-CoV-2, previous comorbidities, immunological activation, psychological and emotional factors [5–7]. The aim of this study is to describe persisting symptoms six months after COVID-19 diagnosis in a prospective cohort in the Northwest Spain. The study population includes mild, moderate, serious or critical cases classified as hospitalized or non-hospitalized patients. Ethics All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This project was approved by the Ethical Committee of Pontevedra-Vigo-Ourense (reference 2021/184). All the patients included in the study belong to the COHVID-GS, which signed an informed consent form at the time of inclusion in the cohort. Statistical analysis For quantitative variables, median and interquartile were calculated. Categorical variables were reported as frequencies and percentages. Subjects were classified in two groups, based on their need for hospitalization due to Covid-19. Symptoms, clinical and epidemiological data were compared in both groups using chi-square test, Fisher’s exact test or U Man-Whitney test as appropriate. P-value less than 0.05 was considered significant. Multivariable logistic regression analysis with backward stepwise elimination was used to identify risk factors for long COVID. Those variables with a p value lesser than 0,1 were considered as independent variables. Statistical analyses were performed with IBM Statistical Product and Service Solutions (SPSS) program, version 22. Procedures All patients included at the COHVID-GS were scheduled for clinical follow-up visits using phone calls and on site visits after 1, 3 and 6 months of the diagnosis. Subjects were classified as hospitalized when they required hospitalization for at least 24 hours. Main hospitalization criteria were pneumonia on chest X-ray and/or respiratory insufficiency. At each visit, patients completed a custom symptom questionnaire (supplementary data). COHVID-GS also includes data about comorbidities, smoke status, weight and height. Pre-existing symptoms that did not change after the diagnosis of COVID-19 were excluded. Symptoms of another suspected origin were also excluded (e.g., iron deficit, thyroid dysfunction). Dyspnoea was classified according to the British Medical Research Council mMRC scale. Brain fog was defined as poor concentration, recent memory disorder or inability to focus. Dyspnoea was classified according to the British Medical Research Council mMRC scale. Brain fog was defined as poor concentration, recent memory disorder or inability to focus. In this study, we only included patients who survived at least six months after the COVID-19 diagnosis and completed all follow-up visits. We excluded patients who died during the study period, those who refused to complete all follow-up visits and those who were unable to assist to the site (e.g., cognitive impairment, physical disability The recruitment period started in March 2020 and ended by May 2021 and included consecutive cases. For the purpose of this study, data was censored in January 2021 when the first wave cases reached 6 months since COVID-19 diagnosis. Page 3/14 Ethics Baseline characteristics of the study population A total of 630 patients were included in the COHVID-GS. However, 81 of them died during the hospitalization period and only 284 subjects who survived completed the six months period of follow-up. Of them, 172 required hospitalization and 76 were never hospitalized. The baseline characteristics of the study population are shown in Table 1. Page 4/14 Page 4/14 Table 1 Table 1 Baseline characteristics of study population Total Hospitalized Non hospitalized p-value Patients in follow-up, n (%) 248 (100%) 172 (69.4%) 76 (30.6%) Demographics Age, in years, median (IQR) 57 (46— 68) 62 (51—71) 47 (34–54) p < 0.001 Sex, male, n (%) 148 (59.7%) 103 (59.9%) 45 (62.5%) p = 1.000 Spanish, n (%) 234 (94.4%) 159 (92.4%) 75 (98.7%) p = 1.000 Latin American, n (%) 11 (4.4%) 10 (5.8%) 1 (1.3%) p = 0.070 European (not Spanish), n (%) 2 (3.8%) 2 (1.2%) 0 p = 0.267 Oceania, n (%) 1 (0.4%) 1 (0.6%) 0 p = 1.000 Severity Admission to critical care, n (%) 29 (11.7%) 29 (16.8%) N/A N/A Invasive mechanical ventilation required, n (%) 23 (9.3%) 23 (13.4%) N/A N/A Comorbidities Obesity, n (%) 75 (30.2%) 62 (36.0%) 13 (17.1%) p < 0.001 Arterial hypertension, n (%) 65 (26.2%) 58 (33.7%) 7 (9.2%) p < 0.001 Diabetes mellitus, n (%) 26 (10.5%) 25 (14.5%) 1 (1.3%) p = 0.001 Asthma, n (%) 21 (8.5%) 7 (4.1%) 14 (18.4%) p = 0.807 Chronic ischaemic heart disease, n (%) 21 (8.5%) 21 (12.2%) 0 p < 0.001 Chronic obstructive pulmonary disease, n (%) 15 (6.0%) 15 (8.7%) 0 p = 0.007 Active cancer, n (%) 5 (2.0%) 4 (2.3%) 1 (1.3%) p = 1.000 Table 1 Baseline characteristics of study population Page 5/14 Page 5/14 Total Hospitalized Non hospitalized p-value HIV infection, n (%) 3 (1.2%) 2 (1.2%) 1 (1.3%) p = 1.000 Chronic kidney disease, n (%) 3 (1.2%) 3 (1.7%) 0 p = 0.555 Liver cirrhosis, n (%) 2 (0.8%) 2 (1.2%) 0 p = 1.000 Tobacco use Active smoker, n (%) 18 (7.3%) 8 (4.6%) 10 (13,9%) p = 0.031 Former smoker, n (%) 105 (42.3%) 75 (43.6%) 35 (48.6%) p = 0.575 Never smoker, n (%) 120 (69.8%) 85 (49.4%) 35 (20.3%) p = 0.582 Overall, the median age was 57 years, however, non-hospitalized patients were significantly younger than those who were hospitalized (62 vs. 47 years, respectively). Baseline characteristics of the study population Moreover, male patients were more frequent in both groups (148 males, 59.7%). Overall, the median age was 57 years, however, non-hospitalized patients were significantly younger than those who were hospitalized (62 vs. 47 years, respectively). Moreover, male patients were more frequent in both groups (148 males, 59.7%). Overall, the median age was 57 years, however, non-hospitalized patients were significantly younger than those who were hospitalized (62 vs. 47 years, respectively). Moreover, male patients were more frequent in both groups (148 males, 59.7%). Most common comorbidities were obesity (n = 75, 30.2%), followed by arterial hypertension (n = 65, 26.2%) and diabetes mellitus (n = 26, 10.1%). Moreover, all comorbidities were more usual in the hospitalized group, except for asthma (4.1% vs. 18.4% respectively). In the hospitalized group, 29 patients (16.8%) required critical care management, including 23 who required invasive mechanical ventilation. Most common comorbidities were obesity (n = 75, 30.2%), followed by arterial hypertension (n = 65, 26.2%) and diabetes mellitus (n = 26, 10.1%). Moreover, all comorbidities were more usual in the hospitalized group, except for asthma (4.1% vs. 18.4% respectively). In the hospitalized group, 29 patients (16.8%) required critical care management, including 23 who required invasive mechanical ventilation. Six months after the COVID-19 diagnosis, 119 patients (48.9%) reported at least one symptom (Table 2). Chest symptoms were more frequent (n = 67, 27%), of which dyspnoea (n = 51, 20.6%), chest pain (n = 15, 6.0%) and cough (n = 11, 4.4%) were the most commonly reported. Baseline characteristics of the study population Page 6/14 Page 6/14 Page 6/14 Table 2 Prevalence of 6 months persisting symptoms Total Hospitalized Non hospitalized p-value Total patients, n (%) 248 (100%) 172 (69.4%) 76 (30.6%) Any symptom, n (%) 119 (48.0%) 90 (52.3%) 29 (38.2%) p = 0.040 Chest symptoms, n (%) 67 (27.0%) 55 (32.0%) 12 (15.8%) p = 0.008 Dyspnoea, n (%) 51 (20.6%) 43 (25.0%) 8 (10.5%) p = 0.010 Grade MMRC 1, n (%) 43 (17.3%) 36 (20.9%) 7 (0.9%) p = 0.029 Grade MMRC 2, n (%) 8 (3.2%) 7 (4.1%) 1 (1.3%) p = 0.441 Chest pain, n (%) 15 (6.0%) 11 (6.4%) 4 (5.3%) p = 0.444 Cough, n (%) 11 (4.4%) 10 (5.8%) 1 (1.3%) p = 0.673 Extra-thoracic symptoms, n (%) 97 (39.1%) 74 (43.0%) 23 (30.3%) p = 0.058 Digestive symptoms, n (%) 10 (4.0%) 7 (4.1%) 3 (3.9%) p = 0.999 Abdominal pain, n (%) 7 (2.8%) 5 (2.9%) 2 (2.6%) p = 1.000 Diarrhoea, n (%) 4 (1.6%) 3 (1.7%) 1 (1.3%) p = 1.000 Musculoskeletal symptoms, n (%) 18 (7.3%) 16 (9.3%) 2 (2.6%) p = 0.068 Myalgia, n (%) 10 (4.0%) 10 (5.8%) 0 p = 0.034 Arthralgia, n (%) 12 (4.8%) 10 (5.8%) 2 (2.6%) p = 0.354 Fatigue, n (%) 40 (16.1%) 36 (20.9%) 4 (5.3%) p = 0.001 Anosmia, n (%) 17 (6.9%) 9 (5.2%) 8 (10.5%) p = 0.054 Ageusia, n (%) 10 (4.0%) 5 (2.9%) 5 (6.6%) p = 0.181 Headache, n (%) 12 (4,8%) 6 (3.5%) 6 (8.0%) p = 0.196 Brain fog, n (%) 9 (3.6%) 9 (5.2%) 0 p = 0.061 Mood disorder, n (%) 10 (4.0%) 5 (2.9%) 5 (6.6%) p = 0.396 Hair loss, n (%) 18 (7.3%) 16 (9.3%) 2 (2.6%) p = 0.068 Sleep disorder, n (%) 9 (3.6%) 9 (5.2%) 0 p = 0.061 Conjunctival chemosis, n (%) 1 (0.4%) 1 (0.6%) 0 p = 1.000 Page 7/14 Extra-thoracic symptoms were described in 97 patients (39.1%) and the most frequently reported were fatigue (n = 40, 16.1%), musculo-skeletal symptoms (n = 18, 7.3%) and hair loss (n = 18, 7.3%). Extra-thoracic symptoms were described in 97 patients (39.1%) and the most frequently reported were fatigue (n = 40, 16.1%), musculo-skeletal symptoms (n = 18, 7.3%) and hair loss (n = 18, 7.3%). Overall, smell disorders were more frequent than taste disorders (6.9% vs. 4.0%, respectively). Persisting Symptoms In Hospitalized And Non-hospitalized Patients Persisting symptoms were more prevalent in the hospitalized group (52.3% vs. 38.2%, p = 0.040). Chest symptoms in this group were more frequently reported compared with the non-hospitalized group (32.0% vs. 15.8%, p = 0.008). However, extra-thoracic symptoms did not differ between both groups (43.0% vs. 30.3%, p = 0.058). The most common chest symptom was dyspnoea, which was more frequently reported in hospitalized patients (25.0% vs. 10.5%, p = 0.010). Most of the subjects reported a mMRC (modified Medical Research Council) grade 1 dyspnoea (n = 43, 17.3%). On the other hand, chest pain (6.4% vs. 5.3%) and persisting cough (5.8% vs. 1.3%) did not vary between both groups. Fatigue (20.9% vs. 5.3%, p = 0.001) and myalgia (5.8% vs. 0%, p = 0.034) were more frequently reported in the hospitalized group than in the non-hospitalized group. Also, hair loss (9.3% vs. 2.6%) and sleep disorders (5.2% vs. 0%) were more common in hospitalized patients, although this difference was not statistically significant. Arthralgia (5.8% vs. 2.6%), ageusia (2.9% vs. 6.6%) and digestive symptoms (4.1% vs. 3.9%) did not differ between both groups. Anosmia was more prevalent in non-hospitalized subjects (5.2% vs. 10.5%, p = 0.054), but did not meet statistical significance. Baseline characteristics of the study population Neurological symptoms six months after the COVID-19 diagnosis, excluding smell and taste disruptions, were uncommon (less than 5.0%). Headache, mood disorder and sleep disturbance were the most common neurological symptoms. Nine patients (3.2%) reported symptoms that met brain fog criteria. Digestive symptoms were also uncommon (n = 10, 4.0%). Persisting Symptoms In Male And Female Patients Overall, persisting symptoms were more common in females than males (59.0% vs. 40.5%, p = 0.004) (Table 3). Dyspnoea (29.0 vs. 14.9%, p = 0.007), headache (10.0% vs. 4.8%, p = 0.004), fatigue (22.0% vs. 12.2%, p = 0.039) and hair loss (15.0% vs. 7.3%, p < 0.001) were all more prevalent in females while anosmia (6.0% vs. 7.4%), taste disturbance (4.0% in both genders) and musculoskeletal symptoms (6.8% vs. 8.0%) did not differ between females and males. Page 8/14 Table 3 Prevalence of persisting symptoms at 6 months based on gender Female Male p-value Any symptom, n (%) 59 (59.0%) 60 (40,5%) p = 0.004 Chest symptoms, n (%) 35 (35.0%) 32 (21,6%) p = 0.020 Dyspnoea, n (%) 29 (29.0%) 22 (14.9%) p = 0.007 Chest pain, n (%) 7 (7.0%) 8 (5.4%) p = 0.600 Cough, n (%) 7 (7.0%) 4 (2.7%) p = 0.125 Extra-thoracic symptoms, n (%) 50 (50.0%) 47 (31.8%) p = 0.004 Neurological symptoms, n (%) 26 (26%) 21 (14.2%) p = 0.022 Anosmia, n (%) 6 (6.0%) 11 (7.4%) p = 0.800 Agueusia, n (%) 4 (4.0%) 10 (4%) p = 1.000 Headache, n (%) 10 (10%) 12 (4.8%) p = 0.004 Brain fog, n (%) 5 (5%) 4 (2.7%) p = 0.491 Diarrhea, n (%) 4 (4.0%) 0 p = 0.025 Myalgia, n (%) 5 (5.0%) 5 (3.4%) p = 0.530 Arthralgia, n (%) 4 (4.0%) 8 (5.4%) p = 0.767 Fatigue, n (%) 22 (22%) 18 (12.2%) p = 0.039 Hair loss, n (%) 15 (15%) 18 (7.3%) p < 0.001 Table 3 Predictors Of Covid-19 Persisting Symptoms We performed a multivariate analysis in order to determine the risk factors for persisting COVID-19 symptoms. We identified Chronic Pulmonary Obstructive Disease (COPD) (OR 5.009; CI 95%, 1.321– 18.997, p = 0.018), female gender (OR 2.700, 95% CI 1.543–4.724, p < 0.001) and tobacco consumption as independent risk factors for remaining symptoms (OR 1.736; CI 95%, 1.002–3.007, p = 0.049) (Table 4). Page 9/14 Table 4 Risk factors for long COVID. Odds Ratio Confidence interval (95%) p-value COPD 5.009 1.321–18.997 p = 0.018 Sex, female 2.700 1.543–4.724 p < 0.001 Hospitalization 1.711 0.951–3.078 p = 0.073 Tobbacco consumption 1.736 1.002–3.007 p = 0.049 COPD: chronic obstructive pulmonary disease Table 4 Table 4 Risk factors for long COVID. COPD was the only comorbidity associated with a higher prevalence of persisting symptoms. Fourteen patients (5.1%) were affected by COPD before COVID-19 diagnosis and 12 of them (80.0%) had at least one persisting symptom. Nine subjects (75.0%) reported new or worsened dyspnoea compared with their perception prior COVID-19 diagnosis. Dyspnoea (60.0% vs. 18.0%, p = 0.001) and chest pain (26.7% vs. 4.7%, p = 0.008) were most commonly reported in COPD group. On the other hand, cough (6.7% vs. 4.3%), fatigue (20.0% vs. 15.9%), myalgia (13.3% vs. 3.4%), arthralgia (6.7% vs. 4.7%) did not differ between COPD and non-COPD subjects. Discussion However, even in the absence of pneumonia, 15% of non-hospitalized patients reported persisting chest symptoms. Recently, Blomberg et al., described long COVID symptoms in Norwegian non-hospitalized and hospitalized population with a slightly higher prevalence than in our study (52.0% vs. 38.2% respectively) [10]. Fatigue was also frequent, reaching up to 20.9% in the hospitalized group. Although this percentage is relevant, it was lower than others reported before [12, 13], around 65–70%. However, these studies had a shorter follow-up period (90 to 100 days after hospitalization). The reason of fatigue remains unknown, but it is probably a result of a combination of factors, including direct nervous and muscle damage due to SARS-CoV-2 replication [5, 6], immune activation and immune dysregulation, or emotional factors [7]. On the other hand, myalgia and arthralgia were less frequent than in previous studies. Sykes et al., described a prevalence of 50% of these symptoms in hospitalized patients at 100 days after discharge [14]. Overall, persisting anosmia was reported in 6.8% of subjects and was more common in the non- hospitalized group (10.5% vs. 5.2%, respectively). This prevalence is lower than published in previous studies [15, 16]. In addition, prior reports have suggested that anosmia may be associated with a mild to moderate COVID-19 and it could be more common in females than males [15]. However, the low prevalence of anosmia observed in our study cannot confirm this hypothesis. At six months, 9 subjects reported unspecified neurological symptoms such as poor concentration, loss of recent memory or inability to focus. These unspecified symptoms are named brain fog by some authors, but its origin or relationship with coronavirus disease remains unknown [5, 17]. Brain fog syndrome after COVID-19 has been reported previously [18, 19], although there is not a validated diagnostic criteria established. The studies focused on neurological sequelae of COVID-19 did not use a unanimous criterion and therefore, the conclusions are very heterogeneous. The lack of knowledge about this type of symptoms makes more difficult to study the real prevalence or to establish a relationship with coronavirus disease. Another neurological symptoms like sleep or mood disorders have been reported with a similar prevalence than in our study [20]. However, psychological conditions, stress and isolation may be associated with these symptoms, alongside with coronavirus infection. Discussion We characterized long COVID in a prospective cohort of patients with mild and severe COVID-19 with a systematic clinical follow-up. The prevalence of any symptom at six months was elevated (48%), with a different distribution between hospitalized and non-hospitalized patients. Persisting symptoms did not differ between those patients who needed critical care and those who did not. Long COVID has been recognized as a public-health problem and a matter of concern for COVID-19 surveys. Several studies have reported information in series of patients in clinical follow-up after the acute infection, but many questions remain unsolved. One of them is if long COVID might be gender- dependent. In our study, females showed a higher prevalence of persisting symptoms in hospitalized and in non-hospitalized groups. Dyspnoea, headache, fatigue and hair loss were all more common in females than males. A prior study performed by Chaoling-Huang et al., also described a higher frequency of long COVID symptoms in females than males [8]. To the best of our knowledge, there are only few studies evaluating long COVID symptoms between males and females, both in hospitalized and non-hospitalized setting [10]. In the recent months, more than 200 persistent symptoms have been described after the acute phase of COVID-19. Most of them are related to the respiratory tract (dyspnoea, cough, chest pain), the main target of SARS-CoV-2. However, many patients report other type of symptoms such as fatigue, cognitive impairment or smell disorders [11]. Page 10/14 Page 10/14 Chest symptoms were the most common reported affection, with greater prevalence in the hospitalized group. This difference may be related to pneumonia, respiratory distress and lung damage, which were more frequent in hospitalized subjects. The prevalence of chest symptoms in this group raised to 52%, slightly higher than observed in previous reports [9]. However, even in the absence of pneumonia, 15% of non-hospitalized patients reported persisting chest symptoms. Recently, Blomberg et al., described long COVID symptoms in Norwegian non-hospitalized and hospitalized population with a slightly higher prevalence than in our study (52.0% vs. 38.2% respectively) [10]. Chest symptoms were the most common reported affection, with greater prevalence in the hospitalized group. This difference may be related to pneumonia, respiratory distress and lung damage, which were more frequent in hospitalized subjects. The prevalence of chest symptoms in this group raised to 52%, slightly higher than observed in previous reports [9]. Declarations Acknowledgments: We would like to thank all the members of COHVID-GS and IISGS Biobank, patients and nursing staff and the Methodology and Statistic Unit. Discussion The higher prevalence on the hospitalized group may be due to the severity of illness, admission to critical care or treatments (e.g., corticosteroids, immunosuppressants). We identified COPD, female gender, tobacco consumption and the need for hospitalization as predictors for long COVID. Female gender has been reported before as a risk factor for hospitalized subjects but there is a lack of information about other predictors of long COVID. Page 11/14 Page 11/14 Our findings suggest that some of the persisting symptoms could be related to a previous lung comorbidity and hospitalization. SARS-CoV-2 damage on respiratory tract could be more intense in patients with previous lung disease, increasing the risk of long-term symptoms. In addition, pneumonia and ARDS may also contribute to develop persisting symptoms. Our findings suggest that some of the persisting symptoms could be related to a previous lung comorbidity and hospitalization. SARS-CoV-2 damage on respiratory tract could be more intense in patients with previous lung disease, increasing the risk of long-term symptoms. In addition, pneumonia and ARDS may also contribute to develop persisting symptoms. This study has few limitations. Firstly, some of the symptoms reported are subjective and based on the patient’s testimony (e.g. fatigue, headache, dyspnoea). Additionally, the lack of validated scales to measure most of the symptoms makes difficult to compare data between subjects or studies. Secondly, most of the symptoms may be affected by personal, psychological or environmental factors. We needed to use a non-validated custom questionnaire, as there is not a specific post-COVID-19 document established. Many symptoms could be higher reported in one group than another for unrelated reasons with coronavirus disease. This is the case for hair loss that may be more noticed in women and therefore more reported, leading to a possible increase of the prevalence in female subjects. In addition, previous comorbidities and age could increase the persisting symptoms in the hospitalized group. For COPD patients, we did not record symptoms that started before COVID-19 diagnosis. Therefore, it is possible that COPD patients reported more frequently chest or respiratory symptoms. Conclusions In summary, a high prevalence of long COVID (48%) was observed in patients after six months of acute SARS-CoV-2 infection. Overall, persistent symptoms were more common in hospitalized patients compared to outpatients (52.3% vs 38.2%). These symptoms were more common in females than males (59.0% vs. 40.5%). COPD, female gender, and tobacco consumption were identified as long COVID predictors. These findings suggest that in those patients that required hospitalization after SARS-CoV-2 infection a close clinical follow-up might be recommended. Special attention should be provided for patients with previous lung diseases, tobacco consumption and females. Funding COVID-19 Cohort Galicia Sur Health Research Institute (COHVID-GS) is funded by the Instituto de Salud Carlos III through the FONDO COVID19 (COV20/00698). Alexandre Pérez is hired under a Rio Hortega contract (CM20/00243). Data availability: The datasets during and/or analyzed during the current study available from the COHVID-GS on reasonable request (https://www.iisgaliciasur.es/apoyo-a-la-investigacion/cohorte- covid19/). Declaration of interests The authors declare no competing interests. Members of COHVID-GS (Galicia Sur Health Research Institute): Alejandro Araujo, Jorge Julio Cabrera, Víctor del Campo, Manuel Crespo, Alberto Fernández, Beatriz Gil de Araujo, Carlos Gómez, Virginia Leiro, María Rebeca Longueira, Ana López-Domínguez, José Ramón Lorenzo, María Marcos, Alexandre Pérez, María Teresa Pérez, Lucia Patiño, Sonia Pérez, Silvia Pérez-Fernández, Eva Poveda, Cristina Ramos, Benito Regueiro, Cristina Retresas, Tania Rivera, Olga Souto, Isabel Taboada, Susana Teijeira, María Torres, Vanesa Val, Irene Viéitez Page 12/14 Page 12/14 References 1. Berenguer, J. et al. Characteristics and predictors of death among 4035 consecutively hospitalized patients with COVID-19 in Spain. Clin Microbiol Infect, 26 (11), 1525–1536 https://doi.org/10.1016/j.cmi.2020.07.024 (2020). 2. Al-Jahdhami, I., Al-Naamani, K. & Al-Mawali, A. The Post-acute COVID-19 Syndrome (Long COVID). Oman Med J, 36 (1), e220–e220 https://doi.org/10.5001/omj.2021.91 (2021). 2. Al-Jahdhami, I., Al-Naamani, K. & Al-Mawali, A. The Post-acute COVID-19 Syndrome (Long COVID). Oman Med J, 36 (1), e220–e220 https://doi.org/10.5001/omj.2021.91 (2021). 3. Fernández-de-las-Peñas, C. et al. Persistent Post-COVID): An Integrative Classification. Int J Environ Res Public Health, 18 (5), 2621 https://doi.org/10.3390/ijerph18052621 (2021). 3. Fernández-de-las-Peñas, C. et al. Persistent Post-COVID): An Integrative Classification. Int J Environ Res Public Health, 18 (5), 2621 https://doi.org/10.3390/ijerph18052621 (2021). 4. Halpin, S., O’Connor, R. & Sivan, M. Long COVID and chronic COVID syndromes. J Med Virol, 93 (3), 1242–1243 https://doi.org/10.1002/jmv.26587 (2021). 4. Halpin, S., O’Connor, R. & Sivan, M. Long COVID and chronic COVID syndromes. J Med Virol, 93 (3), 1242–1243 https://doi.org/10.1002/jmv.26587 (2021). 5. Pezzini, A. & Padovani, A. Lifting the mask on neurological manifestations of COVID-19. Nat Rev Neurol, 16 (11), 636–644 https://doi.org/10.1038/s41582-020-0398-3 (2020). 5. Pezzini, A. & Padovani, A. Lifting the mask on neurological manifestations of COVID-19. Nat Rev Neurol, 16 (11), 636–644 https://doi.org/10.1038/s41582-020-0398-3 (2020). 6. Ferrandi, P. J., Alway, S. E. & Mohamed, J. S. The interaction between SARS-CoV-2 and ACE2 may have consequences for skeletal muscle viral susceptibility and myopathies. J Appl Physiol, 129 (4), 864–867 https://doi.org/10.1152/japplphysiol.00321.2020 (2020). 6. Ferrandi, P. J., Alway, S. E. & Mohamed, J. S. The interaction between SARS-CoV-2 and ACE2 may have consequences for skeletal muscle viral susceptibility and myopathies. J Appl Physiol, 129 (4), 864–867 https://doi.org/10.1152/japplphysiol.00321.2020 (2020). 7. Morgul, E. et al. COVID-19 pandemic and psychological fatigue in Turkey. Int J Soc Psychiatry, 67 (2), 128–135 https://doi.org/10.1177/0020764020941889 (2021). 7. Morgul, E. et al. COVID-19 pandemic and psychological fatigue in Turkey. Int J Soc Psychiatry, 67 (2), 128–135 https://doi.org/10.1177/0020764020941889 (2021). 8. Huang, C. et al. 6-month consequences of COVID-19 in patients discharged from hospital: a cohort study., 397 (10270), 220–232 https://doi.org/10.1016/S0140-6736(20)32656-8 (2021). 8. Huang, C. et al. 6-month consequences of COVID-19 in patients discharged from hospital: a cohort study., 397 (10270), 220–232 https://doi.org/10.1016/S0140-6736(20)32656-8 (2021). 9. Xiong, Q. et al. Clinical sequelae of COVID-19 survivors in Wuhan, China: a single-centre longitudinal study. Clin Microbiol Infect, 27 (1), 89–95 https://doi.org/10.1016/j.cmi.2020.09.023 (2021). 10. Blomberg, B. et al. Long COVID in a prospective cohort of home-isolated patients. References Nat Med, https://doi.org/10.1038/s41591-021-01433-3 (June 2021). 11. Davis, H. E. et al. Characterizing Long COVID in an International Cohort: 7 Months of Symptoms and Their Impact. medRxiv. January 2021:2020.12.24.20248802. doi:10.1101/2020.12.24.20248802 11. Davis, H. E. et al. Characterizing Long COVID in an International Cohort: 7 Months of Symptoms and Their Impact. medRxiv. January 2021:2020.12.24.20248802. doi:10.1101/2020.12.24.20248802 12. Mandal, S. et al. ‘Long-COVID’: a cross-sectional study of persisting symptoms, biomarker and imaging abnormalities following hospitalisation for COVID-19., 76 (4), 396–398 https://doi.org/10.1136/thoraxjnl-2020-215818 (2021). Page 13/14 Page 13/14 13. Carfì, A., Bernabei, R. & Landi, F. Persistent Symptoms in Patients After Acute COVID-19. JAMA, 324 (6), 603 https://doi.org/10.1001/jama.2020.12603 (2020). 14. Sykes, D. L. et al. Post-COVID-19 Symptom Burden: What is Long-COVID and How Should We Manage It?, 199 (2), 113–119 https://doi.org/10.1007/s00408-021-00423-z (2021). 15. Hopkins, C., Surda, P., Whitehead, E. & Kumar, B. N. Early recovery following new onset anosmia during the COVID-19 pandemic – an observational cohort study. J Otolaryngol - Head Neck Surg, 49 (1), 26 https://doi.org/10.1186/s40463-020-00423-8 (2020). 16. D'Ascanio, L. et al. A. Olfactory Dysfunction in COVID-19 Patients: Prevalence and Prognosis for Recovering Sense of Smell.Otolaryngol Head Neck Surg. 2021Jan;164(1):82–86. Doi: 10.1177/0194599820943530. 17. Stefano, G. B. et al. The Pathogenesis of Long-Term Neuropsychiatric COVID-19 and the Role of Microglia, Mitochondria, and Persistent Neuroinflammation: A Hypothesis. Med Sci Monit, 10;27, e933015 https://doi.org/10.12659/MSM.933015 (2021 May). 17. Stefano, G. B. et al. The Pathogenesis of Long-Term Neuropsychiatric COVID-19 and the Role of Microglia, Mitochondria, and Persistent Neuroinflammation: A Hypothesis. Med Sci Monit, 10;27, e933015 https://doi.org/10.12659/MSM.933015 (2021 May). 18. Pinna, P. et al. Neurological manifestations and COVID-19: Experiences from a tertiary care center at the Frontline. J Neurol Sci, 415, 116969 https://doi.org/10.1016/j.jns.2020.116969 (2020). 18. Pinna, P. et al. Neurological manifestations and COVID-19: Experiences from a tertiary care center at the Frontline. J Neurol Sci, 415, 116969 https://doi.org/10.1016/j.jns.2020.116969 (2020). 19. Karadaş, Ã., Öztürk, B. & Sonkaya, A. R. A prospective clinical study of detailed neurological manifestations in patients with COVID-19. Neurol Sci, 41 (8), 1991–1995 https://doi.org/10.1007/s10072-020-04547-7 (2020). 20. Romero-Sánchez, C. M. et al. Neurologic manifestations in hospitalized patients with COVID-19: The ALBACOVID registry. Neurology, 95 (8), e1060–e1070 https://doi.org/10.1212/WNL.0000000000009937 (2020). SupplementaryData.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryData.docx Page 14/14
https://openalex.org/W1971876242	https://ro.uow.edu.au/cgi/viewcontent.cgi?article=3636&context=sspapers	English	null	Communicating about screening	BMJ. British medical journal	2,008	cc-by	4,063	University of Wollongong University of Wollongong Research Online Research Online Faculty of Social Sciences - Papers (Archive) Faculty of Arts, Social Sciences & Humanities 2008 Communicating about screening Communicating about screening Vikki A. Entwistle University of Dundee Stacy Carter University of Sydney, stacyc@uow.edu.au Lyndal Trevena University of Sydney Kathy Flitcroft University of Sydney Les Irwig University of Sydney See next page for additional authors Follow this and additional works at: https://ro.uow.edu.au/sspapers Part of the Education Commons, and the Social and Behavioral Sciences Commons Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au University of Wollongong University of Wollongong Research Online Research Online ial Sciences - Papers (Archive) Faculty of Arts, Social Sciences & Humanities Communicating about screening Communicating about screening Vikki A. Entwistle University of Dundee Disciplines Disciplines Disciplines Disciplines Education \| Social and Behavioral Sciences Education \| Social and Behavioral Sciences Abstract Abstract Informed choice is important for screening, but not everyone wants or is able to analyse research data. Vikki Entwistle and colleagues propose a new approach to communication. Keywords Keywords communicating, about, screening Publication Details Publication Details Publication Details Publication Details Entwistle, V. A., Carter, S. M., Trevena, L., Flitcroft, K., Irwig, L., McCaffery, K. & Salkeld, G. (2008). Communicating about screening. BMJ: British Medical Journal, 337 (7673), 789-791. Analyse and choose Th l d h It is unclear how healthcare providers should communicate about screening in order to support appropriate uptake. And what constitutes appropriate uptake is also contested because of disagreements about the merits of particular tests and tensions between concerns to promote health and to respect autonomy.2-4 Debates about commu nication have tended to consider two types of approach, which we call “be screened” and “analyse and choose.” We consider their problems and propose a third approach, “consider an offer.” The analyse and choose approach is one response to criticisms of the be screened approach. It emphasises respect for auton omy and treats this as a matter of ensuring that competent individuals have sufficient understanding of their options and can make intentional, sufficiently independent choices.9 It assumes that sufficient under standing requires comprehension of detailed research based information about benefits and harms and promotes informed individ ual decision making based on this. The key features of this approach are an emphasis on the importance of individual (sometimes independent) choice and the provision of comparative data about the various outcomes of screening and no screening. The approach is exemplified by decision aids, which seek to present the data in accessible ways. National screening programmes Yes: Offer is not from a source I trust or I have reason not to accept it Yes: Offer is from a source I trust, and I have reason to accept it Reject offer Accept offer No: I need additional information Commercial screening test providers Clinicians Recommendations or offers of screening consistent with the consider an offer approach Now what do I think? Do I have enough information to decide whether or not to accept the offer? Other information relating to screening, ideally including screening literacy campaigns (Ideally) supportive relationships that promote and sustain autonomy Further information or support from other sources More detailed information, including research data about benefits and harms (possibly in a decision aid) from original source Overview of the consider an offer approach There are three main criticisms of the ana lyse and choose approach. Firstly, the impli cation that everyone eligible for screening should consider detailed effectiveness data may be unnecessarily burdensome. This criticism is particularly strong when expert committees acting in the public interest have reviewed the available research, judged the tests to be broadly effective and acceptable, and supported the introduction of government endorsed screening programmes. Be screened and includes a breast awareness code that instructs women to “Go for breast screening every 3 years if you are over 50.” People are offered a wide range of screen ing tests by diverse providers. For example: maternal and child health services screen for genetic conditions and developmen tal problems; general practitioners screen for cardiovascular risk factors; NHS pro grammes screen for bowel, breast, and cer vical cancer; and commercial providers offer various health assessments, including body and gene scans. Provision of tests is not well regulated, and there is a bewildering amount of information of variable accuracy in the public domain.1 The be screened approach aims to persuade people to have screening, usually with a view to promoting health gain, cost effective serv ice provision, or profit.2-4 Its key features are encouragement to be screened; an emphasis on the benefits of screening and de-emphasis of potential harms; and a lack of recognition that it might be reasonable not to be screened. The main criticisms of the be screened approach are that it inadequately reflects the benefit-harm profile of screening tests and fails to respect autonomy because it does not facilitate informed decision making by individuals.6-8 Some communications with features typical of this approach purport to facilitate informed choice, but they present only one option: to be screened. g This approach is found in commercial advertisements and some invitations to par ticipate in government funded screening programmes. For example, the leaflet Breast Cancer: the Facts, from the NHS Breast Cancer Screening Programme presents screening as necessary for women aged over 50.5 It asks, “Should all women have breast screening?” and gives no hint of any scope for a nega tive answer. The leaflet highlights the benefits of mammography and describes the main processes but plays down potential harms. It does not mention that screening may lead to overtreatment or that clinicians and epidemi ologists seriously dispute the value of breast screening. It gives no indication that women might reasonably choose not to be screened, Authors Authors Vikki A. Entwistle, Stacy Carter, Lyndal Trevena, Kathy Flitcroft, Les Irwig, Kirsten McCaffery, and Glenn P. Salkeld This journal article is available at Research Online: https://ro.uow.edu.au/sspapers/2634 ANALYSIS Consider an offer Th d f Information about consequences is an important component of practical reasoning, and people usually want to know (or at least not be misled about) the main reasons for or against healthcare interventions Although prostate cancer kills many men, a lot of men have prostate cancer but do not die from it . . . Prostate specific antigen (PSA) testing will pick up some prostate cancers but will also miss some. And we can’t tell which of the prostate cancers that it does pick up would be life threatening. When a man is found to have prostate cancer after being screened, no one can be sure if current treatments will leave him better off than if he had not been screened. Research is ongoing, but at the moment we really don’t know the overall effects of PSA testing Although prostate cancer kills many men, a lot of men have prostate cancer but do not die from it . . . Prostate specific antigen (PSA) testing will pick up some prostate cancers but will also miss some. And we can’t tell which of the prostate cancers that it does pick up would be life threatening. When a man is found to have prostate cancer after being screened, no one can be sure if current treatments will leave him better off than if he had not been screened. Research is ongoing, but at the moment we really don’t know the overall effects of PSA testing You’re a heavy smoker, which increases your risk of heart disease and stroke. If you’re worried about having a stroke like your uncle, screening might help us think again about the smoking and look at other ways you can reduce that risk Prostate cancer is rare in men under 50 and you have no family history of prostate cancer, so the chances of you having it are relatively low. Analyse and choose Th l d h Secondly, there are concerns that encouraging detailed decision analysis by individuals might not lead to good choices (it might disrupt peo ple’s usual effective decision making proc esses) or deter uptake of effective, appropriate screening.4 7 10 11 Thirdly, some critics think this approach overemphasises rational and More detailed information, including research data about benefits and harms (possibly in a decision aid) from original source More detailed information, including research data about benefits and harms (possibly in a decision aid) from original source Overview of the consider an offer approach BMJ \| 4 october 2008 \| Volume 337 789 ANALYSIS independent decision making—reflecting an inappropriately narrow understanding of autonomy. and trustworthiness of those making it and of its personal relevance); provide or facilitate access to further information if that is required; and acknowledge that the recommendation or offer might reasonably be refused. although others who try to impose their views and discourage competent considera tion of alternatives do tend to undermine it. although others who try to impose their views and discourage competent considera tion of alternatives do tend to undermine it. Neither of the two approaches above con siders the importance of the interests and trustworthiness of those who offer and advise about screening. This failing, together with recent evidence about what patients value about communication with health profes sionals19 and involvement in decision mak ing,20 leads us to suggest a third approach to communication about screening. Neither of the two approaches above con siders the importance of the interests and trustworthiness of those who offer and advise about screening. This failing, together with recent evidence about what patients value about communication with health profes sionals19 and involvement in decision mak ing,20 leads us to suggest a third approach to communication about screening. Beyond a clear statement about who a recommendation or offer applies to, it may be useful to highlight circumstances that can make screening more or less appropriate for particular individuals—eg, age and risk exposures that modify the likelihood of benefit and values that can give people reason to accept or decline screening tests What is the basis of the recommendation, and what are the main benefits and harms of screening? New considerations g y Just what information and how much detail are required will vary across screening tests, contexts, and individuals, but will usually include a summary of the potential benefits and harms of the test considered, considera tion of any known objections to it, informa tion about test providers, and factors that might affect the appropriateness of the test for particular individuals (table). The optional extra information might include detailed data on outcomes and, more controversially, other people’s experiences and preferences, espe cially in value laden contexts such as screen ing for fetal abnormality.7 Recent research into decision making has highlighted some potential downsides of detailed decision analysis and maximising (aiming to make the best possible choice). It suggests that heuristics and “satisficing” (aim ing to make good enough choices) can be less burdensome and yield better decisions and outcomes.12 13 Are there any factors that make the screening test more appropriate for some people than others? Consider an offer Th d f The understanding of autonomy that pre vails in health care has been criticised for focusing too narrowly on discrete decisions, over-idealising rationalism, and inappropri ately assuming that interpersonal collabora tion and trust will compromise rather than promote autonomy.14-16 Although autonomy relates to individuals, it is both developed and exercised in the context of social rela tionships.16-18 People who use “intellectual outsourcing” to help shape their opinions, and who do not process detailed data for themselves before they choose or act, do not necessarily fail to exercise autonomy,18 The consider an offer approach is designed to respect personal autonomy without overbur dening people with unwanted information and decision making tasks and without deterring uptake of effective and personally appropriate screening. Within this approach, communica tors either recommend or offer screening or help people to consider recommendations or offers from others. They openly explain and discuss the basis for the recommendation or offer; encourage and facilitate an individual assessment of the recommendation or offer (including consideration of the potential bias When presented with a screening offer, people might reasonably respond in various ways. Some might judge the trustworthiness and personal relevance of a screening offer on the basis of the initial communication; others might habitually seek and follow the advice of a trusted health professional; and others might want to evaluate research evidence for esponse to a healthy 45 year old’s query bout prostate cancer screening Recommendation of cardiovascular risk assessment Recommendation of cardiovascular risk assessment The National Institute for Health and Clinical Excellence and all the major professional and patient groups have worked together and now recommend that we carry out a cardiovascular risk assessment for most people aged 45 and over Studies following thousands of people over several decades have allowed experts to develop accurate estimates of how likely individuals are to have a heart attack or stroke, based on their age, sex, family history, blood pressure, blood cholesterol, and whether they smoke. Many things can be done to reduce the risk and screening would allow us to discuss options appropriate for your level of risk An explanation of how a recommendation was reached may help people to assess its trustworthiness. Provenance and pee r review: Not commissioned; externally peer reviewed. SPL Sense About Science. 1 Making sense of testing. London, 2008. www.senseaboutscience.org/index.php/site/ project/232. Sense About Science. 1 Making sense of testing. London, 2008. www.senseaboutscience.org/index.php/site/ project/232. Patients offered screening can be helped to consider whether a test is appropriate p j / Rimer BK, Briss PA, Zeller PK, Chan ECY, Woolf SH. 2 Informed decision-making: what is its role in cancer screening? Cancer 2004;101(suppl 5):1214-28. themselves—at least for some tests. It should be feasible to respond to these varied preferences even when communications are necessarily standardised to some extent. For example, mailed invitations from national screening pro grammes can encourage people to consider whether they would like more information and tell them where to find additional resources and personal support. themselves—at least for some tests. It should be feasible to respond to these varied preferences even when communications are necessarily standardised to some extent. For example, mailed invitations from national screening pro grammes can encourage people to consider whether they would like more information and tell them where to find additional resources and personal support. about the adequacy of the information sup plied. The further protection that the analyse and choose approach offers by encouraging rational personal decisions based on detailed data on outcomes is not practical for many. g ; ( pp ) Raffle AE. Information about screening: is it to achieve 3 high uptake or to ensure informed choice? Health Expect 2001;4:92-8. Hargreaves KM, Stewart RJ, Oliver SR. Informed choice 4 and public health screening for children: the case of blood spot screening. Health Expect 2005;8:161-71. NHS Cancer Screening Programme. 5 Breast screening: the facts. 2007. www.cancerscreening.nhs.uk/ breastscreen/publications/ia-02.html. B tt A T L D HM M C ff K U f 6 Hargreaves KM, Stewart RJ, Oliver SR. Informed choice 4 and public health screening for children: the case of blood spot screening. Health Expect 2005;8:161-71. Hargreaves KM, Stewart RJ, Oliver SR. Informed choice 4 and public health screening for children: the case of blood spot screening. Health Expect 2005;8:161-71. NHS Cancer Screening Programme. 5 Breast screening: the facts. 2007. www.cancerscreening.nhs.uk/ breastscreen/publications/ia-02.html. For screening programmes backed by agen cies such as the National Screening Commit tee, the consider an offer approach should not adversely affect uptake of broadly effective tests. Communication consistent with this approach should help people to recognise when providers are trustworthy. Provenance and pee r review: Not commissioned; externally peer reviewed. In contrast, the be screened approach might lead to mis trust over time if people come to realise prac titioners have underplayed the downsides of screening. p g p ; NHS Cancer Screening Programme. 5 Breast screening: the facts. 2007. www.cancerscreening.nhs.uk/ breastscreen/publications/ia-02.html. p g p NHS Cancer Screening Programme. 5 Breast screening: the facts. 2007. www.cancerscreening.nhs.uk/ breastscreen/publications/ia-02.html. p Barratt A, Trevena L, Davey HM, McCaffery K. Use of 6 decision aids to support informed choices about screening. BMJ 2003;329:507-10. Barratt A, Trevena L, Davey HM, McCaffery K. Use of 6 decision aids to support informed choices about screening. BMJ 2003;329:507-10. This approach respects autonomy because it encourages and enables people to consider screening offers carefully. Although it can incorporate strong recommendations, it does not close down opportunities for thoughtful refusals of screening. For example, practition ers will avoid presenting screening as routine16 or necessary and will ensure people feel they can safely say they do not think a test is right for them. The consider an offer approach can facilitate informed decision making about screening, providing summary information about the benefits and harms of screening to all and decision aids with more detailed epidemi ological information to those who want them (figure), but it does not assume that autono mous choice or informed decision making will always require every individual to work through detailed statistics for themselves. g ; Irwig L, McCaffery K, Salkeld G, Bossuyt P. Informed 7 choice for screening: implications for evaluation. BMJ 2006;332:1148-50. g Irwig L, McCaffery K, Salkeld G, Bossuyt P. Informed 7 choice for screening: implications for evaluation. BMJ 2006;332:1148-50. 2006;332:1148 50. Entwistle V. The potential contribution of decision aids t 8 screening programmes. Health Expect 2001;4:109-15. Beauchamp TL, Childress JF. 9 Principles of biomedical ethics. 5th ed. Oxford: Oxford University Press, 2001. J RG H i J Th A W ll D P i ; Entwistle V. The potential contribution of decision aids to 8 screening programmes. Health Expect 2001;4:109-15. Entwistle V. The potential contribution of decision aids to 8 screening programmes. Health Expect 2001;4:109-15. g p g p ; Beauchamp TL, Childress JF. 9 Principles of biomedical ethics. 5th ed. Oxford: Oxford University Press, 2001. ethics. 5th ed. Oxford: Oxford University Press, 2001. Jepson RG, Hewison J, Thompson A, Weller D. Patient 10 perspectives on information and choice in cancer screening: a qualitative study in the UK. Soc Sci Med 2007;65:890-9. Provenance and pee r review: Not commissioned; Provenance and pee r review: Not commissioned; externally peer reviewed. Consider an offer Th d f But if you are particularly anxious about it and concerned to find out, then we should take that into account—but still bearing in mind that the test is not 100% accurate and may lead to unnecessary treatment Information about the financial and other significant interests of the organisations and individuals that recommend, offer, and provide screening are relevant in some contexts Although I wouldn’t really recommend it, I can arrange for a PSA test for you on the NHS… You’ll find private health care providers more enthusiastic about the test—but then they can make money from it in a way that I can’t The income we get from government to help run the practice depends in part on us doing some of these checks, but we recommend it because we think it can help patients. The practice would still care for you if you chose not to be screened People’s information needs vary. Initial communications can usefully encourage people to consider whether they have enough information and enable those who want more to access other potentially useful sources Does that give you enough to go on? I can give you more information, or point you towards a website that explains the assessment in more detail Has that given you enough information, or would you like more detail or more time to think about it? I have a leaflet here that summarises the information we’ve talked about, and it lists a few other sources of information 790 BMJ \| 4 october 2008 \| Volume 337 ANALYSIS SPL Patients offered screening can be helped to consider whether a test is appropriate Program, School of Public Health, University of Sydney Kirsten McCaffery senior research fellow, Screening and Diagnostic Test Evaluation Program, School of Public Health, University of Sydney Glenn Salkeld professor, Screening and Diagnostic Test Evaluation Program, School of Public Health, University of Sydney Vikki A Entwistle professor, Social Dimensions of Health Institute, Universities of Dundee and St Andrews, Dundee, DD1 4HJ v.a.entwistle@dundee .ac.uk Stacy M Carter senior lecturer, Centre for Values, Ethics and Law in Medicine, University of Sydney, Sydney, NSW 2006, Australia and School of Public Health, University of Sydney Lyndal Trevena senior lecturer, School of Public Health, University of Sydney Kathy Flitcroft research fellow, Screening and Diagnostic Test Evaluation Program, School of Public Health, University of Sydney Les Irwig professor, Screening and Diagnostic Test Evaluation Accepted: 21 June 2008 Contributors and sources: The article originated from a seminar VAE gave to the Screening and Test Evaluation Program at the School of Public Health, University of Sydney in September 2007, supported by National Health and Medical Research Council [Australia] program grant 402764 and a University of Sydney international visiting research fellowship. The ideas were developed in subsequent discussions, telephone conference calls, and email exchanges. These drew on the authors’ experiences of developing, evaluating, and thinking critically about information resources relating to screening and on their familiarity with relevant literature. VAE had the original idea for the article and drafted the manuscript. All authors contributed significantly to the content and to improving structure and clarity. VAE is guarantor. Contributors and sources: The article originated from a seminar VAE gave to the Screening and Test Evaluation Program at the School of Public Health, University of Sydney in September 2007, supported by National Health and Medical Research Council [Australia] program grant 402764 and a University of Sydney international visiting research fellowship. The ideas were developed in subsequent discussions The ideas were developed in subsequent discussions, telephone conference calls, and email exchanges. These drew on the authors’ experiences of developing, evaluating, and thinking critically about information resources relating to screening and on their familiarity with relevant literature. VAE had the original idea for the article and drafted the manuscript. All authors contributed significantly to the content and to improving structure and clarity. VAE is guarantor. Provenance and pee r review: Not commissioned; externally peer reviewed. The consider an offer approach is less demanding on those eligible for screening than the analyse and choose approach, but it puts more onus on providers to communicate in a range of ways to meet diverse informa tion needs. Some programmes already use features of consider an offer—for example, information about newborn bloodspot tests presents recommendations and explanations and points out that tests are not compulsory.22 But if the approach is found to be successful, health service agencies will need to develop more resources to support adoption by front line health professionals. Woodrow C, Watson E, Rozmovits L, Parker R, Austoker J. 11 Public perceptions of communicating information about bowel cancer screening. Health Expect 2008;11:16-25. bowel cancer screening. Health Expect 2008;11:16-25. g p Schwartz B. 12 The paradox of choice: why more is less. New York: Harper Collins, 2004. Douglas K, Jones D. Top 10 ways to make better 13 decisions. New Sci 2007;194(2602):35-43. Mackenzie C, Stoljar N, eds. 14 Relational autonomy: feminist perspectives on autonomy, agency and the social self. New York: Oxford University Press, 2000. Kukla R. Conscientious autonomy: displacing decisions 15 in health care. Hastings Cent Rep 2005;35:34-44. Kukla R. How do patients know? 16 Hastings Cent Rep 2007;37:27-35. Meyers DT. 17 Self, society and personal choice. New York: Columbia University Press, 1989. Because it accepts the reality of intellectual outsourcing18 and the importance of trust, the consider an offer approach makes people vulnerable to manipulation: trust can be both inappropriately placed and abused.21 How ever, this vulnerability is arguably no greater than with the be screened approach. Consider an offer provides some protection by discuss ing the basis of recommendations or offers, facilitating assessment of the trustworthiness of those who make them, and raising questions y Appiah KA. 18 The ethics of identity. New Jersey: Princeton University Press, 2005. Burkitt-Wright E, Holcombe C, Salmon P. Doctors’ 19 communication of trust, care, and respect in breast cancer: qualitative study. BMJ 2004;328:864. Entwistle VA, Prior M, Skea ZC, Francis J. Involvement 20 in decision-making: a qualitative investigation of its meaning for people with diabetes. Soc Sci Med 2008;66:362-75. Baier A. Trust and antitrust. 21 Ethics 1986;96:231-60. UK National Screening Programme. 22 Screening tests for your baby. 2007. www.screening.nhs.uk/ anpublications/baby_screening_tests.pdf. Cite this as: BMJ 2008;337:a1591 BMJ \| 4 october 2008 \| Volume 337 791
https://openalex.org/W2918420047	http://ojs.uho.ac.id/index.php/peternakan-tropis/article/download/5450/pdf	Indonesian	null	Kapasitasi Spermatozoa Sapi Peranakan Ongole dalam Berbagai Formulasi Pengencer Air Kelapa Selama Simpan Dingin	JITRO (Jurnal Ilmu dan Teknologi Peternakan Tropis)	2,019	cc-by-sa	3,404	ABSTRACT Coconut water extender is the solution to the difficulty and high cots of semen diluents. The objective of this study were to examine the capacitation of Ongole Crossbreed sperm in various formulations of coconut water extender during storage in 4-5 oC. The study method used laboratory experimental. The experimental design was Randomized Completely Block Design and the data were analyzed by Analyze of Variance. There are three treatments in this study (P1 = Coconut Water + 20% of yolk, P2 = Coconut Water + 20% of yolk + 0,4% white egg + fructose 1 mg/ml, dan P3 = Coconut Water + 20% of yolk + 0,4% white egg + fructose 2 mg/ml) and ten replications of each treatment. There were not significant differences in percentage of sperm uncapacitation, sperm capacitaion and sperm acrosom reaction between various formulations of coconut water diluents during chilled storage at 4-5 oC. Coconut water diluents are able to maintain quality of sperm acrosom up to day 5th with values above 50%. Keywords: acrosom reaction, capacitation, coconut water, liquid semen ABSTRAK Pengencer dasar air kelapa merupakan solusi dari sulit dan mahalnya harga pengadaan bahan baku pembuatan pengencer. Tujuan dari penelitian ini adalah untuk mengetahui kapasitasi spermatozoa sapi Peranakan Ongole dalam berbagai formulasi pengencer air kelapa selama simpan dingin pada suhu 4-5oC. Metodologi yang digunakan adalah eksperimantal laboratorium menggunakan rancangan acak kelompok (RAK). Terdapat tiga perlakuan yaitu, P1 = Pengencer air kelapa + 20% kuning telur, P2 = Pengencer air kelapa + 20% kuning telur + 0,4% putih telur + fruktosa 1000 mg/l. P3 = Pengencer air kelapa + 20% kuning telur + 0,4% putih telur + fruktosa 2000 mg/l, dengan 10 ulangan. Hasil menunjukkan bahwa tidak terdapat perbedaan yang signifikan pada persentase spermatozoa belum kapasitasi, terkapasitasi dan telah rekasi akrosom antara perlakuan formulasi pengencer air kelapa yang berbeda (P1, P2, P3) selama peyimpana suhu 2-5oC. Pengencer berbasis air kelapa mampu mempertahankan kualitas akrosom diatas 50% penyimpanan hari ke lima. Kata kunci : air kelapa, kapasitasi, reaksi akrosom, semen cair Email korespondensi: dedi.pendidik@gmail.com; trinil_susilawati@yahoo.com (Diterima: 19 12 2018; disetujui 10 1 2019) Email korespondensi: dedi.pendidik@gmail.com; trinil_susilawati@yahoo.com (Diterima: 19 12 2018; disetujui 10 1 2019) (Diterima: 19-12-2018; disetujui 10-1-2019) (Diterima: 19-12-2018; disetujui 10-1-2019) Jurnal Ilmu dan Teknologi Peternakan Tropis, Januari 2019, 6(1):85-91 http://ojs.uho.ac.id/index.php/peternakan-tropis Jurnal Ilmu dan Teknologi Peternakan Tropis, Januari 2019, 6(1):85-91 http://ojs.uho.ac.id/index.php/peternakan-tropis Jurnal Ilmu dan Teknologi Peternakan Tropis, Januari 2019, 6(1):85-91 http://ojs.uho.ac.id/index.php/peternakan-tropis Jurnal Ilmu dan Teknologi Peternakan Tropis, Januari 2019, 6(1):85-91 p-ISSN: 2406-7489 e-ISSN: 2406-9337 Terakreditasi Ditjen Penguatan Riset dan Pengembangan, Kemenristekdikti Keputusan No: 21/E/KPT/2018, Tanggal 9 Juli 2018 Terakreditasi Ditjen Penguatan Riset dan Pengembangan, Kemenristekdikti Keputusan No: 21/E/KPT/2018, Tanggal 9 Juli 2018 Dedi Muhammad1, Nurul Isnaini1, Kuswati1, Aulia Puspita Anugra Yekti1, Muchmad Luthfi2, Lukman Affandhy Sunarto2, Trinil Susilawati1 1Fakultas Peternakan Universitas Brawijaya Malang Jl. Veteran, Lowokwaru, Malang, Jawa Timur 65145 2Loka Penelitian Sapi Potong Grati Jl. Pahlawan 2, Grati, Pasuruan, Jawa Timur 67184 Dedi Muhammad1, Nurul Isnaini1, Kuswati1, Aulia Puspita Anugra Yekti1, Muchmad Luthfi2, Lukman Affandhy Sunarto2, Trinil Susilawati1 1Fakultas Peternakan Universitas Brawijaya Malang Jl. Veteran, Lowokwaru, Malang, Jawa Timur 65145 2Loka Penelitian Sapi Potong Grati Jl. Pahlawan 2, Grati, Pasuruan, Jawa Timur 67184 *Email korespondensi: dedi.pendidik@gmail.com; trinil_susilawati@yahoo.com PENDAHULUAN et al., 2018). Bahan pengencer yang ditambah bertujuan untuk memenuhi nutrisi dan menyediakan lingkungan yang sesuai bagi spermatozoa. Disamping itu pengencer juga harus memiliki kemampuan untuk melindungi spermatozoa dari kerusakan akibat suhu, dibuat Metode penyimpanan semen dalam bentuk semen cair telah terbukti mampu menguragi tingkat kerusakan membran spemratozoa. Semen cair dibuat dengan menambahkan pengencer kemudian disimpan pada suhu 2-4 oC (Susilawati 85 Muhammad et al./Jurnal Ilmu dan Teknologi Peternakan Tropis 6(1):85-91 dari bahan-bahan lokal yang mudah didapatkan, dan harganya tidak mahal (Susilawati, 2011). spermatozoa sapi PO (Peranakan Ongole) dalam berbagai formulasi pengencer air kelapa selama simpan dingin. dan harganya tidak mahal (Susilawati, 2011). Air kelapa sebagai bahan lokal yang mudah didapatkan di lingkungan sekitar dapat dijadikan sebagai pengencer semen alternatif. Kandungan beberapa karbohidrat, mineral, serta zat-zat lain dalam air kelapa mampu memenuhi kebutuhan spermatozoa. Vigliar, Sdepanian, and Neto (2006) menyatakan bahwa air kelapa mengandung unsur karbon berupa karbohidrat sederhana, seperti: sukrosa, glukosa, dan fruktosa. Yong et al. (2009) menambahkan bahwa kandungan yang terkandung di dalam air kelapa muda yaitu air 94,180 g/100g, lipid 0,073 g/100g, protein 0,120 g/100g, pH 4,7 dan mengandung beberapa ion yaitu Na, Zn, Fe, P, Ca, Mn, Cu serta beberapa asam amino yaitu arginin, metionin, alanin, lisin, aspartat fenilalanin, glisin, serin, glutamat, prolin, histidin, isoleusin, treonin, valin dan leusin. Rancangan Penelitian Penelitian ini menggunaan Rancangan Acak Kelompok (RAK) dengan 3 perlakuan dan setiap perlakukan diulang 10 ulangan. Pengelompokan dilakukan berdasarkan waktu penampungan. Perlakuan terdiri atas: Perlakuan pertama (P1) = Pengencer air kelapa + 20% kuning telur ayam ras. Perlakuan kedua (P2) = Pengencer air kelapa + 20% kuning telur ayar ras + 0,4% putih telur + fruktosa 1000 mg/l. Dan perlakuan ketiga (P3) = Pengencer air kelapa + 20% kuning telur + 0,4% putih telur + fruktosa 2000 mg/l. Hasil Audia et al. (2017); Mugiyati et al. (2017); Salim et al. (2018) menunjukkan bahwa penggunaan pengencer air kelapa dengan varietas yang berbeda sebagai pengencer semen kambing boer mampu mempertahankan kualitas spermatozoa yang masih baik hingga hari ke 2 pada penyimpanan suhu 4-5oC. Daramola et al. (2016) menambahkan bahwa pengencer air kelapa memiliki kemampuan menjaga intergitas akrosom spermatozoa rusa dengan baik. Hal tersebut memungkinkan spermatozoa terjaga dari kapasitasi dan reaksi akrosom. Semen Sapi PO Semen sapi PO yang digunakan memiliki kriteria motilitas massa minimal 2+, motilitas individu minimal 70%. Frekuensi penampungan paling banyak dua kali per minggu. Semen ditampung menggunakan teknik vagina buatan. Lokasi dan Waktu Penelitian Penelitian dilaksanakan di Laboratorium Loka Penetilian Sapi Potong Grati Pasuruan dan Laboratorium Biomedik Fakultas Kedokteran Universitas Brawijaya pada bulan April 2018. Pengencer Air Kelapa Pengencer air kelapa dibuat dari bahan dasar air kelapa yang diambil dari jenis kelapa hijau (Cocos nucifera) dengan usia buah 5 sampai 8 bulan. Sebelum air kelapa digunakan, terlebih dahulu disaring menggunakan kertas saring halus kemudian dipanaskan dengan suhu 57 oC selama 25 menit. Pamanasan bertujuan untuk menonaktifkan enzim yang ada pada air kelapa. Setelah itu ditambahkan NaHCO3 1 mg/ml sebagai buffer, antibiotik streptomycin sulfate 1 mg/ml dan penicillin 1 mg/ml, 20% kuning telur ayam ras, dan sebagai perlakuan yaitu putih telur ayam 0,4%, fruktosa 10 dan 20 mg/ml. Penambahan bahan pengencer selama proses pengenceran semen dapat menyebabkan turunnya kadar zat-zat yang terkandung dalam plasma semen, misalnya kadar asam amino, lemak dan ion-ion yang dapat mempengaruhi keseimbangan osmolaritas yang dapat mempengaruhi motilitas dan kapasitasi spermatozoa (Adnani, Bebas, dan Budiasa, 2012). Sumber energi berupa fruktosa serta zat makromolekul berupa protein ditambahkan dalam pengencer air kelapa diharapkan mampu menggantikan beberapa zat yang telah hilang dari plasma semen akibat proses pengenceran sehingga mempu mempertahankan kehidupan spermatozoa. Analisis Data Data yang diperoleh dianalisa menggunakan analisis ragam (ANOVA) dengan RAK (Rancangan Acak Kelompok) kemudian dilakukan pengujian lanjut menggunakan Duncan’s Multiple Range Test (Uji Jarak Berganda Duncan). Rata-rata hasil pengamatan volume semen dari sapi PO yang digunakan untuk penelitian adalah 4,67 ± 0,58 ml. Tabel 1. Rata-rata kualitas semen sapi PO sebelum pengenceran Parameter Rata-rata ± simpang baku Makroskopis Volume per ejakulat (ml) 4,67 ± 0,58 Warna Putih susu pH 6,47 ± 0,12 Konsistensi kental Mikroskopis Motilitas Massa ++ Motilitas Individu (%) 71,67 ± 2,89 Viabilitas (%) 91,39 ± 2,38 Abnormalitas (%) 2,81 ± 0,80 Konsentrasi (juta/ml) 1260,00 ± 222,71 Spermatozoa Belum Kapasitasi (%) 86,40 ± 1,97 Spermatozoa Terkapasitasi (%) 9,41 ± 1,35 Spermatozoa Telah Reaksi Akrosom (%) 4,19 ± 1,02 Tabel 1. Rata-rata kualitas semen sapi PO sebelum pengenceran Parameter Rata-rata ± simpang baku Makroskopis Volume per ejakulat (ml) 4,67 ± 0,58 Warna Putih susu pH 6,47 ± 0,12 Konsistensi kental Mikroskopis Motilitas Massa ++ Motilitas Individu (%) 71,67 ± 2,89 Viabilitas (%) 91,39 ± 2,38 Abnormalitas (%) 2,81 ± 0,80 Konsentrasi (juta/ml) 1260,00 ± 222,71 Spermatozoa Belum Kapasitasi (%) 86,40 ± 1,97 Spermatozoa Terkapasitasi (%) 9,41 ± 1,35 Spermatozoa Telah Reaksi Akrosom (%) 4,19 ± 1,02 konsentrasi semen sapi bervariasi dari 1.000- 1.800 juta spermatozoa setiap mililiter atau 800- 2.000 juta spermatozoa setiap mililiter. Rata-rata viabilitas spermatozoa sapi PO yang digunakan untuk penelitian adalah 91,39±2,38 %. Hal tersebut memiliki arti bahwa semen sapi PO yang digunakan dalam penelitian termasuk memenuhi syarat untuk digunakan. Ducha et al. (2013); Ismaya (2014) yang menyatakan bahwa semen yang akan diencerkan paling tidak minimal jumlah spermatozoa yang hidup 70%. Hasil tersebut termasuk rendah jika dibandingkan dengan pernyataan Garner and Hafez (2008) bahwa volume semen sapi hasil penampungan berkisar antara 5-8 ml. Adapun pH semen rata-rata 6,47±0,12. Hasil tersebut memperlihatkan bahwa pH semen yang digunakan untuk penelitian adalah normal. Ax et al., (2008) menjelaskan bahwa pH semen sapi berkisar antara 6,4 sampai 7,8. Rata-rata persentase motilitas massa semen sapi PO yang digunakan untuk penelitian adalah 2+. Persyaratan motilitas massa agar semen dapat diproses minimal 2+ (Ducha et al., 2013). Rataan motilitas individu semen segar sapi PO yang digunakan adalah 71,67±2,89 %. Muhammad et al. (2016) menyatakan bahwa syarat minimal nialai motilitas semen untuk diencerkan adalah 70%. Nilai konsentrasi spermatozoa semen sapi PO yang digunakan adalah 1260,00±222,71 juta/ml. Pemeriksaan Status Akrosom (CTC) Metode pemeriksaan status akrosom menggunakan mikroskop epiflouresence dengan pewarnaan Chlortetracycline (CTC). Prosedur pewarnaan CTC yang digunakan dalam pemeriksaan status akrosom dilakukan dengan cara yaitu 45 µl semen yang diperiksa dimasukkan ke dalam ependrof kapasitas 1,5 ml, tambahkan 45 µl larutan pewarna CTC kemudian goyang - goyang selama 2-3 menit, setelah itu Pengencer dengan bahan dasar air kelapa dengan penambahan kuning telur sebagai bahan krioprotektan, fruktosa sebagai substrat energi tambahan dan putih telur sebagai makromolekul diharapkan dapat mempertahankan kualitas dan status belum kapasitasi spermatozoa seoptimal mungkin. Berdasarkan uraian tersebut perlu dilakukan penelitian tentang kapasitasi 86 Muhammad et al./Jurnal Ilmu dan Teknologi Peternakan Tropis 6(1):85-91 Kualitas Semen Sapi PO Pemeriksaan semen sapi PO sebelum diencerkan meliputi pemeriksaan makroskopis dan mikroskopis. Pemeriksaan makroskopis meliputi pemeriksaan pH, warna, volume, dan konsistensi spermatozoa. Pemeriksaan mikroskopis terdiri dari pemeriksaan motilitas massa, motilitas individu, abnormalitas, viabilitas, konsentrasi, status akrosom spermatozoa. Rata-rata dan simpangan baku kualitas semen sapi PO sebelum dilakukan pengenceran dapat dilihat pada Tabel 1. HASIL DAN PEMBAHASAN ditambahkan 8 µl CTC Fixative dan digoyang lagi selama 1 menit. Dari larutan tersebut diambil 10 µl kemudian ditempatkan pada object glass kemudian ditambahkan 10 µl larutan 1,4- diazabicyclooctane (DABCO) kemudian dicampur rata secara hati-hati, setelah itu ditutup dengan cover glass. Tahap terakhir ditutup dengan tisu yang tebal kemudian ditekan-tekan secara hati-hati, tepi cover glass ditutup menggunakan cutex untuk melindungi preparat dari kerusakan (Morado et al., 2015). Kualitas Semen Sapi PO Spermatozoa Belum Kapasitasi Pemeriksaan status akrosom pada spermatozoa terdiri dari persentase spermatozoa belum kapasitasi, persentase spermatozoa terkapasitasi, dan persentase spermatozoa yang telah rekasi akrosom. Kondisi status akrosom spermatozoa sapi teridentifikasi salah satunya melalu perubahan peredaran kalsium pada sel spermatozoa yang bisa diamati menggunakan mikroskop epi-fluoresens dengan pewarnaan CTC. Hasil pengamatan spermatoza menggunakan mikroskop epi-fluoresens ditunjukkan dalam Gambar 1. Gambar 1. Status Akrosom Spermatozoa Diamati Dengan Mikroskop Epi-fluorescence. a = belum kapasitasi, b: spermatozoa terkapasitasi, c: spermatozoa telah rekasi akrosom. b a c Analisis Data Garner and Hafez (2008) bahwa Rata-rata persentase spermatozoa pada semen sapi PO yang belum mengalami kapasitasi secara berurutan yaitu 86,4±2,0%. Rata-rata persentase spermatozoa yang sudah mengalami kapasitasi yaitu 9,4±1,3%. Dan rata-rata presentase spermatozoa yang telah mengalami reaksi akrosom yaitu 4,2±1,0%. Ervandi, Susilawati, Wahyuningsih (2013) menyatakan bahwa semen sapi memiliki rata-rata 87 Muhammad et al./Jurnal Ilmu dan Teknologi Peternakan Tropis 6(1):85-91 maupun setelah pengenceran ditinjukkan pada Tabel 2. spermatozoa yang belum mengalami kapasitasi sebesar 87,5%, spermatozoa yang telah mengalami kapasitasi 9,4% dan telah mengalami reaksi akrosom 3,0%. Yeste, Briz, Pinart, Sancho, Garcia-Gil, Badia, Bassols, Pruneda, Bussalleu, Casas, Bonet (2008) menyatakan bahwa angka status akrosom semen sapi yaitu spermatozoa belum kapasitasi sekitar 85%, spermatozoa terkapasitasi sekitar 10%, dan spermatozoa telah mengalami reaksi akrosom sekitar 7%. Hasil analisis ragam dari persentase spermatozoa yang belum mengalami kapasitasi selama penyimpanan pada suhu dingin antar formulasi pengencer air kelapa yang berbeda (P1, P2, dan P3) menunjukkan tidak terdapat perbedaan yang nyata (P>0,05) pada peyimpanan hari ke-1 dan kari ke-5. Meskipun demikian, persentase spermatozoa yang belum mengalami kapasitasi tergolong tinggi dari semua perlakuan hingga penyimpananhari ke-5 yaitu di atas 60%. Spermatozoa dalam mempertahankan kondisi sehingga tidak mengalami kapasitasi sangat dipengaruhi oleh kolesterol membran. Moore et al. (2005) melaporkan bahwa kejadian penurunan yang signifikan pada kolesterol membran spermatozoa setelah dilakukan penurunan suhu yang sangat drastis. Kolesterol pada membran memiliki fungsi sebagai agen kapasitasi, jumlah dan distribusi kolesterol pada membran mengindikasikan tahapan-tahapan kapasitasi. Gualtieri (2010) menambahkan bahwa penipisan kolesterol membran spermatozoa menyebabkan peningkatan permeabilitas membran terhadap kalsium dan bikarbonat intraseluler yang pada akhirnya mengurangi persentase spermatozoa yang belum mengalami kapasitasi. Spermatozoa Terkapasitasi a c Kapasitasi adalah proses pelepasan bahan- bahan pelapis membran spermatozoa secara bertahap, terutama pada bagian akrosom (Susilawati, 2011). Persentase spermatozoa yang telah mengalami kapasitasi merupakan salah satu parameter yang perlu diperhatikan untuk menentukan kualitas spermatozoa (Gualtieri, 2010). Rata-rata persentase spermatozoa yang telah mengalami kapasitasi pada kondisi segar, setelah penyimpanan hari ke-1 dan ke-5 ditinjukkan pada Tabel 3. Gambar 1. Status Akrosom Spermatozoa Diamati Dengan Mikroskop Epi-fluorescence. a = belum kapasitasi, b: spermatozoa terkapasitasi, c: spermatozoa telah rekasi akrosom. Hasil analisis ragam terhadap persentase spermatozoa yang telah mengalami kapasitasi selama penyimpanan pada suhu dingin antar formulasi pengencer air kelapa yang berbeda (P1, P2, dan P3) juga menunjukkan Adapun rata-rata dan simpang baku spermatozoa yang belum mengalami kapasitasi pada semen sapi PO baik sebelum pengenceran Tabel 2. Rata-rata spermatozoa belum kapasitasi P Rata-rata spermatozoa belum kapasitasi (%) ± Simpang baku Segar H1 H5 P1 86,02 ± 2,63 83,7 ± 0,56 64,85 ± 0,70 P2 86,02 ± 2,63 83,9 ± 1,23 66,03 ± 0,91 P3 86,02 ± 2,63 83,6 ± 1,45 65,87 ± 0,22 Keterangan : P = Perlakuan, H = Hari penyimpanan. 88 Muhammad et al./Jurnal Ilmu dan Teknologi Peternakan Tropis 6(1):85-91 Hasil analisis ragam persentase spermatozoa yang telah mrngalami reaksi akrosom antar perlakuan formulasi pengencer air kelapa yang berbeda (P1, P2, P3) yang berbeda pada bangsa sapi PO selama simpan dingin pada suhu 4-5oC menunjukkan tidak terdapat perbedaan yang nyata (P >0,05) pada peyimpanan hari ke-1 dan hari ke-5. Persentase keutuhan akrosom tersebut mengindikasikan bahwa tidak terdapat kerusakan spermatozoa yang dapat menurunkan kualitas kemampuan fertilisasi dari spermatozoa selama penyimpanan menggunakan pengencer air kelapa. Hernandez et al. (2012) menyatakan bahwa spermatozoa dikatakan kehilangan kemampuan fertilisasinya apabila kerusakan akrosom melebihi 50%. Kerusakan akrosom spermatozoa tersebut berkaitan dengan fungsi membran sebagai pelindung akrosom spermatozoa. Guyton dan Hall (2007) menambahkan bahwa kolesterol merupakan salah satu komponen penyusun membran spermatozoa dan juga berfungsi memberikan sifat fluiditas pada membran sehingga tidak mudah rusak. Kandungan kolesterol tinggi pada membran akan membuat membran semakin bersifat lentur sebaliknya semakin rendah kandungan kolesterol pada membran akan menyebabkan spermatozoa semakin mudah mengalami kerusakan. tidak terdapat perbedaan yang nyata (P>0,05) pada peyimpanan hari ke-1 dan kari ke-5. Meskipun tidak menunjukkan perbedaan yang nyata antar perlakuan namun berdasarkan hasil pada tabel diketahui bahwa jumlah spermatozoa yang mengalami kapasitasi sebakin hari semakin meningkat. Moore et al. (2005); Guyton dan Hall (2007) menjelaskan terjadinya penurunan yang signifikan pada kolesterol membran spermatozoa setelah pengenceran dan pendinginan. Spermatozoa Terkapasitasi Kolesterol pada membran dapat berfungsi sebagai agen kapasitasi. Jumlah serta distribusi kolesterol membran spermatozoa mengindikasikan tahapan- tahapan kapasitasi. Penipisan kolesterol pada membran merupakan salah satu tahapan pertama dari kejadian kapasitasi yang ditandai oleh penurunan stabilitas membrane. Penipisan kolesterol membran dapat menyebabkan peningkatan kalsium intraseluler dan bikarbonat untuk mengaktifkan AC, menghasilkan cAMP, dan mengaktifkan PKA, selanjutnya protein kinase membuka kanal ion pada membran dan memodulasi peningkatan kalsium intraseluler. Spermatozoa Telah Reaksi Akrosom Rata-rata spermatozoa yang telah mengalami reaksi akrosom pada kondisi segar, setelah penyimpanan hari ke-1 dan ke-5 ditinjukkan pada Tabel 4. Tabel 3. Rata – rata spermatozoa terkapasitasi P Rata-rata spermatozoa belum kapasitasi (%) ± Simpang baku Segar H1 H5 P1 9,99 ± 2,63 10,71 ± 0,56 23,27 ± 0,70 P2 9,99 ± 2,63 10,90 ± 0,60 21,87 ± 0,35 P3 9,99 ± 2,63 10,79 ± 0,31 22,11 ± 1,06 Keterangan : P = Perlakuan, H = Hari penyimpanan. Tabel 3. Rata – rata spermatozoa terkapasitasi Tabel 4. Rata-rata spermatozoa telah reaksi akrosom P Rata-rata spermatozoa belum kapasitasi (%) ± Simpang baku Segar H1 H5 P1 3,99 ± 1,36 5,56 ± 0,20 11,80 ± 0,40 P2 3,99 ± 1,36 5,21 ± 0,64 12,11 ± 0,56 P3 3,99 ± 1,36 5,61 ± 1,14 12,02 ± 0,84 Keterangan : P = Pelakuan, H = Hari penyimpanan. DAFTAR PUSTAKA Guyton A.C. & J.E. Hall. 2007. Fisiologi Kedokteran. Edisi 11. Penerbit Buku Kedokteran EGC. Jakarta. Adnani Luh P.D.H, W. Bebas, & M.K. Budiasa. 2012. Penambahan bovine serum albumin pada pengencer kuning telur terhadap motilitas dan daya hidup spermatozoa anjing. Indonesia Medicus Veterinus 1(4):519-529. Hernandez P.J.E. R.F. Fernandez, S.J.L. Rodriguez, M.Y.G. Soto, J.E.H. Verona, & R.A.D. Garcia. 2012. Post-thaw acrosomal viability and reaction in sperm obtained from equine epididymis tail. Rev Salud Anim. 34(2):84-88. Audia R.P., M.A. Salim, N. Isnaini & T. Susilawati. 2017. Pengaruh perbedaan kematangan air kelapa hijau sebagai bahan pengencer yang ditambah 10% kuning telur terhadap kualitas semen cair kambing Boer. Jurnal Ternak Tropika 18(1):58-68. Ismaya. 2014. Bioteknologi Inseminasi Buatan pada Sapi dan Kerbau. Gadjah Mada University Press. Yogyakarta. Moore A.I., E.L. Squires, & J.K. Graham. 2005. Adding cholesterol to the stallion sperm plasma membrane improve cryosurvival. Cryobiology 51(3):241-249. Ax R., M. Dally, B. Didion, R. Lenz, C. Love, D. Varner, Hafez, & M. Bellin. 2008. Semen Evaluation. In Reproduction in Farm Animal (7th edition). Edited by Hafez, E.S.E. Co. Director. Reproductive Health Kiawah Island. South Carolina. USA. pp 365-370. Morado S., V. Pereyra, E. Breininger, R. Sara, & P. Cetica. (2015). Study of sperm evaluation parameters to estimate cryopreserved bovine semen fertility. Austin Journal of Veterinary Science & Animal Husbandry 2(1):1005. Daramola J.O, E.O. Adekunle, O.E. Oke, O.M. Onagbesan, I.K. Oyewusi, & J.A. Oyewusi. Effects of coconut (Cocos nucifera) water with or without egg-yolk on viability of cryopreserved buck spermatozoa. Anim. Reprod., Belo Horizonte 13(2):57-62. Muhammad D., T. Susilawati, & S. Wahjuningsih. 2016. Pengaruh penggunaan CEP-2 dengan suplementasi kuning telur terhadap kualitas spermatozoa sapi FH (Frisian Holstein) kualitas rendah selama penyimpanan suhu 4-5oC. Jurnal Ternak Tropika 17(1):66-76. Ducha N., T. Susilawati, Aulanni’am, & S. Wahjuningsih. 2013. Motilitas dan viabilitas spermatozoa sapi Limousin selama penyimpanan pada refrigerator dalam pengencer CEP-2 dengan suplementasi kuning telur. Jurnal Kedokteran Hewan 7(1):5-8. Mugiyati, M. A. Salin, N. Isnaini, & T. Susilawati. 2017. Pengaruh air kelapa merah yang muda dan tua sebagai pengencer terhadap kualitas semen kambing Boer selama penyimpanan dingin. Jurnal Ternak Tropika 18(1):20-26. Ervandi M., T. Susilawati, & S. Wahyuningsih. 2013. Effect of Different Diluents on the Quality of Sperm Sexing Cows With a Gradient Albumin (Egg White). Indonesian Journal of Animal and Veterinary 18 (3):177-184. Salim M.A., M.N. Ihsan, N. Isnaini, A.P.A. Yekti, & T. Susilawati. 2018. KESIMPULAN Kemenristek DIKTI yang telah memberikan dana penelitian dengan skema PUPTN dan Lembaga Pengelolah Dana Pendidikan (LPDP) melalui program BPI. Terima kasih juga kepada Loka Penelitian Sapi Potong Grati yang menyediakan tempat penelitian. Tidak terdapat perbedaan pada persentase spermatozoa belum kapasitasi, terkapasitasi, dan telah rekasi akrosom antara perlakuan formulasi pengencer air kelapa yang berbeda (P1, P2, P3) selama peyimpana suhu 2-5oC. Pengencer berbasis air kelapa mampu mempertahankan kualitas akrosom diatas 50% penyimpanan hari ke lima. 89 Muhammad et al./Jurnal Ilmu dan Teknologi Peternakan Tropis 6(1):85-91 DAFTAR PUSTAKA Quality of Boer goad liquid semen on different coconut water diluent (Cocos nucifera) during cold storage. Asian Journal of Microbiology, Biotechnology & Environmental Sciences 20:150-157. Garner D.L. & E.S.E. Hafez. 2008. Spermatozoa and Seminal Plasma. In Reproduction in Farm Animals (7th edition). Edited by E.S.E Hafez and B. Hafez. 2008. Lippincott & Williams. Baltimore, Marryland. USA. pp 96-109. Susilawati, T. 2011. Spermatology. Universitas Brawijaya (UB) Press. Malang. Susilawati T., D. Ratnawati, N. Isnaini, Kuswati & A. P. A. Yekti. 2018. Character of liquid semen motility in various diluents on Balinese cattle during cold storage. Asian Journal of Microbiology, Biotechnology & Environmental Sciences 20(1):166-172. Gualtieri R. 2010. Ability of Sulfated Glycoconjugates and Disulfide Reductants to Release Bovine Epididymal Sperm Bound to the Oviductal Epithelium In Vitro.Theriogenology 73:1037-1043. 90 Muhammad et al./Jurnal Ilmu dan Teknologi Peternakan Tropis 6(1):85-91 Vigliar R., V. L. Sdepanian, & U. Fagundes- Neto. 2006. Biochemical profile of coconut water from coconut palms planted in an Inland Region. Jornal de Pediatria. 82(4):308-312. Yeste, M., M. Briz, E. Pinart, S. Sancho, N. Garcia-Gil, E. Badia, J. Bassols, Pruneda, E. Bussalleu, I. Casas, & S. Bonet. 2008. Hyaluronic acid delays boar sperm capacitation after 3 days of storage at 15 oC. animal reproduction science 109:236- 250. Yong J.W.H., L. Ge, Y.F. Ng & S.N. Tan. 2009. The chemical composition and biological properties of coconut (Cocos nucifera L.) water. Molecules. 14: 5144-5164. 91
https://openalex.org/W2182824184	https://zenodo.org/record/8264973/files/IJBB-V1-No1-p27-33.pdf	English	null	In vivo study on the efficacy of hypoglycemic activity of Spirulina plantesis in long evan rats.	Zenodo (CERN European Organization for Nuclear Research)	2,011	cc-by	3,136	Nurunnahar Fayzunnessa1, M Alam Morshed1,2,3, Azim Uddin3, Anzana Parvin1, Rahman Saifur1 1Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia 7003, Bangladesh. 2Department of Biochemistry and Cell Biology, Bangladesh Institute of Health Sciences (BIHS), Darus Salam, Dhaka 1216, Bangladesh Received: 02 May 2011 Revised: 08 May 2011 Accepted: 09 May 2011 Corresponding author: M Alam Morshed  morshedbt@gmail.com In vivo study on the efficacy of hypoglycemic activity of Spirulina plantesis in long evan rats In vivo study on the efficacy of hypoglycemic activity of Spirulina plantesis in long evan rats Nurunnahar Fayzunnessa1, M Alam Morshed1,2,3, Azim Uddin3, Anzana Parvin1, Rahman Saifur1 Introduction Spirulina, a blue-green alga, is now becoming a health food worldwide. It is a multicellular, filamentous cyanobacterium belonging to algae of the class Cyanophyta (Anitha et al., 2006). The United Nations world food conference declared spirulina as “the best for tomorrow”, and it is gaining popularity in recent years as a food supplement (Kapoor et al., 1993). The spirulina ability as a potent anti-viral (Hayashi et al., 1996; Hayashi et al, 1993; Patterson, 1993; Gustafson et al. 1989), anti–cancer (Babu et al. 1995; Lisheng et al. 1991; Qishen et al. 1988; Schwartz et al. 1986; Schwartz, 1988), hypocholesterolemic (Nakaya et al., 1988; Kato et al., 1984) and health improvement (Annapurna et al., 1991) agent is gaining attention as a nutraceutical and a source of potential pharmaceutical. Diabetes mellitus is a disease due to abnormality of carbohydrate metabolism and it is mainly linked with low blood insulin level or insensitivity of target organs to insulin. It is the most prevalent chronic disease in the world affecting nearly 25% of the population (Ghosh et al., 2001). Hyperglycemia is an important character of diabetes mellitus, an endocrine disorder based disease. In modern medicine, no satisfactory effective therapy is still available to cure diabetes mellitus (Berger, 1985). Though pharmaceutic drugs like sulfonylureas and biguanides are used for the treatment of diabetes but these are either too expensive or have undesirable side effects or contraindications (Berger, 1985; Rang, 1991). In recent years, there has been renewed interest in plant medicine (Dubey, 1994; Prince, 1998; Ladeji, 2003) for the treatment against different diseases as herbal drugs are generally out of toxic effect (Geetha, 1994; Rao, 2003) reported from research work conducted on experimental model animal. Although in human, whether there is any toxic effect are not investigated, isolated studies screened various plants having “folk medicine reputation” by biochemical test for this for antidiabetogenic effect (Vats, 2002) Diabetes mellitus, a metabolic disorder, is becoming a major health problem. Many groups have reported on the antidiabetic activity of Spirulina platensis (Anuradha et al., 2001; Anitha et al., 2006). Despite that, its hypoglycemic potential regarding to its mode of action remains largely untapped owing to the inadequate documentation and validation of its activity against known oral hypoglycemic drugs. In this regard the present study was undertaken to evaluate the hypoglycaemic activity of Spirulina platensis in the animal model. Abstract The ethanol extract of Spirulina platensis was investigated for antihyperglycemic effects in Long Evans rats. Three tests were carried out to assess these activities. The extract caused a dose dependent inhibition of glucose absorption and showed hypoglycemic effects at rats weighing from 110 – 150 gram. The anti-diabetic effects were estimated by measuring the amount of glucose in the samples collected after the experiment. The extract at a dose level of 250mg/kg showed significant result (p<0.05) at 15 minutes and the dose level of 500mg/kg showed significant efficacy (p<0.05) at 10 and 15 minutes and the glucose absorption rates were 38.94±0.21, 34.99±1.91 and 40.86±0.07 respectively. The present study explored the extra pancreatic action of the plant in Long Evans rats. This study suggests that ethanol extract of Spirulina platensis has anti- diabetic effects in a dose dependant manner and these may be effective in the treatment of diabetes. Corresponding author: M Alam Morshed  morshedbt@gmail.com 27 \| Fayzunnessa et al. Introduction In the present study we explored the extra pancreatic action of the cyanobacterium in Long Evans rats. Spirulina is a microscopic blue-green aquatic plant and it is the nature‟s richest and most complete source of organic nutrition. The concentrated nutritional profile of Spirulina occurs naturally, so it is ideal for those preferring a whole food supplement to artificial nutrient sources. Spirulina, the blue-green alga, has a unique blend of nutrients that no single source can provide. It contains a wide spectrum of nutrients that include B-complex vitamins, minerals, good quality proteins, gamma-linolenic acid and the super antioxidants, beta-carotene, vitamin E and trace elements. Spirulina is fast emerging as a whole answer to the varied demands due to its impressive nutrient composition which can be used for therapeutic uses (Venkataraman, 1998). Plant materials and preparation of test samples The powder of Spirulina platensis was collected from Science Laboratory, Dhaka, Bangladesh. The powder was then soaked in 80% ethanol. These suspensions were filtered with thin and clean cloth and then filtered by filter paper. The suspensions were evaporated by Rotary evaporator (Bibby RE-200, Sterilin Ltd., UK) at 680C. In this case, 175mbar (to remove ethanol), 72mbar (to remove water) pressure 28 Fayzunnessa et al. hydrolyze the sucrose and then neutralized with NaOH. The blood glucose level and the amount of glucose liberated from residual sucrose in the gastrointestinal tract were measured. Then the gastrointestinal sucrose content was calculated from the amount of liberated glucose (Goto et al 1995). Glucose was measured by glucose-oxidase (GOD-PAP) method. and 160rpm rotation speed were maintained constantly. Finally, freeze-drier (HETOSICC, Heto Lab Equipment, Denmark) was used to get complete extract from the gummy extract and preserved at +4ºc. Experimental animals The study was conducted with adult (both sex) Long- Evans rats (weighing 110±150g). They were bred at the BIRDEM animal house and maintained at a constant room temperature of 22±50C, 40-70% humidity conditions and the natural day-night cycle with an ad libitum access to food except the day of experimental procedure when animals were used after 12hrs fasting. The influence of circadian rhythms was avoided by starting all experiments at 8.30 a.m. Effects on intestinal glucose absorption An intestinal perfusion technique (Swintosky and Pogonowska-Wala, 1982) was used to study the effects of Spirulina platensis extracts on intestinal absorption of glucose in rats fasted for 36 hours and anesthetized with sodium pentobarbital (50 mg/kg). The plant extracts were added to a kreb‟s solution (g/L 1.02 CaCl2, 7.37 NaCl, 0.20 KCl, 0.065 NaH2PO4.6H2O, 0.6 NaHCO3, pH 7.4), supplemented with glucose (54.0 g/L) and perfused at a perfusion rate of 0.5 mL/min for 30 min through the duodenum. The perfusate was collected from a catheter set at 40 cm. The system was set at a constant temperature of 37 0C. Spirulina platensis extracts were added to Kreb‟s solution to a final conc. of 5mg/ml and 10mg/ml, so that the amount of extract in the perfused intestine is equivalent to the dose of 250 g/kg and 500g/kg body weight respectively. The control group was perfused only with Kreb‟s buffer supplemented with glucose. The results were expressed as percentage of absorbed glucose, calculated from the amount of glucose in solution before and after the perfusion. Effects on intestinal glucose absorption An intestinal perfusion technique (Swintosky and Pogonowska-Wala, 1982) was used to study the effects of Spirulina platensis extracts on intestinal absorption of glucose in rats fasted for 36 hours and anesthetized with sodium pentobarbital (50 mg/kg). Biochemical procedure Biochemical procedure Serum glucose levels were estimated by glucose oxidase (GOD/POD) method (Sera Pak, USA). The absorbance was measured by microplate ELISA Reader (Bio-Tek EL-340, USA). Effect on sucrose absorption from gastrointestinal tract Experiments were carried out on normal rats. Extracts of Spirulina platensis were fed to the rats by using a syringe (3ml) with a metallic tube that was smooth and curved at the end, which led the feed directly to the stomach. Rats were fasted for 12 h before receiving a 50% sucrose solution by gavage (2·5 g/kg/5ml body weight) with (for experimental) or without (for control) ethanolic extract of Spirulina platensis (0·5 g/kg body weight). Blood samples were collected by amputation of the tail tip under mild diethyl ether anesthesia (Mamun et al, 2001). Blood samples were collected at 30 min before sucrose load and at 30, 60, 180 and 360 min after sucrose administration to determine the glucose level. Finally rats were sacrificed to collect the gastrointestinal tract. The gastrointestinal tract was excised and divided into 6 segments: the stomach, the upper 20 cm, middle, and lower 20 cm of the small intestine, the cecum, and the large intestine. Each segment was washed out with ice-cold saline, acidified with H2SO4 and centrifuged at 3000 rpm (1000 g) for 10 min. The supernatant thus obtained was boiled for 2 h to Statistical analysis Data from the experiments were analyzed using the Statistical Package for Social Science(SPSS) software 29 Fayzunnessa et al. for windows version 17 (SPSS Inc., Chicago, Illinois, USA). All the data were expressed as Mean ± SD. Statistical analysis of the results were performed by using one-way analysis of variance (ANOVA) followed by Dunnett's t-test for comparisons. The limit of significance was set at p<0.05. gradually decreased from 20 min to 30 min. The extract at a dose level of 250mg/kg showed significant result at 15 minutes (p<0.05) and the dose level of 500mg/kg showed significant efficacy at 10 and 15 minutes (p<0.05). 0 10 20 30 40 50 60 05 min 10 min 15 min 20 min 25 min 30 min Time (Minute) % Absorption of glucose Control Ext_250 Ext_500 Fig. 2. The effect of Spirulina platensis on intestinal glucose absorption with respect to time compared to control rats. 0 10 20 30 40 50 60 05 min 10 min 15 min 20 min 25 min 30 min Time (Minute) % Absorption of glucose Control Ext_250 Ext_500 Effect on sucrose absorption from gastrointestinal tract The six-segment study was performed to assess the amount of sucrose remaining in the GIT at six different positions. The various data for sucrose absorption in the gastrointestinal tract are presented in the Fig. 1. The amount sucrose unabsorbed in different GIT segments showed that in control rats vs. rats fed with 500mg/kg extract at 30, 60, 180, and 360 minutes in mmol/l were 0.120 vs. 0.149, 0.038 vs. 0.057, 0.003 vs. 0.003 and 0.004 vs. 0.085 respectively. Fig. 2. The effect of Spirulina platensis on intestinal glucose absorption with respect to time compared to control rats. Discussion 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 30 min 60 min 180 min 360 min Time Unabsorbed Sucrose (mmol/l) control extract 500mg/kg 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 30 min 60 min 180 min 360 min Time Unabsorbed Sucrose (mmol/l) control extract 500mg/kg Fig. 1. Graph comparing the total sucrose content in the whole gastrointestinal tract at 30 minutes, 60 minutes, 180minutes, and 360minutes in a group of control rats vs. rats given a gavage with Spirulina platensis extract 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 30 min 60 min 180 min 360 min Time Unabsorbed Sucrose (mmol/l) control extract 500mg/kg Hypoglycemic activity that is found when given with a simultaneous glucose load in diabetic rats indicates that the extracts may interfere with the intestinal glucose absorption in the gut by various mechanisms (Nahar et al., 2000; Vinik and Wing, 1990; Lempcke, 1987). It may be postulated that the plant extract might stimulate glycogenesis in the liver, which is enhanced by feeding (Creutzfeld et al., 1979). Time One of the objectives of the present study was to investigate whether the antihyperglycemic effect is related to the inhibition of carbohydrate absorption in the gut. In order to confirm this hypothesis, we examined sucrose content in six segments of the rat gastrointestinal tract after simultaneous administration of sucrose. The extract of Spirulina platensis suppressed postprandial hyperglycemia after sucrose ingestion till 30 minutes in the stomach and upper 20cm of the small intestine and all throughout the small intestine till 60 minutes. The extract increased the residual sucrose content at these times after sucrose administration. After 180 min, Fig. 1. Graph comparing the total sucrose content in the whole gastrointestinal tract at 30 minutes, 60 minutes, 180minutes, and 360minutes in a group of control rats vs. rats given a gavage with Spirulina platensis extract. Fig. 1. Graph comparing the total sucrose content in the whole gastrointestinal tract at 30 minutes, 60 minutes, 180minutes, and 360minutes in a group of control rats vs. rats given a gavage with Spirulina platensis extract. Results Effect on sucrose absorption from gastrointestinal tract Effect on intestinal glucose absorption As shown in Fig. 2, intestinal glucose absorption in non-diabetic control rats was gradually increased during 30 min of perfusion. Addition of Spirulina platensis to the glucose perfusate resulted an increase in intestinal glucose absorption at 10 min which were 30 Fayzunnessa et al. of selected diabetic patients. Indian J Nutr Dietet 38, 40-44. the concentration of sucrose detected throughout the gut indicated that most of the sucrose was absorbed. This result suggests that the extract can delay sucrose absorption without completely inhibiting it, which might be due to inhibition on rapid digestion and absorption of carbohydrate at the upper part of intestine and undigested carbohydrate is digested and absorbed after 180 min. These findings suggest that the reduction of hyperglycemia by the extract is, at least partly, related to the retardation of carbohydrate absorption in the gut. This was also confirmed in gut perfusion experiment, where the extract reduced intestinal glucose absorption, especially around 5 and 15 minutes, when the dose was administered at 250mg/kg body weight and around 5, 15, and 20 minutes, when the dose was administered at 500mg/kg body weight. Babu M. 1995. Evaluation of chemoprevention of oral cancer with Spirulina fusiformis. Nutr Cancer 24,197-202. Berger W. 1985. Incidence of severe side effects during therapy with sulphonylureas and biguanides. Hormones Metabolic Res.17, 111-5. Berger W. 1985. Incidence of severe side effects during therapy with sulphonylureas and biguanides. Hormones Metabolic Res.17, 111-5. Creutzfeld W. 1979. The incretin concept today. Diabetologia 16, 75-85. Dubey G.P., Dixit S.P., Alok S. 1994. Alloxan- induced diabetes in rabbits and effect of herbal formulation D-400. Indian Journal of Pharmacology 26, 225-226. In conclusion, the present study demonstrated that the extract can delay sucrose absorption without completely inhibiting it in the intestine. The present study has evaluated potential anti-diabetic activity of Spirulina platensis Geetha BS., Biju CM., Augusti KT. 1994. Hypoglycemic effect of leucodelphidin derivative isolated from Ficus bengalensis (Linn.). Indian Journal of Pharmacology 38, 220-222. Ghosh S, Suryawanshi SA. 2001. Effect of Vinca rosea extracts in treatment of alloxan diabetes in male albino rats. Indian Journal of Experimental Biology 39, 748-759. Acknowledgement We would like to show our gratitude to all of the stuffs of the Department of Pharmacology, BIRDEM, Dhaka, Bangladesh. Goto Y, Yamada K, Ohyama T, Matsuo T, Odaka H, Ikeda H. 1995. An alpha-glucosidase inhibitor, AO-128, retards carbohydrate absorption in rats and humans. Diabetes Res Clin Pract 28, 81–87. References Anitha Layam, Chandra Lekha Kasi Reddy. 2006. Antidiabetic property of Spirulina. Diabetologia Croatica 35(2), 29-33. Gustafson K. 1989. AIDS antiviral sulfolipids from cyanobacteria (blue-green algae). J Natl Cancer Inst 81,1254. Annapurna V, Shah N, Bhaskaran P, Bamji SM, Reddy V. 1991. Bioavailability of spirulina carotenes in preschool children. J Clin Biochem Nutr 10, 145-151. Hayashi K. 1993. An extract from Spirulina platensis is a selective inhibitor of herpes simplex virus type 1 penetration into cells. Phytother Res 7, 76-80 Anuradha V, Vidhya D. 2001. Impact of administration of spirulina on the blood glucose levels 31 Fayzunnessa et al. Hayashi T, Hayashi K. 1996. Calcium spirulan, an inhibitor of enveloped virus replication, from a bluegreen alga Spirulina platensis. J Nat Prod 59, 83- 87. Nakaya N, Homma Y, Goto Y. 1988. Cholesterol lowering effect of spirulina. Nutr Rep Int 37, 1329- 1337. Patterson RW. 1993. Antiviral activity of blue- green cultures. J Phycol 29, 125-130. Kapoor R, Mehta U. 1993. Effect of supplementation of blue green algae on outcome of pregnancy of rats. Plants Food Hum Nutr 43, 131-148. Prince PS, Menon VP, Pari L. 1998. Hypoglycemic activity of Syzigium cuminii seeds: effect on lipid peroxidation in alloxan diabetes rats. Journal of Ethnopharmacology 61, 1-7. Kato T, Takemoto K, Katayama K, Kuwahara Y. 1984. Effect of Spirulina platensis to alimentary hypercholesterolemia in rat. Jpn Nat Food Assoc J 37, 323. Qishen P. 1988. Enhancement of endonuclease activity and repair DNA synthesis by polysaccharide of Spirulina platensis. Acta Genet Sinica (Chin J Genet) 15, 374-381. Ladeji O., Omekarah I., Solomon M. 2003. Hypoglycemic properties of aqueous bark extract of Ceiba pentandra in streptozotocin-induced diabetic rats. Journal of Ethnopharmacology 84, 139-142. Rang HP, Dale MM, Rittar JM. 1991. The endocrine system Pharmacology. In: Pharmacology. Longman Group Ltd., UK. 504-508. Lempcke B. 1987. Control of absorption: Delaying absorption as a therapeutic principle. In: Structure and function of the Z small Intestine. Cospary WF (ed). New York, Elsevier, pp 263-80 Rao BK, Sudarshan PR, Rajasekhar MD, Nagaraju N, Rao CA. 2003. Antidiabetic activity of Terminalia pallida fruit in alloxan-induced diabetic rats. Journal of Ethnopharmacology 85, 169-172. Lisheng L. 1991. Inhibitive effect and mechanism of polysaccharide of Spirulina platensis on transplanted tumour cells in mice. Marine Sci. 5, 33-38. Lisheng L. 1991. Inhibitive effect and mechanism of polysaccharide of Spirulina platensis on transplanted tumour cells in mice. Marine Sci. 5, 33-38. Schwartz J. 1986. Inhibition of experimental oral carcinogenesis by tropical beta carotene. Carcinogenesis 7, 711-715. Mamun MIR, Rokeya B, Choudhury NS, Muniruzzaman M, Nahar N, Ahmed MU, Mosihuzzaman M, Ali L, Khan AKA, Khan SH. 2001. Anti-hyperglycemic effect of Pterospermum acerifolium Wild and Pterospermum semisagittatum Ham. Diabetes Res. 35, 163-70. Schwartz J, Shklar G. 1988. Prevention of experimental oral cancer by extracts of Spirulina – Duraliella algae. Nutr. Cancer 11, 127-134. Swintosky JV, Pogonowskawala E. 1982. The in-situ rat gut technique. Pharm Int. 3, 163–167. Nahar N, Rokeya B, Ali L, Hassan Z, Nur-e- Alam M, Choudhury NS, Khan AKA, Mosihuzzaman M. 2000. Effect of three medicinal plants on blood glucose levels in nondiabetic and diabetic model rats. Diabetes Res. 35, 41-49. Vats V., Grover JK., Rathi SS. 2002. Evaluation of antihyperglycemic effect of Trigonella foenum- graecum Linn, Occium sanctum Linn. and Pterocarpus marsupium Linn., in normal and 32 \| Fayzunnessa et al. 32 of Venkataraman LV. 1998. Spirulina: global reach of a health care product. Souvenir, IFCON „98, 4th International Food Convention pp175. Venkataraman LV. 1998. Spirulina: global reach of a health care product. 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https://openalex.org/W3079127461	https://www.frontiersin.org/articles/10.3389/fphar.2020.01249/pdf	English	null	Resveratrol Modulates the Gut Microbiota and Inflammation to Protect Against Diabetic Nephropathy in Mice	Frontiers in pharmacology	2,020	cc-by	9,843	Citation: Cai T-T, Ye X-L, Li R-R, Chen H, Wang Y-Y, Yong H-J, Pan M-L, Lu W, Tang Y, Miao H, Snijders AM, Mao J-H, Liu X-Y, Lu Y-B and Ding D-F (2020) Resveratrol Modulates the Gut Microbiota and Inﬂammation to Protect Against Diabetic Nephropathy in Mice. Front. Pharmacol. 11:1249. doi: 10.3389/fphar.2020.01249 Resveratrol Modulates the Gut Microbiota and Inﬂammation to Protect Against Diabetic Nephropathy in Mice Ting-Ting Cai 1†, Xiao-Long Ye 1†, Ru-Run Li 1, Hui Chen 1, Ya-Yun Wang 1, Hui-Juan Yong 1, Ming-Lin Pan 1, Wei Lu 1, Ying Tang 1, Heng Miao 1, Antoine M. Snijders 2, Jian-Hua Mao 2, Xing-Yin Liu 3, Yi-Bing Lu 1* and Da-Fa Ding 1* Keywords: diabetic nephropathy, resveratrol, gut microbiome, inﬂammation, gut–kidney axis Edited by: Peng Chen doi: 10.3389/fphar.2020.01249 Specialty section: This article was submitted to Gastrointestinal and Hepatic Pharmacology, a section of the journal Frontiers in Pharmacology Specialty section: This article was submitted to Gastrointestinal and Hepatic Pharmacology, a section of the journal Frontiers in Pharmacology Received: 13 January 2020 Accepted: 29 July 2020 Published: 19 August 2020 Received: 13 January 2020 Accepted: 29 July 2020 Published: 19 August 2020 ORIGINAL RESEARCH published: 19 August 2020 doi: 10.3389/fphar.2020.01249 1 Department of Endocrinology, The Second Afﬁliated Hospital of Nanjing Medical University, Nanjing, China, 2 Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States, 3 Department of Pathogen Biology-Microbiology Division, Key Laboratory of Pathogen of Jiangsu Province, Nanjing Medical University, Nanjing, China Edited by: Peng Chen Edited by: Peng Chen, Southern Medical University, China Reviewed by: Yi Duan, University of California, San Diego, United States Liwei Xie, Guangdong Academy of Science, China Edited by: Peng Chen, Southern Medical University, China Reviewed by: Yi Duan, University of California, San Diego, United States Liwei Xie, Guangdong Academy of Science, China Oral administration of resveratrol is able to ameliorate the progression of diabetic nephropathy (DN); however, its mechanisms of action remain unclear. Recent evidence suggested that the gut microbiota is involved in the metabolism therapeutics. In the current study, we sought to determine whether the anti-DN effects of resveratrol are mediated through modulation of the gut microbiota using the genetic db/db mouse model of DN. We demonstrate that resveratrol treatment of db/db mice relieves a series of clinical indicators of DN. We then show that resveratrol improves intestinal barrier function and ameliorates intestinal permeability and inﬂammation. The composition of the gut microbiome was signiﬁcantly altered in db/db mice compared to control db/m mice. Dysbiosis in db/db mice characterized by low abundance levels of Bacteroides, Alistipes, Rikenella, Odoribacter, Parabacteroides, and Alloprevotella genera were reversed by resveratrol treatment, suggesting a potential role for the microbiome in DN progression. Furthermore, fecal microbiota transplantation, derived from healthy resveratrol-treated db/m mice, was sufﬁcient to antagonize the renal dysfunction, rebalance the gut microbiome and improve intestinal permeability and inﬂammation in recipient db/db mice. These results indicate that resveratrol-mediated changes in the gut microbiome may play an important role in the mechanism of action of resveratrol, which provides supporting evidence for the gut–kidney axis in DN. Correspondence: Da-Fa Ding dingdafa@njmu.edu.cn Yi-Bing Lu luyibing2004@126.com †These authors have contributed equally to this work Correspondence: Da-Fa Ding dingdafa@njmu.edu.cn Yi-Bing Lu luyibing2004@126.com †These authors have contributed equally to this work Specialty section: This article was submitted to Gastrointestinal and Hepatic Pharmacology, a section of the journal Frontiers in Pharmacology Received: 13 January 2020 Accepted: 29 July 2020 Published: 19 August 2020 Citation: Cai T-T, Ye X-L, Li R-R, Chen H, Wang Y-Y, Yong H-J, Pan M-L, Lu W, Tang Y, Miao H, Snijders AM, Mao J-H, Liu X-Y, Lu Y-B and Ding D-F (2020) Resveratrol Modulates the Gut Microbiota and Inﬂammation to Protect Against Diabetic Nephropathy in Mice. Front. Pharmacol. 11:1249. INTRODUCTION Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. Several molecular mechanisms are involved in the pathophysiology of DN, including oxidative stress, the renin‐angiotensin system, transforming growth factor‐b (TGF‐b) activation, and the inﬂammatory response (Arora and Singh, 2013). The August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org Resveratrol Modulates the Gut Microbiota Cai et al. group were set up housing two to three mice per cage. The db/m+resveratrol and db/db+resveratrol mice were treated with resveratrol by oral gavage at a dose of 10 mg/kg/day for 12 weeks. The db/m and db/db groups received an equivalent amount of saline by oral gavage for the same period. The dosage was adjusted for body weight changes every week for the entire study period. During the experiment, all mice was received the same standard chow. Fasting blood sugar level (FBS) was measured by a glucometer (Abbot, Alameda, CA, USA) every 2 weeks in all animals. A BP-2000 blood pressure analysis system (Visitech Systems, Apex, NC) was used to assess systolic blood pressure (SBP) by tail cuff plethysmography in conscious mice. SBP was measured 10 times per day for 4 consecutive days, and a mean value was calculated for each mouse. Urine was collected from the metabolic cage after housing individual mouse for 24-h and was stored at -80°C until analysis. At the end of the experiment, blood samples were collected by the retro-orbital plexus for biochemical assays. Kidneys, intestine and fecal samples were also collected.All animal experiments were conducted in accordance with the committee guidelines of the Nanjing Medical University and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University, No.14030134. inﬂammatory response is strongly correlated with the onset and progress of renal damage in diabetic subjects (Navarro-Gonzalez et al., 2011), but its precise role in the progression of DN is still obscure. In recent years, the modiﬁcation of gut microbiota (GM) and activation of inﬂammation pathways have been implicated in the development of DN, with the conception of a gut–kidney axis being proposed, although the causative relationship remains to be elucidated (Yang et al., 2018). Indeed, the GM has been identiﬁed as a promoter or mediator of systemic inﬂammation. Dysbiosis,intestinalbarrierdysfunction,andbacterialtranslocation can cause inﬂammation in chronic kidney disease (CKD) (Andersen et al., 2017). Biochemical Analyses and Cytokine Measurements The serum levels of creatinine (SCr) and blood urea nitrogen (BUN) were assessed using an automated biochemical analyzer (Hitachi 7060, Tokyo, Japan). In order to evaluate the level of inﬂammation in mice, ELISA was used to measure serum levels of endotoxin lipopolysaccharide (LPS), pro-inﬂammatory cytokines interleukin(IL)-6 and interleukin (IL)-1b, tumor necrosis factor (TNF)-a, and interferon (IFN)-g), according to the manufacturer’s instructions (ELISA, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Twenty-four-hour microalbuminuria was also assessed by ELISA, according to the manufacturer’s instructions. INTRODUCTION Therefore, the onset of early kidney complications associated with diabetes may be prevented by modulating the GM. Resveratrol, a stilbene polyphenolic compound, is widely present in grapes, mulberries, red wines and peanut skins, which has been studied in various animal model of human diseases, such as diabetes and coronary heart disease (Baur et al., 2006; Singh et al., 2015). Resveratrol can inhibit oxidative stress and has anti-inﬂammatory and anti-apoptotic properties (Madeo et al., 2019). Increased research attention in the ﬁeld of DN has focused on its potential value in protecting kidney damage. Our previous studies in diabetic mouse models have indicated that orally administered resveratrol can have nephroprotective effects (Ding et al., 2010; Huang et al., 2017). However, the pathophysiologic mechanisms underlying the effects of resveratrol on DN are still obscure. Owing to the low bioavailability of resveratrol, it has been postulated that a mechanism of resveratrol efﬁcacy is mediated through its interaction with the gut. In fact, several mechanisms have been proposed by which resveratrol can exert its health effects through modulating the composition of the gut microbiota (Bird et al., 2017; Komaroff, 2017). It has been reported that ingestion of resveratrol induced changes in the gut microbiome composition and improvements in glucose homeostasis of obese and diabetic mice (Sung et al., 2017b; Chaplin et al., 2018). However, the effect of resveratrol on the GM and the development of DN remains largely unclear. Histopathological Analysis and Immunohistochemistry In the present study we investigate whether resveratrol exerts anti-DN effects by modulating the composition of gut microbiome, intestinal barrier dysfunction, and the inﬂammatory response. y Kidney and small intestine tissue samples were sent to the Pathology laboratory of The Second Afﬁliated Hospital of Nanjing Medical University for H&E or PAS staining. For immunohistochemical staining, sections were deparafﬁnized, heated in citrate-buffered solution (pH 6.0) for antigen retrieval, and incubated with anti-mouse primary antibody against IFN-g (Abcam 175878, USA), TNF-a (Abcam ab6671, USA), ZO-1 protein (Invitrogen, 61-7300 Shanghai, China), or Claudin-1 protein (Invitrogen, 32-5600 Shanghai, China) overnight at 4°C. Sections were incubated with biotinylated secondary antibodies, followed by adding diaminobenzidine tetrahydrochloride substrate to visualize the antibody staining. A pathologist reviewed kidney histology and staining in a blinded manner. Histological scoring was performed by measuring intestine damage as previously described (Maslowski et al., 2009). Fecal Microbiota Transplantation p Fresh fecal matter was collected from mice treated with resveratrol or control db/m mice as the donor of fecal microbiota transplantation (FMT). Meanwhile, the fresh transplant material was prepared on the same day of FMT within 10 min before oral gavage to prevent changes in bacterial composition. A separate cohort of male db/db mice (8 weeks old, n=16) underwent adaptive feeding for 1 week, then received a cocktail of antibiotics in their drinking water for three consecutive days (vancomycin, 100 mg/kg; neomycin sulfate, 200 mg/kg; metronidazole, 200 mg/kg; and ampicillin, 200 mg/kg). After completion of the antibiotics treatment, mice were fasted overnight (day 0) and the fasting blood glucose (FBS) and the systolic blood pressure (SBP) were measured. Then mice were randomly assigned to two groups, one group of mice received FMT by using a fecal suspension from donor db/m mice (C, control-FMT, n = 8), the other group received FMT by using a fecal suspension from donor db/m+resveratrol mice (R, resveratrol-FMT, n = 8) by oral gavage (200 ml; each FMT dose was prepared from an average weight of 40 mg of fecal matter, equivalent to 2 fresh fecal pellets from the donor). FMT of db/db mice were repeated six additional times on consecutive days (without fasting) for a total of seven FMT per mouse. During this time, all recipient mice continued to have access to The 16S sequences were analyzed using a combination of software mothur (version 1.33.3),UPARSE (usearch version v8.1.1756, http://drive5.com/uparse/), and R (version 3.6.1). A principal component analysis (PCA) using the package ade4 implemented in R software (R 3.6.1) was performed. The a- diversity of gut microbiota and the community composition of the samples was drawn using R. The statistical signiﬁcance of differences in bacterial composition among the different samples was assessed by either the Wilcox or the Kruskal-Wallis test. Intestinal Permeability standard chow. The changes in body weight and blood sugar were recorded daily for four weeks after FMT treatment. After treatment, the recipient mice were euthanized and blood, intestines and kidneys were collected as described above. Intestinal permeability was determined by measuring plasma levels of ﬂuoresceinisothiocyanate conjugated-dextran (FD-4). Mice were orally gavaged with 0.5 ml of FD-4 (22 mg/ml, molecular mass 4.4 kDa) 5 h prior to euthanasia. Plasma was harvested at the time of euthanasia and 50ml of plasma was diluted with an equivalent volume of phosphate-buffered saline (PBS pH 7.4), and the concentration of FD-4 was measured spectroﬂuorometrically (NanoDrop 3300, Thermo Scientiﬁc, Wilmington, DE) as described previously (Ding et al., 2010). Serially diluted samples were used as standards, and all samples were analyzed in triplicate. Data Collection, 16S rRNA Sequencing, and Quality Control y The DNA was extracted from 200 mg samples using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Isolated DNA was stored at −20˚C for further study. The V4–V5 region of 16S rRNA gene was ampliﬁed in each sample using universal primers 515F(5’- GTGCCAGCMGCCGCGGTAA-3’) and 926R (5’-CCGT CAATTCMTTTGAGTTT-3’). The PCR products from different samples were indexed and mixed at equal ratios for sequencing by 2300 bp paired-end sequencing on the MiSeq platform using MiSeq v4 Reagent Kit (Illumina) at TinyGen Bio- Tech (Shanghai) Co., Ltd. Gut Microbiota Analysis The raw fastq ﬁles were demultiplexed and pair-end reads for all samples were processed through Trimmomatic (version 0.35) to remove low quality base pairs using the following parameters: SLIDINGWINDOW: 50:20 MINLEN: 50. Trimmed reads were then further merged using the FLASH program (version 1.2.11) with default parameters. The low quality contigs were removed based on screen.seqs command using the following ﬁltering parameters, maxambig=0, minlength = 200, maxlength =580, maxhomop= 8. After removing low-quality reads a total of 733,481 high-quality reads remained (range from 17,557 to 38,441 reads per sample). The demultiplexed reads were clustered into 510 operational taxonomic units (OTUs) at 97% sequence identity using the UPARSE pipeline, which were then assigned for taxonomy against the Silva 119 database by the “classify.seqs” command in mothur with a conﬁdence score ≥ 0.8. OTU taxonomies (from Phylum to Species) were determined based on NCBI. Based on the results of the optimized sequence OTUs, clustering analysis, a variety of diversity index analysis (alpha diversity analysis in the sample) and detection of sequencing depth was performed. Statistical analysis of community structure at each classiﬁcation level and beta diversity analysis between samples were also performed. IL-1, forward: 5′-TCTTTGAAGTTGACGGACCC-3′, reverse: 5′- TGAGTGATACTGCCTGCCTG-3′; TNF-a, forward: 5′-GAAGTTCCCAAATGGCCTCC-3′ reverse: 5′-GTGAGGGTCTGGGCCATAGA-3′; IFN-g, forward: 5′-TGTCATCGAATCGCACCTGA-3′ reverse: 5′-TCAGCACCGACTCCTTTTCC-3′; Total RNA was isolated from mice kidneys with TRIzol (Invitrogen, Carlsbad, CA) reagent according to the manufacturer’s instructions. Gene expression was calculated as △CT using GAPDH as a reference, and was expressed relative to the control group normalized to a value of 1. Total RNA was isolated from mice kidneys with TRIzol (Invitrogen, Carlsbad, CA) reagent according to the manufacturer’s instructions. Gene expression was calculated as △CT using GAPDH as a reference, and was expressed relative to the control group normalized to a value of 1. Animal Experiments Animal Experiments The Male C57BL/KsJ diabetic db/db and db/m mice were used in this study. Db/db mice are a genetic model of early stage type 2 DN with hyperglycemia and urinary albumin excretion enhancement, while db/m mice served as the control (Madeo et al., 2019). The mice were housed in well-ventilated plastic cages with stainless steel grid tops at 22 ± 2°C with a 12 h light/ dark cycle. At 8 weeks of age, the mice were randomly assigned into four groups (db/m, n=6; db/m+resveratrol, n=7; db/db, n=6; and db/db+resveratrol, n=7). Multiple cages for each treatment August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 2 Resveratrol Modulates the Gut Microbiota Cai et al. Quantitative Real-Time PCR Quantitative PCR was performed using the StepOne Plus Real- Time PCR system (Applied Biosystems, Thermo Fisher Scientiﬁc, Inc). Primer sequences for the targeted mouse genes were as follows: IL-6, forward: 5′-ACAACCACGGCCTTCCCTACTT-3′, reverse: 5′-CACGATTTCCCAGAGAACATGTG-3′; Frontiers in Pharmacology \| www.frontiersin.org Effects of Resveratrol on Gut Microbial Structure in db/m Mice and db/db Mice To elucidate the mechanism of the renoprotective effect of resveratrol treatment, we investigated the impact of resveratrol on the gut microbiota in db/db mice. 16S rDNA sequencing was used to assess changes in the fecal microbiota of db/m and db/db mice with or without resveratrol treatment. From 26 samples, after removing low-quality reads total 733,481 high-quality reads remained (range from 17,557 to 38,441 reads per sample) and 510 OTUs were obtained.The a-diversity was signiﬁcantly lower in db/db mice compared to db/m mice (Figures 3A, B, Table S1), whereas resveratrol treatment increased bacterial richness and a-diversity in db/db mice, but not in db/m mice (Figures 3A, B). Principal-component analysis (PCA) of 16S rRNA sequence data of feces contents showed a clear separation based on community structure between db/db and db/m mice (Figure 3C), and between saline- and resveratrol-treated db/db mice (Figure 3C). The two most abundant phyla across the four treatment groups were Firmicutes and Bacteroidetes. Resveratrol treatment decreased the relative abundance of Firmicutes and increased abundance of Bacteroidetes in both db/db and db/m mice (Figure 3D), which was further quantiﬁed by calculating the Firmicutes/Bacteroidetes (F/B) ratio (Figure 3E). The overall image of bacterial composition at the species level in four groups Next, we examined whether resveratrol administration inﬂuenced the renal morphology in db/db mice. Renal tissues of db/db mice (Figure 1H) showed a series of indicators of DN, including glomerulus hypertrophy, capillary basement membrane thickening, and mesangial matrix expansion, as compared to db/m mice (Figure 1H). After resveratrol treatment, glomerular lesion formation was remarkably alleviated (Figure 1H). Moreover, resveratrol treatment signiﬁcantly decreased the expression of mice (Figures 1I–L). Together, these results indicate that resveratrol ameliorates the DN-associated functional abnormalities, renal morphological changes, and renal inﬂammatory markers in db/db mice. Resveratrol Prevents the Progression of Diabetic Nephropathy in db/db Mice p p y To investigate the potential therapeutic effect of resveratrol on DN in a db/db diabetic mouse model. As expected, body and kidney weights of db/db mice were signiﬁcantly higher than control db/m mice (Figures 1A, B). In comparison to saline- treated db/db mice, resveratrol treatment of db/db mice signiﬁcantly reduced body (Figure 1A) and kidney (Figure 1B) weights and signiﬁcantly decreased levels of serum creatinine (Cr) (Figure 1C), blood urea nitrogen (BUN) (Figure 1D) and urine 24-h microalbuminuria (UAER) (Figure 1E). The fasting blood sugar (FBS) (Figure 1F) and the systolic blood pressure (SBP) (Figure 1G) were not signiﬁcantly affected by resveratrol treatment in the db/db mice group. In contrast, resveratrol-treatment did not affect the body and kidney weights and the biochemical parameters in db/m mice (Figures 1A–G). Data Availability All the 16S rRNA sequencing data required to assess the conclusions of this research are available without restrictions from the Sequence Read Achieve (SRA) at the National Center August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 3 Resveratrol Modulates the Gut Microbiota Cai et al. for Biotechnology Information (NCBI) under BioProject accession PRJNA628114. for Biotechnology Information (NCBI) under BioProject accession PRJNA628114. a scoring system that takes into account mucosal ulceration and depth of injury (Chaplin et al., 2018). The histological score was signiﬁcantly higher in the db/db mice group compared to the db/ m mice group, while the histological score was signiﬁcantly lower in the resveratrol-treated db/db mice than in saline treated db/db mice (Figures 2A, B). Intestinal permeability was monitored by measuring the plasma levels of 4 kDa FITC-dextran 4. As shown in Figure 2C, a higher level of 4 kDa FITC-dextran 4 was observed in db/db mice compared to db/m mice, which showed that diabetic mice had an increased gut leakiness. Interestingly, resveratrol treatment signiﬁcantly decreased the level of 4 kDa FITC-dextran in db/db mice, indicating that resveratrol ameliorates intestinal permeability. Furthermore, we conﬁrmed the impaired gut barrier in db/db mice by immunostaining for tight-junction proteins ZO-1 and claudin- 1. Decreased expression of ZO-1 and claudin-1 was detected in db/db mice compared to db/m mice. Importantly, resveratrol treatment substantially recovered ZO-1 and claudin-1 protein expression in db/db mice (Figure 2A). In contrast, the intestinal damage and permeability were not signiﬁcantly different between resveratrol-treated and untreated db/m mice. To further demonstrate the impact of resveratrol on intestinal inﬂammation, immunostaining showed that the levels of IFN-g and TNF-a are signiﬁcantly higher in db/db mice when compared to db/m mice (Figure 2A). Resveratrol treatment signiﬁcantly decreased the levels of IFN-g and TNF-a in the small intestine of db/db mice (Figure 2A). We also conﬁrmed that resveratrol decreased plasma levels of LPS, IFN-g, TNF-a, and IL-6 in db/db mice (Figures 2D–H). Together these results suggest that resveratrol can protect against gut barrier dysfunction, mucosal inﬂammation and endotoxemia. Statistical Analysis Statistical signiﬁcance of the differences between the groups of mice was assessed using R 3.6.1 and GraphPad Prism software v 5.0 (GraphPad Software, San Diego, CA, USA). PCA were performed using SIMCA-P software to cluster the sample plots across groups. Differential abundances of genera were tested by one-way analysis of variance (ANOVA) and non-parametric tests, including the Wilcoxon rank-sum test and Mann– Whitney U test. Data are presented as the means ± SEM for the indicated number of independently performed experiments, and p values <0.05 were considered statistically signiﬁcant. Resveratrol Improves Intestinal Barrier Function and Ameliorates Intestinal Permeability and Inﬂammation The progression of kidney disease could be mediated through an impaired gut-kidney axis. We therefore examined the effect of resveratrol on intestinal permeability, barrier function and intestinal inﬂammation in db/db and db/m mice. The intestinal histopathology was evaluated by H&E staining using August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 4 Resveratrol Modulates the Gut Microbiota Cai et al. Cai et al. A B D E F G I H J K L C FIGURE 1 \| Resveratrol relieves a series of indicators of DN and improves glomerular histological abnormalities in db/db mice. (A–G) Quantitative analysis of bod weight, kidney weight, BUN, Cr, UAER FBS and SBP in db/db and db/m control mice treated with either saline or resveratrol. (H) Representative photomicrogra of HE-stained and PAS-stained kidney sections (magniﬁcation: 400×). (I–L) Kidney mRNA levels of TNF-a, IFN-g, IL-6, IL-1b relative to GAPDH Bar graphs show densitometry analysis of speciﬁc bands expressed and was expressed relative to the control group normalized to a value of 1. Data is representative of multiple m per group (db/m: n=6, db/m+Res: n=7, db/db: n=6, db/db+Res: n=7). Data are presented as mean ± SEM. p<0.001; p<0.01; p<0.05; and ns: not signiﬁca C A B D E F E D H G H G K I I J L J L K I J FIGURE 1 \| Resveratrol relieves a series of indicators of DN and improves glomerular histological abnormalities in db/db mice. (A–G) Quantitative analysis of body weight, kidney weight, BUN, Cr, UAER FBS and SBP in db/db and db/m control mice treated with either saline or resveratrol. (H) Representative photomicrographs of HE-stained and PAS-stained kidney sections (magniﬁcation: 400×). (I–L) Kidney mRNA levels of TNF-a, IFN-g, IL-6, IL-1b relative to GAPDH Bar graphs show densitometry analysis of speciﬁc bands expressed and was expressed relative to the control group normalized to a value of 1. Data is representative of multiple mice per group (db/m: n=6, db/m+Res: n=7, db/db: n=6, db/db+Res: n=7). Data are presented as mean ± SEM. p<0.001; p<0.01; p<0.05; and ns: not signiﬁcant. showed in Figure S1. An increased F/B ratio has been associated with diabetes and obesity. However, the abundance of Proteobacteria was low, with no signiﬁcant difference between groups. At the genus level, we identiﬁed six OTUs that were particularly responsive to the interaction between mice genotype and resveratrol treatment. Resveratrol Improves Intestinal Barrier Function and Ameliorates Intestinal Permeability and Inﬂammation Indeed, as shown in Figures 3F–K, the fecal microbiota of resveratrol-treated db/db mice was enriched for the following six genera: Bacteroides, Alistipes, Rikenella, Odoribacter, Parabacteroides, and Alloprevotella when compared to the db/db mice group (Table S2). Importantly, Bacteroides, Alistipes and Parabacteroides exhibit anti- inﬂammatory properties and were markedly increased in the resveratrol-treated db/db mice group, with an increase of 21-fold, 2-fold and 2.8-fold respectively (Figures 3F–H) August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 5 Resveratrol Modulates the Gut Microbiota Cai et al. Cai et al. A B D E F G H C FIGURE 2 \| Resveratrol improves intestinal barrier function and ameliorates intestinal permeability and inﬂammation. (A) Representative photomicrographs of HE- stained small intestine sections and photomicrographs of immunoﬂuorescence for ZO-1, Claudlin-1, IFN-g, and TNF-a in db/db and db/m control mice treated with either saline or resveratrol (Magniﬁcation: 200×). (B) Histological score of small intestine damage in db/db and db/m control mice treated with either saline or resveratrol. (C) Plasma FITC-dextran 4 (DX, 4,000 molecular weight) levels after oral challenge in db/db and db/m control mice treated with either saline or resveratrol. (D) The levels of plasma endotoxin (LPS), EU: endotoxin units, in db/db and db/m control mice treated with either saline or resveratrol. (E–H) The levels of serum TNF-a, IFN-g, IL-6, IL-1b in db/db and db/m control mice treated with either saline or resveratrol. Data is representative of multiple mice per group: db/m: n=6, db/m+Res: n=7, db/db: n=6, db/db+Res: n=7). Data are presented as the mean ± SEM. *p<0.001; p<0.05; and ns: not signiﬁcant. B A B C C D H D F E G F G E H H FIGURE 2 \| Resveratrol improves intestinal barrier function and ameliorates intestinal permeability and inﬂammation. (A) Representative photomicrographs of HE- stained small intestine sections and photomicrographs of immunoﬂuorescence for ZO-1, Claudlin-1, IFN-g, and TNF-a in db/db and db/m control mice treated with either saline or resveratrol (Magniﬁcation: 200×). (B) Histological score of small intestine damage in db/db and db/m control mice treated with either saline or resveratrol. (C) Plasma FITC-dextran 4 (DX, 4,000 molecular weight) levels after oral challenge in db/db and db/m control mice treated with either saline or resveratrol. (D) The levels of plasma endotoxin (LPS), EU: endotoxin units, in db/db and db/m control mice treated with either saline or resveratrol. Resveratrol Improves Intestinal Barrier Function and Ameliorates Intestinal Permeability and Inﬂammation (E–H) The levels of serum TNF-a, IFN-g, IL-6, IL-1b in db/db and db/m control mice treated with either saline or resveratrol. Data is representative of multiple mice per group: db/m: n=6, db/m+Res: n=7, db/db: n=6, db/db+Res: n=7). Data are presented as the mean ± SEM. **p<0.001; p<0.05; and ns: not signiﬁcant. (Chung et al., 2012). These results suggest that the gut microbiota composition in db/db mice was signiﬁcantly altered compared to db/m mice and that resveratrol could at least partially rescue this change in db/db mice. single-housed and antibiotics-pretreated db/db mouse by oral gavage daily for 7 days (Figure 4A). The use of antibiotics treatment is to remove the original gut microbiota of db/db mice, and to construct a relatively sterile environment to ensure the effect of FMT. After FMT treatment, mice were followed up for 4 weeks (Figure 4A). 16S rRNA sequencing analysis of fecal samples from FMT recipient mice showed that the a-diversity of the microbiota in resveratrol-FMT recipient mice was not signiﬁcantly different compared to control-FMT db/db mice, as reﬂected by the Chao, Shannon and Simpson indexes (Figures 4B–D, Table S3). On the other hand, 16S rRNA sequencing analysis veriﬁed that resveratrol-FMT reconstituted the gut microbiome of db/db mice, principle component analysis Transplantation of the Resveratrol- In order to conﬁrm whether resveratrol-mediated changes in the intestinal microbiota contribute to improvement of renal function, fecal slurries collected from saline- (control-FMT) or resveratrol- treated (resveratrol-FMT) db/m mice were transplanted into August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 6 Resveratrol Modulates the Gut Microbiota Cai et al. Cai et al. A B D E F G I H J K C FIGURE 3 \| Effects of resveratrol on gut microbiota in db/m and db/db mice. Male db/db and db/m control mice were treated with either saline or resveratrol: db/m (n=6), db/m+resveratrol (db/m+Res,n=7), db/db(n=6, db/db+resveratrol(db/db+Res,n=7). Microbiome composition was determined by 16S rRNA gene sequencing. (A, B) Chao and Shannon alpha-diversity index in db/m groups, db/m+Res, db/db, db/db+Res mice. (C) Principal-component analysis (PCA) of fecal microbiome in db/m mice, db/db and db/db+Res mice (db/m: blue square; db/db: red dots;db/db+Res: green triangle). (D) Gut microbe relative abundance at the phylum level in db/m, db/m+Res, db/db, and db/db+Res mice. (E) Firmicutes/Bacteroidetes ratio in db/m, db/m+Res, db/db, and db/db+Res mice. (F–K) Relative species abundance at the genus level for Bacteroides, Alistipes, Parabacteroides, Rikenella, Odoribacter and Alloprevotella in db/m, db/m+Res, db/db, and db/db+Res mice Data are represented as the mean ± SEM. *p<0.01; p<0.05; and ns: not signiﬁcant. B C A E D E D G F H F G H J I K I I J K FIGURE 3 \| Effects of resveratrol on gut microbiota in db/m and db/db mice. Male db/db and db/m control mice were treated with either saline or resveratrol: db/m (n=6), db/m+resveratrol (db/m+Res,n=7), db/db(n=6, db/db+resveratrol(db/db+Res,n=7). Microbiome composition was determined by 16S rRNA gene sequencing. (A, B) Chao and Shannon alpha-diversity index in db/m groups, db/m+Res, db/db, db/db+Res mice. (C) Principal-component analysis (PCA) of fecal microbiome in db/m mice, db/db and db/db+Res mice (db/m: blue square; db/db: red dots;db/db+Res: green triangle). (D) Gut microbe relative abundance at the phylum level in db/m, db/m+Res, db/db, and db/db+Res mice. (E) Firmicutes/Bacteroidetes ratio in db/m, db/m+Res, db/db, and db/db+Res mice. (F–K) Relative species abundance at the genus level for Bacteroides, Alistipes, Parabacteroides, Rikenella, Odoribacter and Alloprevotella in db/m, db/m+Res, db/db, and db/db+Res mice. Data are represented as the mean ± SEM. *p<0.01; p<0.05; and ns: not signiﬁcant. August 2020 \| Volume 11 \| Article 1249 7 Frontiers in Pharmacology \| www.frontiersin.org Resveratrol Modulates the Gut Microbiota Cai et al. Cai et al. Transplantation of the Resveratrol- A B D E F G I H J K C IGURE 4 \| Gut microbiome composition of db/db fecal microbiota transplantation recipients from donor db/m mice fed a resveratrol or control diet. (A) Male db/db mice (8 weeks old, n=16) were acclimated for one week, then treated with an antibiotics cocktail for three days. Microbiome depleted mice were randomly assigned o one of two groups receiving Fecal Matter Transplantations (FMTs) from either db/m mice fed a control chow diet (C, control-FMT, n = 8) or db/m mice fed a esveratrol diet (R, resveratrol-FMT, n = 8) for seven consecutive days. After the completion of antibiotics treatment before the ﬁrst FMT, Fasting Blood Sugar (FBS) nd Systolic Blood Pressure (SBP) were measured. Following an overnight fast (day 0), fecal matter from db/m mice was transplanted via oral gavage once a day for consecutive days. All mice receiving FMT had access to standard chow at all times during and after FMT. Fecal samples were collected and SBP, FBG, CR, BUN, REA levels were measured four weeks after completion of FMT. Tissues were collected after a 5–6 h fast on day 4 in the fourth week. (B–D) Chao, Shannon and impson alpha-diversity index in db/db mice following control-FMT (n = 8) or resveratrol-FMT (n = 8). (E) Principal-component analysis (PCA) of 16S rRNA fecal microbiome of db/db mice following control-FMT (n = 8) or resveratrol-FMT (n = 8) (control-FMT: red dots; resveratrol-FMT: blue dots). (F) Gut microbiome relative bundance at the phylum level in db/db mice following control-FMT (n = 8) or resveratrol-FMT (n = 8). (G–K) Relative species abundance at the genus level for listipes, Enterocuccus, Turicbacter, Odoribacter and Rikenella in control-FMT (n = 8) or resveratrol-FMT (n = 8) mice. *p<0.01; p<0.05; and ns: not signiﬁcant. et al. Resveratrol Modulates the Gut Microbio A E F B D C C B G D C F B E D B D C G I H J K I J K H I J K G H G FIGURE 4 \| Gut microbiome composition of db/db fecal microbiota transplantation recipients from donor db/m mice fed a resveratrol or control diet. (A) Male db/db mice (8 weeks old, n=16) were acclimated for one week, then treated with an antibiotics cocktail for three days. Transplantation of the Resveratrol- Microbiome depleted mice were randomly assigned to one of two groups receiving Fecal Matter Transplantations (FMTs) from either db/m mice fed a control chow diet (C, control-FMT, n = 8) or db/m mice fed a resveratrol diet (R, resveratrol-FMT, n = 8) for seven consecutive days. After the completion of antibiotics treatment before the ﬁrst FMT, Fasting Blood Sugar (FBS) and Systolic Blood Pressure (SBP) were measured. Following an overnight fast (day 0), fecal matter from db/m mice was transplanted via oral gavage once a day for 7 consecutive days. All mice receiving FMT had access to standard chow at all times during and after FMT. Fecal samples were collected and SBP, FBG, CR, BUN, UREA levels were measured four weeks after completion of FMT. Tissues were collected after a 5–6 h fast on day 4 in the fourth week. (B–D) Chao, Shannon and Simpson alpha-diversity index in db/db mice following control-FMT (n = 8) or resveratrol-FMT (n = 8). (E) Principal-component analysis (PCA) of 16S rRNA fecal microbiome of db/db mice following control-FMT (n = 8) or resveratrol-FMT (n = 8) (control-FMT: red dots; resveratrol-FMT: blue dots). (F) Gut microbiome relative abundance at the phylum level in db/db mice following control-FMT (n = 8) or resveratrol-FMT (n = 8). (G–K) Relative species abundance at the genus level for Alistipes, Enterocuccus, Turicbacter, Odoribacter and Rikenella in control-FMT (n = 8) or resveratrol-FMT (n = 8) mice. *p<0.01; p<0.05; and ns: not signiﬁcant. (PCA) differentiated resveratrol-FMT mice from the control-FMT db/db mice (Figure 4E). At the phylum level, Firmicutes and Bacteroidetes dominated the gut microbiome of both groups similar to the gut microbiome composition of db/m and db/db mice (Figure 4F). Db/db mice receiving resveratrol- FMT were characterized by decreased proportions of Firmicutes, Tenericutes and Deferribacteres as well as increased proportions of Proteobacteria compared to levels in control-FMT db/db mice. At the genus level, resveratrol-FMT db/db mice exhibited increased abundance levels of Alistipes, Turicibacter, Odoribacter and Rikenella genus and lower abundance of Enterococci compared to control-FMT mice (Figures 4G–K, Table S4), August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 8 Resveratrol Modulates the Gut Microbiota Cai et al. which is similar to our results obtained with oral resveratrol treatment in db/db mice. Transplantation of the Resveratrol- These ﬁndings were also conﬁrmed by heatmap analysis of the bacterial taxons, which could signiﬁcantly separate the resveratrol-FMT recipient mice from the control-FMT db/db mice (Figure S2). These results indicated that resveratrol-FMT could signiﬁcantly inﬂuence the structure and composition of the gut microbiome. We observed no difference in body weight between resveratrol- FMT and control-FMT db/db mice (Figure 5A). Importantly, in comparison to the control-FMT mice, the resveratrol-FMT mice showed signiﬁcantly decreased urine 24-h microalbuminuria (UAER), serum creatinine (Cr), blood urea nitrogen (BUN) and fasting blood sugar (FBS) levels (Figures 5B–E). The Systolic blood pressure (SBP) was lower in the resveratrol-FMT mice, but was not signiﬁcantly different compared to control-FMT mice (Figure 5F). Finally, we discovered that resveratrol-FMT mice showed signiﬁcantly lower kidney mRNA levels of TNF-a, IFN-g, IL-6, and IL-1b compared to control-FMT recipients (Figure 5H), suggesting that reduced inﬂammation by resveratrol-modiﬁed fecal microbial community may be a key mechanism for protecting the renal function of DN. resveratrol-FMT and pre-FMT db/db mice. First, the intestinal histopathology was evaluated by H&E staining. Resveratrol-FMT mice had a lower histological score for mucosal ulceration and depth in the small intestine compared to control-FMT recipients (Figures 6A, B). Intestinal permeability measured by the plasma levels of orally administered 4 kDa FITC-dextran 4 showed decreased permeability in Resveratrol-FMT mice compared to control-FMT and pre-FMT mice (Figure 6C), which was further conﬁrmed by immunohistochemical staining using ZO-1 and claudin-1 of small intestine samples (Figure 6A). To further demonstrate the impact of resveratrol-FMT on intestinal inﬂammation, immunostaining showed that the levels of IFN-g and TNF-a were lower in resveratrol-FMT mice compared to control-FMT mice (Figure 6A). Similarly, when compared to control-FMT recipients or pre-FMT mice, resveratrol-FMT recipients showed signiﬁcantly lower levels of plasma LPS and serum IFN-g, TNF-a, IL-6, and IL-1b demonstrating that transplantation of the resveratrol-modiﬁed fecal microbial community also reduced systemic inﬂammation (Figures 6E– H). These results suggest that the anti-DN effects of resveratrol are at least partly mediated through modulation of the gut microbiota. Transplantation of the Resveratrol- Modiﬁed Fecal Microbial Community Improved Intestinal Characteristics and Decreased Inﬂammatory Response DISCUSSION Increasing evidence supports the causative role of gut microbiota in DN and CKD development and progression. The underlying mechanisms involve diabetes induction, inﬂammatory response Next we analyzed intestinal characteristics, gut inﬂammation and serum inﬂammatory response between control-FMT, A B D E F G C FIGURE 5 \| Fecal microbiota transplantation from db/m mice fed a resveratrol diet to db/db recipient mice improved renal function. (A–F) Quantitative analysis of body weight, Cr, BUN, 24h UAER, FBS, and SBP in control-FMT and resveratrol-FMT mice. (G) Representative kidney mRNA levels of TNF-a, IFN-g, IL-6, and IL-1b by RT-PCR analysis in control-FMT (n=8) and resveratrol-FMT (n=8) mice. Data are represented as the mean ± SEM *p<0.01; p<0.05; and ns: not signiﬁcant. D D C A C B D E F F E G F E G FIGURE 5 \| Fecal microbiota transplantation from db/m mice fed a resveratrol diet to db/db recipient mice improved renal function. (A–F) Quantitative analysis of body weight, Cr, BUN, 24h UAER, FBS, and SBP in control-FMT and resveratrol-FMT mice. (G) Representative kidney mRNA levels of TNF-a, IFN-g, IL-6, and IL-1b by RT-PCR analysis in control-FMT (n=8) and resveratrol-FMT (n=8) mice. Data are represented as the mean ± SEM *p<0.01; p<0.05; and ns: not signiﬁcant. August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 9 Cai et al. Resveratrol Modulates the Gut Microbiota Cai et al. A B D E F G H C FIGURE 6 \| Transplantation of the resveratrol-modiﬁed fecal microbial community improved intestinal characteristics and decreased inﬂammatory response in db/db mice. (A) Representative photomicrographs of H&E-stained small intestine sections and immunoﬂuorescence for ZO-1, Claudlin-1, IFN-g and TNF-a (magniﬁcation: 200×). (B) Representative histological scoring of small intestine damage. (C) Plasma FITC-dextran 4 (DX, 4,000 molecular weight) levels after oral challenge in control post-FMT (n=8) or resveratrol post-FMT (n=8) mice. (D) Plasma levels of LPS (endotoxin units/ml). (E–H) Serum levels of TNF-a, IFN-g, IL-6, and IL-1b by ELISA in control post-FMT (n=8) or resveratrol post-FMT (n=8). *p<0.001; p<0.01; p<0.05; and ns: not signiﬁcant. B A B D C E C E H F G H F G FIGURE 6 \| Transplantation of the resveratrol-modiﬁed fecal microbial community improved intestinal characteristics and decreased inﬂammatory response in db/db mice. (A) Representative photomicrographs of H&E-stained small intestine sections and immunoﬂuorescence for ZO-1, Claudlin-1, IFN-g and TNF-a (magniﬁcation: 200×). (B) Representative histological scoring of small intestine damage. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION (C) Plasma FITC-dextran 4 (DX, 4,000 molecular weight) levels after oral challenge in control post-FMT (n=8) or resveratrol post-FMT (n=8) mice. (D) Plasma levels of LPS (endotoxin units/ml). (E–H) Serum levels of TNF-a, IFN-g, IL-6, and IL-1b by ELISA in control post-FMT (n=8) or resveratrol post-FMT (n=8). p<0.001; p<0.01; *p<0.05; and ns: not signiﬁcant. activation, and metabolism deregulation (Knauf et al., 2019). Our present study using an animal model of diabetic nephropathy demonstrated that the dysbiosis mediated-inﬂammatory response is involved in the development of diabetes-associated kidney disease. In addition, we showed for the ﬁrst time that the protective effect of resveratrol on DN is mediated via modulation of the intestinal microbiota and related gut-kidney axis activation, counteracting the inﬂammasome initiation response as well as renal dysfunction. This is consistent with previous reports, which showed that resveratrol ameliorated type 2 diabetes-induced renal failure diseases, through its antioxidant, anti-inﬂammatory mechanisms (Palsamy and Subramanian, 2011; Li et al., 2018). Chronic low-grade inﬂammation is a pathophysiological feature of diabetes. Intestinal barrier dysfunction and increased intestinal permeability promotes a chronic blood inﬂammation response, resulting in increased exposure of host tissues, including the kidneys, to endotoxins (Sabatino et al., 2017). In this study, we found higher intestinal permeability in db/db mice compared to db/m mice, which showed that diabetes in mice promoted gut leakiness. Importantly, we ﬁrst showed that resveratrol treatment decreased intestinal permeability in db/db mice. This is similar to previous reports, which showed that an In particular, the increased pro-inﬂammation factors play a critical role in the progression of renal dysfunction in DN. Resveratrol, administered orally, relieved a series of indicators of diabetic nephropathy, such as the level of serum BUN, CR, 24- hour urine microalbumin, and the renal inﬂammation response. August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 10 Resveratrol Modulates the Gut Microbiota Cai et al. in the abundance of Alistipes, Odoribacter and Alloprevotella genera changes very similar to those in the resveratrol-treated db/ db mice. The increased abundance of the Alloprevotella in the db/ db+resveratrol has previously been linked to beneﬁcial intestinal health and to aberrant production of metabolic endotoxin (Larsen et al., 2010). Thus, the anti-inﬂammatory effect of resveratrol may be seen to modulate the bacterial composition of these candidates in db/db mice. The Odoribacters, which are known for their butyrate-producing capabilities in the human gut, were increased by the resveratrol treatment. DISCUSSION Within the fecal microbiota analysis, our ﬁndings showed that transplantation of the resveratrol-modiﬁed fecal microbiota from donor mice increased the abundance of Proteobacteria and decreased the abundance of Firmicutes. This trend of Firmicutes level is similar to the microbiota change in the resveratrol-mediated db/db mice. Similarly, at the genus level, the increased abundances of Alistipes, Odoribacter and Rikenella in resveratrol-FMT mice are consistent with resveratrol administration in db/db mice. Importantly, resveratrol-FMT signiﬁcantly increased the abundance of Turicibacter in db/db mice, which is inconsistent report that resveratrol decreased the abundance of Turicibacter in obese mice (Sung et al., 2017b). However, Turicibacter can modulate inﬂammatory responses and exert an anti- inﬂammatory effect in inﬂammatory bowel disease (IBD) (Loh and Blaut, 2012), contributing ultimately to metabolic dysfunction. We also observed a signiﬁcant change in fecal abundance of Odoribacter in resveratrol-FMT mice, which may in part be the reason for the resveratrol-FMT mice having decreased glucose levels (Gomez-Arango et al., 2016). In previous studies, the number of aerobic bacteria, such as Enterobacteria and Enterococci, was dramatically higher in hemodialysis patients, resulting in an increase in some uremic toxins (Perna et al., 2019). In the current study, the transplantation of resveratrol-altered gut microbiota signiﬁcantly lowered the abundance in the Enterococcus genus, which previously has been linked to beneﬁcial anti-inﬂammatory There is considerable evidence to show that the role of the gut microbiota on intestinal barrier dysfunction and bacterial translocation is to cause and perpetuate inﬂammation (Thevaranjan et al., 2017). Furthermore, animal studies in which the gut microbiota is manipulated, as well as observational studies in patients with CKD, have provided considerable evidence that dysbiosis contributes to the process of CKD (Andersen et al., 2017). Although previous reports have found that resveratrol modulated the composition of the gut microbiota, suggesting that the microbiome plays a critical role in the progression of diabetes and obesity (Sung et al., 2017b; Wang et al., 2020), the beneﬁcial role of resveratrol on the microbiota speciﬁcally in DN is unclear. In this study, we found that the ratio of Firmicutes/ Bacteroidetes in the db/db mice group was increased compared with that in the control group, while resveratrol restored the ratio. Previous studies have also shown that the ratio of Firmicutes/ Bacteroidetes plays an important role in metabolic diseases, such as diabetes and obesity (Abdallah et al., 2011). DISCUSSION Odoribacters are also capable of tryptophan metabolism and glycolysis processing (Gomez-Arango et al., 2016). Therefore, we propose that resveratrol may promote the growth of bacteria involved in glycolysis in db/db mice, resulting in the improvement of glucose homeostasis. increase in gut permeability caused by the common mycotoxin deoxynivaenol could be arrested by resveratrol (Ling et al., 2016). The increase in intestinal permeability was also associated with decreased expression of tight-junction, and increased small intestinal lesions (Etxeberria et al., 2015). A major concern with intestinal permeability is the increased passage of LPS from the gut lumen to the plasma. Indeed, LPS was increased in the blood of diabetic individuals, and it has been implicated in induction of systemic inﬂammation, insulin resistance, renal dysfunction, and uremic toxins in DN (Mahmoodpoor et al., 2017; Koppe et al., 2018). However, whether alteration of the intestinal environment by resveratrol in the small intestine is associated with amelioration of the progression in DN is not known. Here, we show that addition of resveratrol prevents the increase in both the level of plasma LPS and the levels of serum inﬂammation markers. Our ﬁndings provide important information on how resveratrol protects against gut barrier dysfunction, with consequences for inﬂammatory-mediated endotoxemia in db/db mice. Previous studies have suggested that resveratrol exerts protective effect on gut barrier function by inhibiting MAPK, NF-B, and JAK/STAT pathways, thus suppressing the generation of proinﬂammatory. Resveratrol also protected inﬂammatory bowel disease and improved gut barrier function by downregulating the expression of HIF-1, mTOR, and STAT3 pathway (Nunes et al., 2018). Diabetes and CKD are known to be associated with a signiﬁcant decrease in microbiota diversity, as well as with altered barrier function and increased permeability of the epithelium (Evenepoel et al., 2017). By contrast, dysbiosis, intestinal barrier dysfunction, and the perpetuation of inﬂammation are ascribed to renal dysfunction in DN (Andersen et al., 2017). In the current study, and to conﬁrm a causal relationship between changes in the fecal microbiome and the observed renal dysfunction, we transplanted the resveratrol- altered gut microbiota from db/m mice to db/db mice. The results of our fecal microbiota transplantation experiments supported our hypothesis that the resveratrol-mediated microbiota is a driving force in improving renal function, improving glucose homeostasis, restoring gut permeability, and reducing inﬂammatory markers. Frontiers in Pharmacology \| www.frontiersin.org DATA AVAILABILITY STATEMENT TABLE S3 \| Alpha-diversity index in db/db mice following control-FMT (n = 8) or resveratrol-FMT (n = 8). The datasets generated for this study can be found in NCBI BioProject PRJNA628114. The datasets generated for this study can be found in NCBI BioProject PRJNA628114. TABLE S4 \| Relative species abundance at the genus level in control-FMT (n = 8) or resveratrol-FMT (n = 8) mice FIGURE S1 \| Bacterial composition at the species level of each sample in four groups. Gut microbe relative abundance at the species level of each sample in db/ m, db/m+Res, db/db, and db/db+Res mice. ETHICS STATEMENT The animal study was reviewed and approved by Nanjing Medical University and approved by the Institutional Animal Care and The animal study was reviewed and approved by Nanjing Medical University and approved by the Institutional Animal Care and FIGURE S2 \| Microbiome composition in control-FMT and resveratrol-FMT db/db mice. The heatmap of relative species abundance at the genus level in control-FMT (n = 8) or resveratrol-FMT (n = 8) db/db mice. Use Committee of Nanjing Medical University, No. 14030134. Use Committee of Nanjing Medical University, No. 14030134. SUPPLEMENTARY MATERIAL Resveratrol treatment is a potentially beneﬁcial therapeutic intervention against the progression of DN, by improving the gut environment and reducing the inﬂammation response, and that these changes may be an important mechanism by which resveratrol mediates its beneﬁcial renal function effects. These ﬁndings provide supporting evidence for the gut–kidney axis in DN. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020. 01249/full#supplementary-material TABLE S1 \| Alpha-diversity index in db/m groups, db/m+Res, db/db, db/db+Res mice. TABLE S2 \| Relative gut microbiota abundance at the genus level in four groups. FUNDING This work was supported by the grants from Major Research and Development Project of Jiangsu (BE2016800), Jiangsu Youth Medical Talents Project (QNRC2016674) and Nanjing Science Technology Plan Project (201715015). ACKNOWLEDGMENTS We thank the entire staff at the Biotech Treatment Center, The Second Afﬁliated Hospital of Nanjing Medical University, Nanjing, China for their excellent technical assistance with this research. We thank the entire staff at the Biotech Treatment Center, The Second Afﬁliated Hospital of Nanjing Medical University, Nanjing, China for their excellent technical assistance with this research. AUTHOR CONTRIBUTIONS effects in targeted intervention studies, both in rodents and in humans (Daillere et al., 2016). Moreover, we found that the abundance of the Parabacteroides genera in resveratrol-FMT mice were higher than this in the control-FMT mice, which were consistent with previous studies showing that resveratrol increased Parabacteroides genera (Sung et al., 2017b; Wu et al., 2019).These bacteria may turn out to be new candidate phylotypes for predicting and treating renal dysfunction in DN. Conceptualization, D-FD, XL, and Y-BL. Methodology, T-TC, X- LY, R-RL, HC, Y-YW, WL. Software, X-YL, M-LP. Validation, YT and X-LY. Formal Analysis, D-FD, T-TC, X-LY. Investigation, H-JY. Resources, HM and Y-BL. Data Curation, Y-BL, T-TC, and X-LY. Writing—Original Draft Preparation, D- FD, T-TC. Writing—Review and Editing, X-YL, AS, J-HM. Supervision, T-TC. Project Administration, D-FD. Funding Acquisition, D-FD. Our study has a number of limitations. Our study did not measure resveratrol or its metabolites in the fecal slurries used for FMT studies, thus it remains possible that the beneﬁcial effects of the FMT observed in our study was confounded by residual resveratrol. In addition, we used fecal samples from db/ m mice as a donor for our FMT studies. Future studies will need to be conducted to compare beneﬁcial effects of FMT from RSV- treated db/db and db/dm mice. Nevertheless, the results from our studies suggest that So changing the fecal microbiota composition by resveratrol is only one of a variety oﬁs one mechanism by which resveratrols that protects against the development of DN (Wenzel and Somoza, 2005; Kim et al., 2018). However, in human study, the speciﬁc role of resveratrol on the gut microbiota and the development of DN are not yet clear. More clinic trials are needed to conﬁrm these and other possible effects and mechanisms. DISCUSSION In addition, the resveratrol-treated group showed an increased abundance of Bacteroides, Alistipes, Rikenella, Odoribacter, Parabacteroides, and Alloprevotella genera when compared to the db/db mice group. In particular, Bacteroides, Alistipes, and Parabacteroides have been signiﬁcantly associated with anti-inﬂammatory factors (Troy and Kasper, 2010). Similarly, several studies in mice showed that resveratrol caused gut microbiota remodeling with an increase in the relative abundance of Bacteroides and Parabacteroides, which attenuated trimethylamine-N-oxide (TMAO) induced atherosclerosis and improved exercise performance and skeletal muscle oxidative capacity in heart failure (Chen et al., 2016; Sung et al., 2017a).Within db/m mice, we observed a signiﬁcant increase August 2020 \| Volume 11 \| Article 1249 Frontiers in Pharmacology \| www.frontiersin.org 11 Resveratrol Modulates the Gut Microbiota Cai et al. REFERENCES Inﬂammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat. Rev. Nephrol. 7, 327–340. doi: 10.1038/nrneph.2011.51 hropathy. Nat. Rev. Nephrol. 7, 327–340. doi: 10.1038/nrneph.2011 Nunes, S., Danesi, F., Del, R. D., and Silva, P. (2018). Resveratrol and inﬂammatory Nunes, S., Danesi, F., Del, R. D., and Silva, P. (2018). Resveratrol and inﬂammatory bowel disease: The evidence so far. Nutr. 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The gut microbiota and the brain-gut-kidney axis in hypertension and chronic kidney disease. Nat. Rev. Nephrol. 14, 442–456. doi: 10.1038/s41581-018-0018-2 Li, J., Li, L., Wang, S., Zhang, C., Zheng, L., Jia, Y., et al. (2018). Resveratrol Alleviates Inﬂammatory Responses and Oxidative Stress in Rat Kidney Ischemia-Reperfusion Injury and H2O2-Induced NRK-52E Cells via the Nrf2/ TLR4/NF-kB Pathway. Cell. Physiol. Biochem. 45, 1677–1689. doi: 10.1159/ 000487735 Conﬂict of Interest: 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. Conﬂict of Interest: 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. Ling, K. H., Wan, M. L., El-Nezami, H., and Wang, M. (2016). 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https://openalex.org/W2487234017	https://eprints.gla.ac.uk/154711/1/154711.pdf	English	null	Direct Transcriptional Consequences of Somatic Mutation in Breast Cancer	Cell reports	2,016	cc-by	12,788	Direct Transcriptional Consequences of Somatic Mutation in Breast Cancer Resource Resource Authors Correspondence adam.shlien@sickkids.ca (A.S.), pc8@sanger.ac.uk (P.J.C.) Correspondence adam.shlien@sickkids.ca (A.S.), pc8@sanger.ac.uk (P.J.C.) Shlien et al., 2016, Cell Reports 16, 2032–2046 August 16, 2016 ª 2016 The Authors. http://dx doi org/10 1016/j celrep 2016 07 028 Shlien et al., 2016, Cell Reports 16, 2032–2046 August 16, 2016 ª 2016 The Authors. http://dx.doi.org/10.1016/j.celrep.2016.07.028 Direct Transcriptional Consequences of Somatic Mutation in Breast Cancer Direct Transcriptional Consequences of Somatic Mutation in Breast Cancer Graphical Abstract Authors Adam Shlien, Keiran Raine, Fabio Fuligni, ..., Kornelia Polyak, Michael R. Stratton, Peter J. Campbell Authors Adam Shlien, Keiran Raine, Fabio Fuligni, ..., Kornelia Polyak, Michael R. Stratton, Peter J. Campbell Authors Adam Shlien, Keiran Raine, Fabio Fuligni, ..., Kornelia Polyak, Michael R. Stratton, Peter J. Campbell Graphical Abstract In Brief Shlien et al. quantify ﬁrst-order transcriptional consequences across all classes of somatic mutation in well- annotated breast cancers. They ﬁnd that cancer cells are more transcriptionally active than stromal cells, especially in ER- negative cancers. Rearrangements that break genes or place them in opposite orientation are an unexpectedly potent source of fusions. Highlights d Greater transcriptional activity in cancer than stromal cells, particularly when ER-ve d Intron mutations only infrequently affect splicing, even at essential splice sites d Distinctive RNA effects of sense-to-antisense and gene-to- intergenic rearrangements d Exhaustive pipeline for identifying aberrant transcripts from RNA-sequencing data Cell Reports Resource Direct Transcriptional Consequences of Somatic Mutation in Breast Cancer Campbell1,13,14,* 1Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK 2D t t f H G ti U i it f L 3000 L B l i 5Department of Pathology, Oslo University Hospital, 0450 Oslo, Norway 6Institut Curie, 75005 Paris, France Synergie Lyon Cancer, Centre Le´ on Be´ rard, 69008 Lyon Dana-Farber Cancer Institute, Boston, MA 02215, USA Correspondence: adam.shlien@sickkids.ca (A.S.), pc8@sanger.ac.uk (P.J.C.) http://dx.doi.org/10.1016/j.celrep.2016.07.028 Correspondence: adam.shlien@sickkids.ca (A.S.), pc8@sanger.ac.uk (P.J.C.) http://dx.doi.org/10.1016/j.celrep.2016.07.028 Correspondence: adam.shlien@sickkids.ca (A.S.), pc8@sanger.ac.uk (P.J.C.) http://dx.doi.org/10.1016/j.celrep.2016.07.028 integration of transcriptome with genome data re- veals the rules by which transcriptional machinery in- terprets somatic mutation. integration of transcriptome with genome data re- veals the rules by which transcriptional machinery in- terprets somatic mutation. Disordered transcriptomes of cancer encompass direct effects of somatic mutation on transcription, coordinated secondary pathway alterations, and increased transcriptional noise. To catalog the rules governing how somatic mutation exerts direct tran- scriptional effects, we developed an exhaustive pipeline for analyzing RNA sequencing data, which we integrated with whole genomes from 23 breast cancers. Using X-inactivation analyses, we found that cancer cells are more transcriptionally active than intermixed stromal cells. This is especially true in estrogen receptor (ER)-negative tumors. Overall, 59% of substitutions were expressed. Nonsense mutations showed lower expression levels than ex- pected, with patterns characteristic of nonsense- mediated decay. 14% of 4,234 rearrangements caused transcriptional abnormalities, including exon skips, exon reusage, fusions, and premature polyadenylation. We found productive, stable tran- scription from sense-to-antisense gene fusions and gene-to-intergenic rearrangements, suggesting that these mutation classes drive more transcriptional disruption than previously suspected. Systematic 2032 Cell Reports 16, 2032–2046, August 16, 2016 ª 2016 The Authors. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Direct Transcriptional Consequences of Somatic Mutation in Breast Cancer Adam Shlien,1,16, Keiran Raine,1 Fabio Fuligni,15 Roland Arnold,15 Serena Nik-Zainal,1 Serge Dronov,1,17 Lira Mamanova,1 Andrej Rosic,15 Young Seok Ju,1 Susanna L. Cooke,1 Manasa Ramakrishna,1 Elli Papaemmanuil,1 Helen R. Davies,1 Patrick S. Tarpey,1 Peter Van Loo,1,2 David C. Wedge,1 David R. Jones,1 Sancha Martin,1 John Marshall,1 Elizabeth Anderson,1 Claire Hardy,1 ICGC Breast Cancer Working Group, Oslo Breast Cancer Research Consortium, Violetta Barbashina,3 Samuel A.J.R. Aparicio,4 Torill Sauer,5 Øystein Garred,5 Anne Vincent-Salomon,6 Odette Mariani,6 Sandrine Boyault,7 Aquila Fatima,8 Anita Langerød,9,10 A˚ ke Borg,11 Gilles Thomas,7,17 Andrea L. Richardson,8 Anne-Lise Børresen-Dale,9,10 Kornelia Polyak,12 Michael R. Stratton,1 and Peter J. Campbell1,13,14,* 1Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK 2Department of Human Genetics, University of Leuven, 3000 Leuven, Belgium 3Breakthrough Breast Cancer, The Institute of Cancer Research, London SM2 5NG, UK 4British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada 5Department of Pathology, Oslo University Hospital, 0450 Oslo, Norway 6Institut Curie, 75005 Paris, France 7Synergie Lyon Cancer, Centre Le´ on Be´ rard, 69008 Lyon, France 8Dana-Farber Cancer Institute, Boston, MA 02215, USA 9Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, 0379 Oslo, Norway 10K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, 0316 Oslo, Norway 11Department of Oncology, Lund University, SE-221 00 Lund, Sweden 12Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA 13Department of Haematology, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK 14Department of Haematology, University of Cambridge, Cambridge CB2 1TN, UK 15Department of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada 16Present address: Department of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada 17Deceased Correspondence: adam shlien@sickkids ca (A S ) pc8@sanger ac uk (P J C ) Adam Shlien,1,16, Keiran Raine,1 Fabio Fuligni,15 Roland Arnold,15 Serena Nik-Zainal,1 Serge Dronov,1,17 Lira Mamanova,1 Andrej Rosic,15 Young Seok Ju,1 Susanna L. Cooke,1 Manasa Ramakrishna,1 Elli Papaemmanuil,1 Helen R. Davies,1 Patrick S. Tarpey,1 Peter Van Loo,1,2 David C. Wedge,1 David R. Jones,1 Sancha Martin,1 John Marshall,1 Elizabeth Anderson,1 Claire Hardy,1 ICGC Breast Cancer Working Group, Oslo Breast Cancer Research Consortium, Violetta Barbashina,3 Samuel A.J.R. Aparicio,4 Torill Sauer,5 Øystein Garred,5 Anne Vincent-Salomon,6 Odette Mariani,6 Sandrine Boyault,7 Aquila Fatima,8 Anita Langerød,9,10 A˚ ke Borg,11 Gilles Thomas,7,17 Andrea L. Richardson,8 Anne-Lise Børresen-Dale,9,10 Kornelia Polyak,12 Michael R. Stratton,1 and Peter J. 2032 Cell Reports 16, 2032–2046, August 16, 2016 ª 2016 The Authors. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). *Correspondence: adam.shlien@sickkids.ca (A.S.), pc8@sanger.ac.uk (P.J.C.) http://dx.doi.org/10.1016/j.celrep.2016.07.028 INTRODUCTION Somatic mutation underpins the development of cancer, and most solid tumors have thousands to tens of thousands of point mutations, coupled with tens to hundreds of genomic rearrange- ments and copy-number changes (Garraway and Lander, 2013; Stratton et al., 2009). Small numbers of these, known as driver mutations, dysregulate the fundamental cellular processes involved in normal tissue homeostasis, and they confer a selec- tive advantage to the clone. A critical point is that Darwinian se- lection acts on phenotype, and so, for a somatic mutation to drive cancer, it must manifest a phenotypic effect. Transcription is the primary conduit by which changes in the genomic code are translated into cellular phenotype, with the corollary that it is a necessary criterion of driver mutations that they directly induce a change in transcript structure. Altered transcript structure can take many forms, including the creation of fusion genes by genomic rearrangement, interference with RNA splicing at mutated splice sites, alteration of the codon sequence for missense substitutions, and over- or under-expression of genes through copy-number alterations or mutation in regulatory regions. 2032 Cell Reports 16, 2032–2046, August 16, 2016 ª 2016 The Authors. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Beyond the primary and direct effects of somatic mutation on transcript structure, there may be a series of downstream, secondary alterations in the transcriptome occurring as a consequence of the primary effect. Most studies of the tran- scriptome in cancer, including those from large-scale efforts such as The Cancer Genome Atlas (TCGA) (Kandoth et al., 2013; Cancer Genome Atlas Network, 2012a), have evaluated these second-order effects, concentrating predominantly on the magnitude of gene expression using microarray technology (Curtis et al., 2012; Perou et al., 2000; Sørlie et al., 2001) or RNA sequencing (RNA-seq) (Shah et al., 2009; Cancer Genome Atlas Research Network, 2012b). They have revealed large-scale disturbances of transcriptional regulation in most cancers, with expression proﬁles for many hundreds of genes differing from proﬁles of normal cellular counterparts. Within a tumor type, similarities in transcriptional proﬁles across indi- viduals allow the disease to be sub-classiﬁed into several groups, many of which have biological, therapeutic, and prog- nostic signiﬁcance. In some cases, these changes can be correlated with underlying driver mutations, such as ERBB2 ampliﬁcation in breast cancer (Sørlie et al., 2001) or speciﬁc fusion genes in acute myeloid leukemia (Valk et al., 2004). INTRODUCTION While these studies have concentrated on mRNA proﬁles, similar observations are beginning to emerge from studies of microRNA transcription (Dvinge et al., 2013), long non-coding RNA levels, and even expression of pseudogenes (Kalyana- Sundaram et al., 2012). samples to 303 coverage, with somatically acquired substitu- tions, insertions or deletions (indels), genomic rearrangements, and copy-number changes called by a suite of in-house algo- rithms. The whole-genome sequencing for the 14 primary breast cancer samples has been described previously (Nik-Zainal et al., 2012a, 2012b), although improvements in our bioinfor- matics algorithm allowed us to update the list of genomic rear- rangements (Table S1). The high-coverage genome-sequencing data for eight breast cancer cell lines are reported for the ﬁrst time here (somatic mutations in Table S2); for the other line (HCC2157), we used exome and low-coverage whole-genome data reported previously (Nik-Zainal et al., 2012b; Stephens et al., 2009). RNA-seq was performed on the 23 breast cancer samples together with eight organoids freshly isolated from uncultured normal breast milk ducts (Choudhury et al., 2013). We developed a suite of algorithms to exhaustively characterize the cancer transcriptome; in so doing, we aimed to wring maximum detail on the structure of cancer transcripts from RNA-seq data. Previ- ous work has examined gene and mutation expression alone or has focused exclusively on one facet of transcript structure (such as fusion genes or alternative splicing) without allowing for the discovery of multiple or complex events or the involvement of the antisense strand. We implemented a seed-and-extend map- ping algorithm to ﬁnd reads that span different regions of the genome, and then we developed a discordant pair analysis algo- rithm, drawing these results together with a set of methods to arrange the results into biologically meaningful categories (described in detail in the Experimental Procedures and Figure S1). Although it is a necessary criterion for a driver mutation to directly induce modiﬁcation of transcript structure, it is not sufﬁ- cient. Many mutations that do not confer selective advantage, so-called passenger mutations, also generate phenotypic con- sequences, but consequences of no beneﬁt to the cell. Cells Tumors are comprised of a complex admixture of clonal cancer cells and polyclonal stromal cells. In breast cancer, the propor- tion of cells deriving from the malignant clone is typically 30%– 70%, although the remaining cells encompass endothelial cells, supporting connective tissue, inﬂammatory cells, lymphocytes, and normal breast epithelium. RNA samples extracted from pri- mary breast cancers, therefore, represent an amalgam of gene expression signatures derived from multiple cell lineages, com- pounding interpretation. INTRODUCTION Initial studies correlating RNA-seq data with genomic change in can- cer have reported some of these direct effects, especially for coding point mutations or canonical fusion transcripts (Shah et al., 2009), but there has been little systematic effort to describe, measure, and quantify ﬁrst-order transcriptional con- sequences across all classes of somatic mutation found in well-annotated cancer genomes. The primary advantage of our software pipeline, which we call RNA Architect, is the comprehensive detection of transcriptional alterations, including events missed by other methods. These would include compound events present in cis, such as fusion transcripts involving alternative splice forms and exon skips with cryptic splice sites; internal exon shufﬂing (reusage); post- transcriptional modiﬁcations, such as early polyadenylation sites; and non-canonical transcript junctions, for example, fu- sions between the sense and antisense of different genes or those involving lowly expressed transcripts that are not present in reference databases. While there exists a number of methods for aligning RNA-seq and detecting fusions (Asmann et al., 2011; Chen et al., 2012; Kim and Salzberg, 2011; McPherson et al., 2012; Swanson et al., 2013; Torres-Garcı´a et al., 2014), there have been few efforts to simultaneously characterize the cancer transcriptome for multiple types of alterations. Here, we report a comprehensive analysis of the primary transcriptional alterations induced by somatic mutation in a set of 23 breast cancers. We found that the genomic variants carried by the cancer cells can have subtle or profound effects on the transcriptome, many of which could not easily be pre- dicted from the genome, many of which amalgamate several in cis mutations, and many of which are stably expressed at high levels. Whole-Genome Sequencing and RNA-Seq from 23 Breast Cancer Samples To understand the inter-relationships between somatic muta- tion and the transcriptome, we matched RNA-seq data to whole-genome-sequencing data in 23 breast cancer samples. Of these, 14 were primary breast cancers and nine were matched breast cancer cell lines. For the genomes, tumor sam- ples were sequenced to 403 coverage and matched normal Cell Reports 16, 2032–2046, August 16, 2016 2033 A B C D E G F (legend on ne A A A B C B C F F D D E E G G (legend on next page) 2034 Cell Reports 16, 2032–2046, August 16, 2016 2034 Cell Reports 16, 2032–2046, August 16, 2016 In females, a randomly selected X chromosome is inactivated in each cell of the inner cell mass of the early blastocyst, and this choice is transmitted through every subsequent cell division. Since cancer cells are derived from a single ancestral cell, all have the same X chromosome inactivated (Fialkow et al., 1981), whereas the polyclonal stromal tissue has a broadly equivalent fraction of cells with maternal or paternal X chromo- somes inactivated. As a result, genes undergoing X inactivation with heterozygous germline SNPs are monoallelically expressed in the cancer cells and biallelically expressed in stromal cells (Figure S2). cells. We also could integrate all the data for a given patient to estimate the overall fraction of transcripts derived from tu- mor cells, and we could compare this to the overall fraction of cancer cells in the sample estimated from the genomic DNA (Figure 1E). This indicates that cancer cells contribute a higher fraction of transcripts in the RNA sample than expected for their cellular proportion, indicating that they are more tran- scriptionally active than the stromal cells. Thus, even though cancer cells comprise, on average, 30%–70% of all cells in a breast tumor, they contribute 70%–90% of all RNA molecules. Strikingly, it appeared that the magnitude of the difference between transcriptional output of cancer cells and stromal cells was greater in estrogen receptor-negative (ER-ve) tumors than in ER-positive tumors (Figure 1E). The difference in ER-ve also was seen in an independent set of primary breast cancers (661 ER positive and 176 ER-ve), using a larger set of variants (38,337 somatic substitutions; Figure 1F; cosmic census genes with high variant allele fraction difference in ER-ve can- cers; Table S3). Whole-Genome Sequencing and RNA-Seq from 23 Breast Cancer Samples Further, it appears that the number of muta- tions expressed in a breast tumor, a measure of its transcrip- tional output, is signiﬁcantly associated with the amount of estrogen receptor it expresses (0.2433, p < 0.0001). That is, tumors with high levels of ER express fewer mutations than cancers with low ER. We formally modeled this relation- ship and determined that, for every 1% decrease in ESR1 expression, 15 more mutations are expressed in breast cancer (Figure 1G). We identiﬁed heterozygous germline SNPs in expressed re- gions of the X chromosome from the genomic sequencing data across the 14 primary breast cancers, excluding regions that were not diploid in the cancer. From the RNA-seq data, we extracted the number of reads expressing each allele. In PD4120a, for example, 385 heterozygous SNPs on the X chro- mosome were expressed. The observed reference/variant ratio in the RNA-seq data at each position ranged from transcripts whose expression was exclusively monoallelic through tran- scripts with skewed ratios to genes that had an approximately equal expression of both alleles (Figure 1A). Respectively, these three scenarios represent genes expressed exclusively in cancer cells, genes expressed in both cancer and stromal cells, and genes expressed exclusively in stromal cells. We developed a statistical algorithm based on a Bayesian hi- erarchical Dirichlet process to model the fraction of transcripts along the X chromosome derived from cancer cells (Experi- mental Procedures). For each heterozygous SNP, the algorithm estimates what fraction of reads covering that base derived from cancer cells, allowing for the uncertainty of whether the refer- ence or variant allele is inactivated in the tumor (Figure 1B; Fig- ure S2B). When amalgamated across SNPs from the whole X chromosome, we can estimate the relative contribution of stro- mal and cancer cells to transcription as a general distribution (Figure 1C). Figure 1. Separating Expression of X-Linked Genes into Stromal and Tumor Compartments (A) Fraction of RNA-seq reads reporting reference allele of heterozygous germline SNPs on the X chromosome in one of the patients (PD4120a). The depth of color reﬂects the level of expression. (B) Fraction of transcripts derived from tumor cells for each heterozygous germline SNP shown in (A), estimated with a Bayesian Dirichlet process, is shown. (C) Estimated distribution and 95% posterior intervals for relative gene expression in cancer versus stromal cells for PD4120a. The y axis reports the estimated density of genes; the x axis reports the fraction of transcripts for each gene deriving from cancer cells. Thus, the transcripts for most genes in PD4120a are 80%– 100% derived from cancer cells and 0%–20% from stromal cells, with only a small peak of genes predominantly expressed from stromal cells. (D) Distributions for several selected primary cancers are shown, as for (C). (E) Overall fraction of transcripts derived from cancer cells (y axis) compared to the estimated proportion on tumor cells in the sample (x axis, estimated from genomic DNA using copy-number proﬁles) is shown. (F) Increased expression of the mutated allele in ER as compared to ER+ breast cancer transcriptomes (plotted relative to the genome). Primary breast cancers sequenced as part of TCGA are shown. Plotted on the y axis is the variant allele fraction in the transcriptome, relative to the genome (VAFdiff). (G) Inverse relationship between each tumors’ expression of Estrogen Receptor 1 (ESR1) and the overall expression of its point mutations (shown as VAFdiff; 0.2433, p < 0.0001). Using linear regression analysis to model this relationship, we determined that, for every 1% drop in ESR1, 15 additional point mutations are expressed. Effects of Point Mutations on Structure of the Transcriptome We identiﬁed all somatically acquired base substitutions in the 23 breast cancers that were in expressed regions, and we compared the fraction of sequencing reads reporting the mutant allele in the transcriptome to that expected from the genome (Figures S3A and S3B). As anticipated, there was a strong overall correlation between the genomic and transcriptomic variant allele fraction (r2 = 0.59; p < 0.0001). Overall, 6,980 substitutions were found in exons, of which 4,751 were expressed to a sufﬁ- cient degree that ﬁve or more sequencing reads covered the base. Of the 6,980 variants identiﬁed in exonic regions of the 23 samples, 4,152 (59%) had discernible expression in the cor- responding transcriptome. We found a considerable portion of transcripts that were exclusively expressed in cancer cells among the 14 patients in which primary breast cancer samples were sequenced (Fig- ure 1D; Figure S2C). Strikingly, many tumors had a set of tran- scripts that were 80%–90% derived from cancer cells and 10%–20% from stromal cells, whereas there were only small numbers of genes expressed predominantly from stromal Figure 1. Separating Expression of X-Linked Genes into Stromal and Tumor Compartments (A) Fraction of RNA-seq reads reporting reference allele of heterozygous germline SNPs on the X chromosom reﬂects the level of expression. (B) Fraction of transcripts derived from tumor cells for each heterozygous germline SNP shown in (A), estimated with a Bayesian Dirichlet process, is shown. (C) Estimated distribution and 95% posterior intervals for relative gene expression in cancer versus stromal cells for PD4120a. The y axis reports the estimated density of genes; the x axis reports the fraction of transcripts for each gene deriving from cancer cells. Thus, the transcripts for most genes in PD4120a are 80%– 100% derived from cancer cells and 0%–20% from stromal cells, with only a small peak of genes predominantly expressed from stromal cells. (D) Distributions for several selected primary cancers are shown, as for (C). s derived from cancer cells (y axis) compared to the estimated proportion on tumor cells in the sample (x axis, estimated from ber proﬁles) is shown. (F) Increased expression of the mutated allele in ER as compared to ER+ breast cancer transcriptomes (plotted relative to the genome). Primary breast cancers sequenced as part of TCGA are shown. Plotted on the y axis is the variant allele fraction in the transcriptome, relative to the genome (VAFdiff). Effects of Point Mutations on Structure of the Transcriptome (G) Inverse relationship between each tumors’ expression of Estrogen Receptor 1 (ESR1) and the overall expression of its point mutations (shown as VAFdiff; 0.2433, p < 0.0001). Using linear regression analysis to model this relationship, we determined that, for every 1% drop in ESR1, 15 additional point mutations are expressed. ch tumors’ expression of Estrogen Receptor 1 (ESR1) and the overall expression of its point mutations (shown as VAFdiff; gression analysis to model this relationship, we determined that, for every 1% drop in ESR1, 15 additional point mutations Cell Reports 16, 2032–2046, August 16, 2016 2035 A B C A A Figure 2. The Effect of Somatic Point Muta- tions on Expression and Aberrant Splicing (A) Comparison of the variant allele fractions in the transcriptome to the genome, for all classes of point mutation. The squared correlation coefﬁ- cient between the genome and transcriptome is in parentheses. Only expressed coding changes are shown (ﬁve or more times coverage). (B) Variant allele fractions in the transcriptome relative to the genome. Nonsense mutations >50 bp from the terminal 30 exon-intron junction are the only variants to show a signiﬁcant difference. (C) Positional effect of mutations on aberrant splicing is shown. Generally, stop signals in the last exon are considered proper, whereas those appearing >50–55 bp upstream of the last exon-exon junction, and therefore upstream of the exon-junction complex, are more likely to be targeted for nonsense-mediated decay (Nagy and Maquat, 1998). We did ﬁnd evidence for nonsense-mediated decay, since the decreased allele fraction in transcriptome relative to genome was signiﬁcantly more pronounced for nonsense mutations if they were >50 bp upstream of the last exon-exon splice junction (p = 0.003; Figure 2B). B B C C Another possible explanation for the low expression of nonsense mutations is that they are tolerated only in genes not expressed in the cancer cells; those occurring in important genes would be subject to negative selection. To explore this possibility, we compared the expres- sion levels from the organoids of normal breast epithelium for genes mutated in the cancer samples. We found no clear- cut differences across the mutation cate- gories for whether the mutated genes were expressed in normal breast epithe- lial cells (Figure S3C), suggesting that this reason does not explain the lower expression levels of nonsense mutations. Therefore, it appears as if only nonsense-mediated decay ex- plains the lower expression of these mutations. Within-Gene Rearrangements Genomic rearrangements contribute to cancer development through several mechanisms, including changing the copy number of a gene or genes, altering the regulatory apparatus of a gene, and reorganizing the exon sequence within a gene or between two genes. To evaluate effects of genomic rear- rangements on transcriptome structure, we classiﬁed somati- cally acquired structural variants across two variables: type of rearrangement (deletion, tandem duplication, inverted, or inter- chromosomal) and whether genes were involved at either side of the breakpoint (gene-to-gene, same or opposite orientation; gene-to-intergenic; within gene, same or different introns; or local genomic complexity, where more than one rearrangement affected one or other gene). For each rearrangement, we identi- ﬁed any aberrant transcript arising from the genes involved, excluding any splice form seen in the normal breast organoids or the Ensembl database. Across the 23 breast cancer samples studied, we identiﬁed 631 intrachromosomal rearrangements conﬁned to a single intron of a gene, mostly deletions and tandem duplications (358 and 192, respectively). Of these, we believe that very few had discernible consequences on transcriptome structure, since there was no apparent excess of such rearrangements generating the highest rank for transcriptional aberration across samples (Figure 3A). For the 38 rearrangements with highest rank, the most common effect on transcript structure was to skip an exon (69%; Figure 3B). As expected, rearrangements that went across different in- trons of the same gene had a considerably greater effect on tran- script structure than those conﬁned to one intron (Figure 3A). In general, the transcriptome reﬂected the rearranged gene struc- ture in an entirely predictable way, with deletions causing exon skips and tandem duplications causing exon reusage (Figure 3B). Of 341 genomic rearrangements involving different introns of the same gene, 84 had the highest rank for transcriptional abnormal- ity. Of these 84, 23 (27%) caused multiple disruptions in the tran- scriptome at the same gene, mostly alternative splice isoforms. Particularly common were exon skips of not just deleted exons but neighboring exons as well. We found a complex transcrip- tional abnormality in the histone H3-lysine-4 (H3K4)-methyl- transferase MLL3 (KMT2C) involving an exon reusage using a cryptic donor (Figure S6A). Follow-up experiments using TCGA data revealed an additional 13 samples with abnormalities in MLL3 (Figure S6B). Within-Gene Rearrangements Even in cancer samples without rearrangements affecting a given gene, we often found evidence for previously undocu- mented transcripts, such as novel splice forms, read-through transcripts, and non-canonical splice acceptor or donor sites. It is, therefore, difﬁcult to argue categorically for a given rear- rangement that an abnormal transcript arises as a direct conse- quence of the genomic change. Instead, since we are more inter- ested in the overall patterns of abnormal transcription caused by somatic mutation, we studied the excess of aberrant tran- scription associated with the different categories of genomic rearrangement. The normalized expression level of aberrant transcripts was ranked for the sample in which the rearrange- ment was found, relative to aberrant transcripts in the other 22 cancer samples (Figure 3A). If a given rearrangement had no effect on transcription, then the ranking would be effectively uni- formly distributed across ranks 1–23, whereas those rearrange- ments that caused signiﬁcant alterations to transcriptome struc- ture would garner the highest rank. Effects of Point Mutations on Structure of the Transcriptome rearrangements identiﬁed in these samples (Supplemental Experimental Procedures); that is, 11.6% of somatically ac- quired genomic rearrangements affecting genes are associated with evidence of aberrant transcription beyond the background rate in breast cancer. This varied by the pattern of genes at the breakpoint, with particularly high rates observed for intragenic rearrangements leading to alterations of exon order but minimal evidence for aberrant transcription arising from rearrangements conﬁned to a single intron within a gene. rearrangements identiﬁed in these samples (Supplemental Experimental Procedures); that is, 11.6% of somatically ac- quired genomic rearrangements affecting genes are associated with evidence of aberrant transcription beyond the background rate in breast cancer. This varied by the pattern of genes at the breakpoint, with particularly high rates observed for intragenic rearrangements leading to alterations of exon order but minimal evidence for aberrant transcription arising from rearrangements conﬁned to a single intron within a gene. We observed a number of different patterns of aberrant tran- scription (Figure 3B). These included fusion transcripts between two genes, alternative splicing events, exon reusage, and pre- mature polyadenylation. To some extent, the alterations in tran- script structure could be predicted by the underlying genomic rearrangements, such as exon skips caused by intragenic dele- tions, but in many cases the abnormalities were rather surprising. In the following sections, we review the transcriptional conse- quences associated with each of the major classes of genomic rearrangement. Effects of Point Mutations on Structure of the Transcriptome There were some differences in the transcription levels of base substitutions according to the predicted consequence on the protein (Figure 2A). We found that silent, missense, and UTR mutations have the same strong correlation between variant allele fractions in the genome and transcriptome, whereas nonsense mutations have a weaker relationship. Indeed, nonsense mutations had a signiﬁcantly lower expres- sion than predicted from the genome compared to other classes of mutation (p < 0.0001). Point mutations can directly affect RNA splicing, leading to retention of introns (especially for splice donor site mutations), exon skipping (splice acceptor site variants), or enhancement of alternative splice sites (other exonic or intronic variants). We assessed the frequency of alternative splicing events related to somatic base substitutions, where the splice isoform was not present in the normal breast organoids (Figure 2C). We found no excess of abnormal splice isoforms associated with muta- tions in exons near splice sites. We found that mutations affecting the essential splice sites at +1, +2, and 1 into the Several reasons could explain the lower expression of nonsense mutations. Nonsense-mediated decay could selec- tively target transcripts with nonsense mutations for degradation. Nonsense-mediated decay depends on the cell distinguishing a premature termination codon from a proper termination codon. 2036 Cell Reports 16, 2032–2046, August 16, 2016 intron were the most strongly associated with altered splicing in the given sample (p = 0.002, p = 0.0001, and p = 0.0005, respec- tively, compared to intronic mutations >100 bp from the nearest exon). Nonetheless, despite this enrichment, the actual fraction of such mutations at essential splice sites that generated detect- able abnormal splice isoforms was <25%, suggesting that most such variants do not affect splicing or the transcripts that result are rapidly degraded. Further into the introns, there were some positions at which mutations caused signiﬁcantly more splicing abnormalities than expected (49, p = 0.04; +23, p = 0.02; +46, p = 0.01; and +60, p = 0.003). Strikingly, several of these isolated positions coincided with sites of reduced germline polymorphism. For example, the regions from +21 to +26 and from +45 to +50 both showed strongly signiﬁcant reductions in genetic variation in the germline (Lomelin et al., 2010), suggest- ing that functional motifs regulating splicing may reside in these sites. Gene-to-Gene Rearrangements in the Same Transcriptional Orientation We identiﬁed 205 somatic rearrangements that juxtaposed one or more exons of two protein-coding genes in the same tran- scriptional orientation; these would be predicted to generate fusion genes. Overall, 70 (34%) of these were expressed (Fig- ure 4A). As seen with the within-gene rearrangements, the tran- scriptome structure was generally as predicted from the genomic rearrangement, although more than one splice isoform There is a clear excess of genomic rearrangements with the maximum ranking for aberrant transcription. Using maximum likelihood methods, we estimated that this excess represents 11.6% (95% conﬁdence interval, 10.4%–12.8%) of 4,234 genic Cell Reports 16, 2032–2046, August 16, 2016 2037 was present in 20 of the 70 rearrangements, increasing the range of transcripts observed (Figure 4B). The only recurrent fusion A B other. In the forme tional start site of b A A A Figure 3. The Transcriptional Conse- quences of Structural Rearrangement All rearrangement types and their position with respect to genes are shown as a matrix in both panels. Transcriptional disruptions caused by each rearrangement type are shown within the matrix. (A) Number of rearrangements causing aberrant transcription. Normalized aberrant transcription levels were contrasted between the sample that contained the rearrangement and all others. Plotted is the aberrant transcription ranking of the rearranged sample relative to all others for the same genes (red bars). The pie charts show the fraction of all rearrangements of that type that are excess in the ﬁnal rank compared to the number expected under a uniform distribution. (B) Types of aberrant transcriptional events caused by rearrangements are shown. B dicted to be out of frame or involve the non-coding UTR, we found evidence for in-frame transcripts in 25 (7%). In many cases, the in-frame transcript was more heavily expressed than the canonical, out-of-frame transcript, suggesting that nonsense-mediated decay may be acting on the latter (Figure 4C). Overall then, these data indicate that 60 of 501 (12%) of genomic rearrangements reordering exons of one or two genes in the same orientation have the potential to generate transcripts encoding in-frame proteins. Many of these are expressed at appre- ciable levels, mostly driven by the up- stream regulatory apparatus. B Figure 4. Rearrangements between and within Genes Figure 4. Rearrangements between and within Genes A (A) Fusions caused by rearranged genes in the same orientation are shown. (B) Proportion of rearrangements predicted to lead to an in-frame event contrasted to the proportion actually expressing in-frame transcripts (top). Characteristics of expressed fusions (bottom) are shown. (C) Many fusions are expressed in multiple iso- forms. ated transcripts linking the sixth exon into a multiply spliced antisense tran- script of SYNE1 (Figure 5C). Fusion tran- scripts involving the same intron of ESR1 are recurrent in breast cancer, and there is evidence they have important func- tional consequences, largely conferred by the C-terminally truncated estrogen receptor (Li et al., 2013). There is also a rearrangement that fuses the ﬁrst 20 exons of the transcriptional co- activator CREBBP to the antisense strand of CLUAP1. CREBBP is a well- known cancer gene that can be targeted by inactivating point mutations (Pasqua- lucci et al., 2011) or, in leukemias, involved in canonical fusion genes (Ca- mo´ s et al., 2006). ated transcripts linking the sixth exon into a multiply spliced antisense tran- script of SYNE1 (Figure 5C). Fusion tran- scripts involving the same intron of ESR1 are recurrent in breast cancer, and there is evidence they have important func- tional consequences, largely conferred by the C-terminally truncated estrogen receptor (Li et al., 2013). There is also a rearrangement that fuses the ﬁrst 20 exons of the transcriptional co- activator CREBBP to the antisense strand of CLUAP1. CREBBP is a well- known cancer gene that can be targeted by inactivating point mutations (Pasqua- lucci et al., 2011) or, in leukemias, involved in canonical fusion genes (Ca- mo´ s et al., 2006). C C C C Rearrangements We identiﬁed 473 genomic rearrange- ments that joined the 50 portion of a gene to an intergenic region and 461 rearrangements linking 30 ends of genes to intergenic space (Figure 6A). As seen with the 30-to-30 gene-to-gene fusions, in the absence of promoters, only one 30 gene-to-intergenic rearrangement led to a detectable RNA transcript. In contrast, 16 (3.4%) of 50 gene-to-intergenic rearrangements led to stable expression of abnormal transcripts related to the rearrangement. novel transcribed sequence from the antisense strand of the distal gene mapped to intronic regions, although a few fusions did generate transcripts that partially or fully overlapped with exons (Figure 5B). This is to be expected since splice sites are directional, so the GT.AG structure of an intron is not reca- pitulated on the antisense strand. However, where one might have expected the reads derived from the antisense strand to be rather scattered, the antisense exons were, in fact, surpris- ingly ﬁxed (Figure 5C). That is, the antisense component of the fusion transcript tended to reuse the same latent splice acceptor and donor sites on the antisense strand. These were almost always associated with consensus GT-AG splice sig- nals. For a small number of examples, multiple antisense exons were recurrently included in the transcript. None of these novel antisense exons was seen in the absence of the given 50-to-50 rearrangement, suggesting that it is the genomic rearrangement that unmasks the latent transcriptional potential of these regions. novel transcribed sequence from the antisense strand of the distal gene mapped to intronic regions, although a few fusions did generate transcripts that partially or fully overlapped with exons (Figure 5B). This is to be expected since splice sites are directional, so the GT.AG structure of an intron is not reca- pitulated on the antisense strand. However, where one might have expected the reads derived from the antisense strand to be rather scattered, the antisense exons were, in fact, surpris- ingly ﬁxed (Figure 5C). That is, the antisense component of the fusion transcript tended to reuse the same latent splice acceptor and donor sites on the antisense strand. These were almost always associated with consensus GT-AG splice sig- nals. For a small number of examples, multiple antisense exons were recurrently included in the transcript. Cell Reports 16, 2032–2046, August 16, 2016 2039 Gene-to-Gene Fusions in Opposite Transcriptional Orientation We would expect half of the genomic re- arrangements linking two genes to join them in opposing orientation, which would be split equally between gene pairs pointing inwardly at each other and gene pairs pointing away from each other. In the former, the 50 regulatory apparatus and transcrip- tional start site of both genes would be retained, and they could start transcripts that would extend into the partner gene on the antisense strand. We identiﬁed 171 somatic rearrangements generating gene-to-gene fusions in opposite orientation, of which 114 were pointing inward (50-to-50 orientation). While there was not much evidence of aberrant transcription arising from 30-to-30 fusions, we found an unexpectedly high frequency of stable transcription at gene pairs pointing inwardly (Figure 5A). was present in 20 of the 70 rearrangements, increasing the range of transcripts observed (Figure 4B). The only recurrent fusion transcript that we observed in this cohort was between NCOA7 and TRMT11, adjacent genes on chromosome 6, caused by tandem duplications in HCC1954 and PD4005a (Figure S4). We examined the protein-reading frame of transcripts arising from gene fusions and within-gene rearrangements that spanned more than one intron (Figure 4B). In 133 of 501 (27%) such events, the resulting exon structure would be predicted to generate an in-frame gene from transcript isoforms reported in Ensembl; we found RNA-seq reads supporting these in-frame transcripts in 35 of 133 (26%). Of the 368 rearrangements pre- In total, 50 (44%) of all 50-to-50 rearrangements generated transcripts that fused the sense portion of one gene with novel exons on the antisense strand of the partner gene. Mostly, the 2038 Cell Reports 16, 2032–2046, August 16, 2016 A C B A B A B B A Regions of Local Complexity We deﬁned a region of local complexity as any gene footprint that contained two or more genomic rearrangements. Typically, these represented sites of extensive genomic ampliﬁcation, such as around ERBB2 or CCND1, or they were regions of chro- mothripsis, a mutational process generating tens to hundreds of localized genomic rearrangements in a one-off catastrophic event (Stephens et al., 2011). Given the complexity of the genomic changes in many of these regions, a surprising number of rearrangements led to measurable transcriptional conse- quences (Figure 7A). Indeed, when compared with genes hit by simple rearrangements, the fractions of rearrangements from regions of local genomic complexity giving aberrant tran- scripts were broadly similar. This suggests that the regulatory apparatus enabling transcription initiation remains at least partially intact in many of these heavily rearranged regions and that the genomic structure supports the production of sta- ble transcripts. One striking conclusion of the analysis is that transcription, once started, will attempt to complete. We found an unexpect- edly high number of transcripts resulting from structural variants that sow the 50 seeds of a gene, namely upstream enhancers, promoter, and ﬁrst few exons, into seemingly infertile ground, such as intergenic space or the antisense strand of another gene. Indeed, in our data, the fraction of such events generating productive transcription was not dissimilar to that observed for rearrangements predicted to cause canonical gene fusions. In the case of gene-to-intergenic rearrangements, the transcrip- tional machinery can scan many tens of kilobases in search of a splice acceptor site, often contributed by the second exon of a downstream intact gene in the same orientation. For sense- to-antisense fusions, the sense transcript often splices into novel exons within the gene footprint of the antisense gene. In one example, this generated a truncated version of the estrogen re- ceptor gene ESR1. Recently, it has been reported that fusion transcripts arising from breaks in the same intron of ESR1 are recurrent in breast cancer and can confer resistance to endo- crine therapy (Li et al., 2013). We found that the transcripts that arose in these regions often represented an integration across multiple rearrangements (Fig- ure 7B; Figure S5). In PD4107a, for example, we found a fusion transcript that linked QKI to the antisense strand of TRPS1 (blue arc, Figure 7B), which was, in fact, driven by two in cis genomic rearrangements linking QKI to ANKRD11 and then ANKRD11 to TRPS1. Rearrangements In some ways, this is the most straightforward analysis of a cancer transcriptome to perform; the causation chain is short and, in theory, predictable. caused by gene-to-intergenic rearrangements that were poten- tially in frame (Figure 6B). The length of the novel intron created by these transcribed gene-to-intergenic fusions was typically in the 50- to 100-kb range, but it could be as high as 250 kb (Figure 6C). Regions of Local Complexity Due to the massive number of rearrange- ments sometimes found in these regions of local complexity, there can be a considerable degree of aberrant transcription. In PD4103a, for example, among the hundreds of clustered rearrangements localized to a small number of genomic regions, we found 12 different fusion transcripts as well as seven alterna- tively spliced isoforms driven by within-gene rearrangements (Figure S5). Another important observation is that the transcriptional output of breast cancer cells is greater than the surrounding stro- mal cells. Using a method that accounts for differences in tumor purity, we found a striking anti-correlation between ER levels and the number of expressed mutations. That is, tumors with high levels of ER express fewer mutations than cancers with low ER. We formally modeled this relationship and determined that, for every 1% decrease in ER expression, 15 more mutations are expressed. As a breast cancer loses estrogen receptor expression and becomes more transcriptionally active, it is more likely to actually express its complement of somatic muta- tions. For example, we found that TP53 mutations are more likely to be expressed in ER than ER+ breast cancers (Figure S7B). While speculative, this is of interest to researchers in the ﬁeld of immunotherapy, since somatic mutations can act as neoanti- gens that trigger host immune responses. There are studies Figure 5. Antisense Expression Caused by Rearranged Genes in Opposite Orientation (A) Stacked bar plot shows the number of expressed transcripts per sample resulting from gene fusions in opposite orientation. (B) The diversity of chimeric transcripts produced by gene-to-gene rearrangements. The expression level of each transcript is plotted on the y axis. Tail-to-tail gene pairs (green) are rarely expressed, whereas, surprisingly, sense-to-sense and sense-to-antisense fusions show similar levels of expression (blue and red, respectively). Transcripts are placed on the x axis according to the type of read joining the two genes. Genes adjoined by exonic reads are plotted to the right on the x axis, and genes brought together only by exon-to-intron reads are on the left. (C) Examples of productive, stable antisense fusion transcripts. Plotted on the y axis are the read depths supporting the fusion. Hatched lines indicate re- arrangement breakpoints. In most cases, we observed a single donor gene, which expresses sequence from its sense strand (yellow), and a single acceptor gene, which expresses sequence from its antisense strand (red). Rarely are both promoters used, leading to reciprocal sense-antisense fusions (both genes express sense and antisense sequence). The fusions SZT2-SLC6A9 and SLC6A9-SZT2 are examples of a reciprocal pair. In general, antisense transcripts display features of traditional exons: they are stably expressed, around 200 bp, and are frequently spliced at GT-AG splice sites (asterisks). Rearrangements None of these novel antisense exons was seen in the absence of the given 50-to-50 rearrangement, suggesting that it is the genomic rearrangement that unmasks the latent transcriptional potential of these regions. The predominant transcripts that resulted from these 50 gene-to-intergenic rearrangements were fusions linking the 50 portion of the broken gene to exon 2 of the ﬁrst intact, sense gene downstream of the breakpoint (Figure 6B). Occasionally, splicing into novel intergenic exons or into exon 1 of the down- stream gene was observed, but, compared to splicing into exon 2, these transcripts were infrequent and represented minor RNA species. In general, the ﬁrst exon of a gene commences with the transcription start site and, therefore, does not contain a splice acceptor site, explaining why 50 gene-to-intergenic rearrange- ments fuse into exon 2. Since the ﬁrst exon of many genes carries the ATG that initiates translation, many of these gene- to-intergenic rearrangements could translate into bona ﬁde fusion proteins. Indeed, we identiﬁed three fusion transcripts It is unclear what functional potential these sense-to- antisense gene fusions might have. Notably, we found an example involving the estrogen receptor, ESR1, which gener- Cell Reports 16, 2032–2046, August 16, 2016 2039 A B C A A B C (legend on ne A B C (legend on next Cell Reports 16, 2032–2046, August 16, 2016 B B B C (legend on next C C (legend on next page) (legend on next page) 2040 Cell Reports 16, 2032–2046, August 16, 2016 loss of transcriptional ﬁdelity, manifesting as shorter 30 UTRs (Mayr and Bartel, 2009), retained introns, trans-splicing (Li et al., 2008), and so on. Here we have concentrated on dissecting the immediate impact that the repertoire of somatic mutations has on the transcriptome in breast cancer, exploring the rules that govern how the transcriptional machinery interprets somatic mutation. In some ways, this is the most straightforward analysis of a cancer transcriptome to perform; the causation chain is short and, in theory, predictable. loss of transcriptional ﬁdelity, manifesting as shorter 30 UTRs (Mayr and Bartel, 2009), retained introns, trans-splicing (Li et al., 2008), and so on. Here we have concentrated on dissecting the immediate impact that the repertoire of somatic mutations has on the transcriptome in breast cancer, exploring the rules that govern how the transcriptional machinery interprets somatic mutation. DISCUSSION We found that 59% of exonic point muta- tions are expressed and 11 6% of genomic rearrangements B some in a given sam of reads across SN Then yi Binðni; pi of reads reporting mixture model depe Xa or Xi. B B however, such a high proportion of genomic rearrangements generating sta- ble fusion transcripts, novel exons, and splicing isoforms could readily provide a substrate for further genomic evolution over many generations. Statistics Statistics Statistical Model for Analyzing Allele-Speciﬁc Expression of Heterozygous Germline SNPs on the X Chromosome DISCUSSION The disturbed transcriptional landscape of cancer cells results from three main forces: (1) direct, primary consequences of somatic mutation; (2) coordinated, secondary gene expression changes resulting from altered cellular signaling, transcrip- tional regulation, and chromatin landscape; and (3) general Figure 5. Antisense Expression Caused by Rearranged Genes in Opposite Orientation Figure 5. Antisense Expression Caused by Rearranged Genes in Opposite Orientation (A) Stacked bar plot shows the number of expressed transcripts per sample resulting from gene fusions in opposite orientation. (B) The diversity of chimeric transcripts produced by gene-to-gene rearrangements. The expression level of each transcript is plotted on the y axis. Tail-to-tail gene pairs (green) are rarely expressed, whereas, surprisingly, sense-to-sense and sense-to-antisense fusions show similar levels of expression (blue and red, respectively). Transcripts are placed on the x axis according to the type of read joining the two genes. Genes adjoined by exonic reads are plotted to the right on the x axis, and genes brought together only by exon-to-intron reads are on the left. (C) Examples of productive, stable antisense fusion transcripts. Plotted on the y axis are the read depths supporting the fusion. Hatched lines indicate re- arrangement breakpoints. In most cases, we observed a single donor gene, which expresses sequence from its sense strand (yellow), and a single acceptor gene, which expresses sequence from its antisense strand (red). Rarely are both promoters used, leading to reciprocal sense-antisense fusions (both genes express sense and antisense sequence). The fusions SZT2-SLC6A9 and SLC6A9-SZT2 are examples of a reciprocal pair. In general, antisense transcripts display features of traditional exons: they are stably expressed, around 200 bp, and are frequently spliced at GT-AG splice sites (asterisks). Cell Reports 16, 2032–2046, August 16, 2016 2041 A C A B C A (C) Genes involved in non-canonical fusions. We observed 18 fusions where a broken gene (donor) splices to another gene (acceptor) that is itself unbroken and often distant. These fusions can be highly expressed (width of line) and cause in-frame transcripts (red line). reporting strong associations between the number of neoanti- gens and response to immunotherapy, and our data suggest that such mutations are more likely to be expressed in ER-ve or ER-low tumors. It is a necessary condition of a somatic mutation to be onco- genic that it induces some transcriptional consequence, but it is far from sufﬁcient. Figure 6. Non-canonical Fusions Caused by Gene-to-Intergenic Breakpoints (A) Percentage of gene-to-intergenic rearrange- ments causing fusions is shown. (B) Length of the intron created is shown. (C) Genes involved in non-canonical fusions. We observed 18 fusions where a broken gene (donor) splices to another gene (acceptor) that is itself unbroken and often distant. These fusions can be highly expressed (width of line) and cause in-frame transcripts (red line). li = 1; if reference allele is on Xa 0; if reference allele is on Xi: Statistics Statistical Model for Analyzing Allele-Speciﬁc Expression of Heterozygous Germline SNPs on the X Chromosome For every heterozygous SNP on the X chromosome, we have an observed count of transcripts expressing the refer- ence allele and a count for those express- ing the variant allele. For each SNP, we do not know whether the reference allele is on the active X chromosome (Xa) or the inactive X chromosome (Xi) in the tumor cells. We further assume that, in the contaminating stromal cells, the ex- pected proportion of cells with the refer- ence allele on Xa is 50%. We model these data using a hierarchical Bayesian model, where the distribution of the fraction of reads deriving from tumor cells from across all genes follows a Dirichlet process. We deﬁne N as the number of heterozy- gous germline SNPs on the X chromo- some in a given sample and ni; i = 1; .; N as the total number of reads across SNP i, of which yi report the reference allele. Then yi Binðni; piÞ, where pi is the expected proportion of reads reporting the reference allele. Here pi follows a mixture model depending on whether the reference allele is on Xa or Xi. reporting strong associations between the number of neoanti- gens and response to immunotherapy, and our data suggest that such mutations are more likely to be expressed in ER-ve or ER-low tumors. It is a necessary condition of a somatic mutation to be onco- genic that it induces some transcriptional consequence, but it is far from sufﬁcient. We found that 59% of exonic point muta- tions are expressed and 11.6% of genomic rearrangements (balanced and unbalanced) hitting a gene footprint generate aberrant transcripts. These aberrant transcripts are polyadeny- lated, stable, and have the potential to generate protein prod- ucts. In the case of cancer, even those that generate proteins will be mostly inconsequential to cell biology, although there will be some that are oncogenic. In the case of species evolution, fðpiÞ = lipi + ð1 piÞ=2; li = 1; if reference allele is on Xa 0; if reference allele is on Xi: li = 1; if reference allele is on Xa 2042 Cell Reports 16, 2032–2046, August 16, 2016 A B re 7. Regions of Local Complexity Give Rise to Unique Transcriptional Consequences ion of local complexity is any gene footprint that contains two or more genomic rearrangements. Statistics Statistical Model for Analyzing Allele-Speciﬁc Expression of Heterozygous Germline SNPs on the X Chromosome Local complexity can occur in regions of chromothrip A A B ure 7. Regions of Local Complexity Give Rise to Unique Transcriptional Consequences egion of local complexity is any gene footprint that contains two or more genomic rearrangements. Local complexity can occur in regions of chromothripsis h-level ampliﬁcation. (legend continued on next pa B B 7. Regions of Local Complexity Give Rise to Unique Transcriptional Consequences n of local complexity is any gene footprint that contains two or more genomic rearrangements. Local complexity can occur in regions of chromothrips vel ampliﬁcation. B B Figure 7. Regions of Local Complexity Give Rise to Unique Transcriptional Consequences Complexity Give Rise to Unique Transcriptional Consequences y gene footprint that contains two or more genomic rearrangements. Local complexity can occur in regions of chromothripsis and g p y q p q local complexity is any gene footprint that contains two or more genomic rearrangements. Local complexity can occur in regions o ampliﬁcation. (legend continued on next page) (legend continued on next page) Cell Reports 16, 2032–2046, August 16, 2016 2043 Cell Reports 16, 2032–2046, August 16, 2016 2043 Then the importance ratio for the jump from pðt1Þ to the pro- posed p is given by We let li Bernð0:5Þ as the prior, with pi DPðaP0Þ. We use the stick-breaking representation of the Dirichlet process as follows: Prðp j Þ Prðpðt1ÞÞ g pðt1Þ p gðp j pðt1ÞÞ: P = X N h = 1 uhdph; with ph P0; Step 4: Updating the Indicator Variables of Whether the Reference Allele Is on Xa or Xi. With a little algebra, this can be shown to follow where dp is a point mass at p, and uh is the weight of the hth gene expression cluster (that is, effectively the proportion of genes for which the fraction of transcripts deriving from tumor cells is p. To capture the stick-breaking formulation, we let kh = Vh Q l < hð1 VlÞ, with Vh Betað1; aÞ. We set a practical maximum number of clusters, C, as 40. As priors, we set P0 Uð0; 1Þ and a Gð0:01; 0:01Þ. Prðli = 1jÞ = 1 , 1 + 1 ph 1 + ph 2yini! : Step 5: Updating the Hyperparameter. Allele Fractions in the Genome and Transcriptome for Different Classes of Somatic Substitution Allele Fractions in the Genome and Transcriptome for Different Classes of Somatic Substitution We ﬁtted linear mixed-effects models to the variant allele frac- tions. The variant allele fraction of reads reporting the mutant allele was the dependent variable. The random effect was the patient from whom the RNA sample derived, allowing for inter-in- dividual differences in both the intercept and slope of the line correlating genomic with transcriptomic variant allele fraction. The ﬁxed effects were the genomic variant allele fraction and class of variant, with the main hypothesis of interest being whether the relationship between genomic and transcriptomic allele fraction differed across the classes of variant. This was tested by adding class of variant-by-slope interaction terms to the model, using likelihood ratio tests to assess for improved ﬁt. Models were ﬁtted using maximum likelihood methods. We ﬁtted linear mixed-effects models to the variant allele frac- tions. The variant allele fraction of reads reporting the mutant allele was the dependent variable. The random effect was the patient from whom the RNA sample derived, allowing for inter-in- dividual differences in both the intercept and slope of the line correlating genomic with transcriptomic variant allele fraction. where h = 1; 2; .; C and ph;i = liph + ð1 phÞ=2. where h = 1; 2; .; C and ph;i = liph + ð1 phÞ=2. Step 2: Updating the Stick-Breaking Weights. These are condi- ; Step 2: Updating the Stick-Breaking Weights. These are condi- tionally conjugate beta posterior distributions as follows: ðVhjÞ Beta 1 + X N i = 1 1ðSi = hÞ; a + X N i = 1 1ðSi > hÞ ! ; where h = 1; .; C 1 and VC = 1. Step 3: Updating the Fraction of Transcripts Deriving from Tumor Cells. We want to generate draws from the posterior distribu- tion of ðph j Þ. We use a Metropolis-Hastings algorithm with beta proposal distribution to do this. So, Statistics Statistical Model for Analyzing Allele-Speciﬁc Expression of Heterozygous Germline SNPs on the X Chromosome The posterior distribu- tion for a is To model the posterior distribution of the Dirichlet process, we use Gibbs sampling as described below. ðajÞ G C + A 1; B X C1 l = 1 logð1 VlÞ ! ; Step 1: Allocating Each Gene to One of the Clusters. We set indicator variables, Sief1; 2; .; Cg, to denote allocation of gene i to a cluster. The posterior distribution of these variables is therefore where the prior is a GðA; BÞ. Statistical Analysis of Relationship between Variant Allele Fractions in the Genome and Transcriptome for Different Classes of Somatic Substitution Statistical Analysis of Relationship between Variant Allele Fractions in the Genome and Transcriptome for Different Classes of Somatic Substitution PrðSi = hjÞ = Vh P l < hð1 VlÞ ni yi pyi h;i 1 ph;i niyi PC r = 1 Vr P l < rð1 VlÞ ni yi pyi r;i 1 pr;i niyi ; EXPERIMENTAL PROCEDURES Detection of Changes to Gene Transcript Structure Detection of Changes to Gene Transcript Structure (E) Fusions genes. A junction between the exons of two protein-coding genesthatisnotfoundincontrolsamples.Wehadtwoapproachesto ﬁnding fusion gene pairs: (1) using aberrant junctions that were split across a breakpoint joining two genes, and (2) using read pairs that were fully mapped (i.e., not split) to both genes. The ﬁrst approach used the seed-and-extend algorithm outlined above. The second approach used a bespoke discordant-pair analysis algorithm, which involved remapping the initially unmapped reads as singletons, re- pairing them together, and evaluating whether there existed clusters of read pairs aligning to two genes in a consistent manner. (4) Merge and extend. K-mers that map to adjacent positions were merged into a single fragment. The k-mers that did not map are those that spanned a breakpoint (inter- or intragenic). We then extended mapped fragments into their unmapped neighbors, one base at a time. We iteratively added bases to the k-mers, and we removed them from the unmapped neighbors, until all fragments mapped to a unique location and we successfully resolved the breakpoints. At this point we allowed mismatches (SNPs, substitutions, or indels) only if they were proceeded by a 2-bp perfect match to the reference. (4) Merge and extend. K-mers that map to adjacent positions were merged into a single fragment. The k-mers that did not map are those that spanned a breakpoint (inter- or intragenic). We then extended mapped fragments into their unmapped neighbors, one base at a time. We iteratively added bases to the k-mers, and we removed them from the unmapped neighbors, until all fragments mapped to a unique location and we successfully resolved the breakpoints. At this point we allowed mismatches (SNPs, substitutions, or indels) only if they were proceeded by a 2-bp perfect match to the reference. We only reported high-conﬁdence fusion genes. These had to have been de- tected by both the seed-and-extend algorithm and the discordant-pair algo- rithm or detected by the discordant-pair algorithm alone with extremely high ranking. This ranking was determined by evaluating the following: how many reads map to other locations in the transcriptome (multi-mappings), the consistency of the mapping positions (percentage of coefﬁcient of varia- tion <25), the number of unique reads in each gene (more than ﬁve), and the consistency of the discordant read pairs’ orientation (<5% inconsistent). We excluded reads where one end maps to the mitochondria. AUTHOR CONTRIBUTIONS A.S., M.R.S., and P.J.C. designed the study, analyzed the data, and wrote the manuscript. K.R., F.F., R.A., S.N.-Z., S.D., A.R, Y.S.J., S.L.C., M.R., E.P., H.R.D., P.S.T., P.V.L., D.C.W., and J.M. contributed towards genomic and transcriptomic tool development and analysis. L.M. developed RNA-seq methods. S.M. was project coordinator. E.A., C.H., V.B., S.A.J.R.A., T.S., O.G., A.V.-S,. O.M., S.B., A.F., A.L., A˚ .B., G.T., A.L.R., A-L.B.-D., and K.P. contributed towards analyses. All authors discussed the results and com- mented on the manuscript. (B) Exon reusages. A junction between two non-adjacent exons from the same transcript, not found in normal organoids (less than two reads) or in Ensembl, where the ﬁrst exon is 3 prime to the second exon. Exon reusages were reported only if there were no Ensembl transcripts that would explain the junction between the exons. We recorded the number of exons reused and required a split forward read and a split reverse read to call an exon reusage. We subcate- gorized exon reusages into those that involved the canonical edges of the exons and those that involved a cryptic splice site (either 50 or 30 of the donor or acceptor site). (B) Exon reusages. A junction between two non-adjacent exons from the same transcript, not found in normal organoids (less than two reads) or in Ensembl, where the ﬁrst exon is 3 prime to the second exon. Exon reusages were reported only if there were no Ensembl transcripts that would explain the junction between the exons. We recorded the number of exons reused and required a split forward read and a split reverse read to call an exon reusage. We subcate- gorized exon reusages into those that involved the canonical edges of the exons and those that involved a cryptic splice site (either 50 or 30 of the donor or acceptor site). SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven ﬁgures, and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.07.028. (A) Exon skips. A junction between two non-adjacent exons from the same transcript, not found in normal organoids (less than two reads) or in Ensembl, where the ﬁrst exon is 5 prime to the second exon. Exon skips were annotated to the Ensembl transcript that involved the fewest number of exons lost. We required a split for- ward read and a split reverse read to call an exon skip. We subca- tegorized exon skips into those that involved the canonical edges of the exons and those that involved a cryptic splice site (either 50 or 30 of the donor or acceptor site). Detection of Changes to Gene Transcript Structure We subcategorized fusion genes based on the relative orientation of the genes involved (same orientation, or in opposite orientation [antisense]) and whether the fusion also involved a cryptic splice site. (5) Clean. Most fusions and aberrant splice breakpoints were resolved us- ing the methods outlined above. However, if a fragment remained un- mapped, we tried to map it using modiﬁed parameters. If there existed homologous sequences between both edges of the breakpoints, lead- ing to ambiguity over where the breakpoint should be positioned, we chose the breakpoint pair that yielded a junction spanning canonical donor-acceptor sequences (GT-AG). (6) Annotate. Having resolved the breakpoints of all fusion genes, aberrant splice forms, and compound events, Architect writes junction coordi- nates into a database (SQLite) and the split reads into a binary align- ment ﬁle (BAM). Each event, which represents the junction of two or more expressed sequences that are not normally found to be adjacent, was ranked across a number of measures, including whether the event is seen in normal samples or in the reference genome; the number of reads supporting the event (total and unique); the sequence context of the junction (i.e., whether canonical donor-acceptor sequences are used); the average number of unique bases per read; and the num- ber of reads mapped in the direct and reverse complemented orienta- tion. We then implemented separate processes that used these mea- sures to arrange the events into the following biologically meaningful categories: ACCESSION NUMBERS The accession numbers for the raw sequence data reported in this paper are The European Genome-phenome Archive under accession numbers EGAD00001002236 and EGAD00001002237 (https://www.ebi.ac.uk/ega/, hosted by the European Bioinformatics Institute [EBI]). Detection of Changes to Gene Transcript Structure read pairs if one end mapped to the mitochondria or if it contained two or more unknown nucleotides (N). The pre-ﬁltering step was performed by aligning all reads to a transcriptome reference containing known exons derived from Ensembl (using a modiﬁed version of BWA 0.5.9). donor and acceptor sites can involve the extension of the exon into the adjacent intron or the reduction of the exon. The junction must not befoundinnormalorganoids(lessthan two reads) orinEnsembl, and it must involve canonical donor-acceptor sequences (GT-AG). donor and acceptor sites can involve the extension of the exon into the adjacent intron or the reduction of the exon. The junction must not befoundinnormalorganoids(lessthan two reads) orinEnsembl, and it must involve canonical donor-acceptor sequences (GT-AG). (D) Early polyadenylationsites.Ajunctionbetweenthe exonofa protein- coding gene and a non-templated run of adenines (or thymines if mapped to the opposite strand), which does not occur at the canon- icaledgeofthegene’sUTR.Thejunctionsmustbe>10bpawayfrom the canonical edge, and they must not be found in normal organoids (less than two reads) or in Ensembl. We subcategorized early polya- denylation sites into those in UTRs and those in coding exons. (2) Index. Having removed all of the reads involving known transcriptional events, we ran a sensitive alignment of the remaining reads. We ﬁrst scanned the human genome, and we built an index containing the po- sitions of all exons of all protein-coding genes, pseudogenes (including polymorphic pseudogenes), and processed transcripts. We used a word size of 9 bp as this provided the best balance between sensitivity and run time. (3) Shatter reads and align k-mers. We then shattered the 75-bp reads into k-mers (13 bp), yielding ﬁve k-mers of equal size and one short k-mer containing the 30 end of the read (the lowest quality portion of Illumina reads). All k-mers were then aligned to the indexed genes without al- lowing for any mismatches. (E) Fusions genes. A junction between the exons of two protein-coding genesthatisnotfoundincontrolsamples.Wehadtwoapproachesto ﬁnding fusion gene pairs: (1) using aberrant junctions that were split across a breakpoint joining two genes, and (2) using read pairs that were fully mapped (i.e., not split) to both genes. The ﬁrst approach used the seed-and-extend algorithm outlined above. The second approach used a bespoke discordant-pair analysis algorithm, which involved remapping the initially unmapped reads as singletons, re- pairing them together, and evaluating whether there existed clusters of read pairs aligning to two genes in a consistent manner. Detection of Changes to Gene Transcript Structure PrðphjÞa 2 64 ðð1 + phÞ=2Þ P i:Si = h liyi x ðð1 phÞ=2Þ P i:Si = h ð1liÞyi x ðð1 + phÞ=2Þ P i:Si = h ð1liÞðniyiÞ x ðð1 phÞ=2Þ P i:Si = h liðniyiÞ 3 75 = ðð1 + phÞ=2Þ P i:Si = h ð2liyilini + niyiÞ ðð1 phÞ=2Þ P i:Si = h ðyi2liyi + liniÞ We developed a suite of tools for the analysis of cancer RNA-seq data, called RNA Architect, comprised of several algorithms. RNA Architect’s central component is a seed-and-extend algorithm used to ﬁnd reads that span disparate regions of the genome. These are junction reads that provide the breakpoint between non-adjacent loci of fusion genes or alter- native splice forms. We describe below how we obtained these highly informa- tive split reads by pre-ﬁltering; grouping the reads into common events; and then classifying, annotating, and ﬁltering the high-conﬁdence events (Figures S1A and S1B). and the proposal distribution is gðx j x0Þ = Betaðx0d; dð1 x0ÞÞ; where d is a scale parameter to be ﬁne-tuned by trial and error to achieve a reasonable acceptance/rejection proportion. and the proposal distribution is gðx j x0Þ = Betaðx0d; dð1 x0ÞÞ; where d is a scale parameter to be ﬁne-tuned by trial and error to achieve a reasonable acceptance/rejection proportion. (1) Pre-ﬁlter. We ﬁrst removed any read that mapped normally to a genic region in the human genome (build 37). That is, the read was fully con- tained in an exon or was split across adjacent exons. We also excluded (A) Proportion of simple and complex rearrangements that lead to an expressed transcript, grouped by sample, is shown. (B) Regions of local complexity and their transcriptional consequences. Two samples’ regions of complexity are shown as pairs of Circos plots. The genomic events one would predict to be expressed are highlighted (blue arcs). Often the tumors do not express these events, or they amalgamate multiple cis rearrangements and express a transcript that combines genes only indirectly linked to another. 2044 Cell Reports 16, 2032–2046, August 16, 2016 2044 Cell Reports 16, 2032–2046, August 16, 2016 read pairs if one end mapped to the mitochondria or if it contained two or more unknown nucleotides (N). The pre-ﬁltering step was performed by aligning all reads to a transcriptome reference containing known exons derived from Ensembl (using a modiﬁed version of BWA 0.5.9). 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S.D., S.M., and A.L. are funded through the BASIS project, which is a European research (C) Alternative donors and acceptors. A junction between two exons thatbegins 50 or30 fromthecanonicalexon-intron border.Alternative (C) Alternative donors and acceptors. A junction between two exons thatbegins 50 or30 fromthecanonicalexon-intron border.Alternative Cell Reports 16, 2032–2046, August 16, 2016 2045 Li, S., Shen, D., Shao, J., Crowder, R., Liu, W., Prat, A., He, X., Liu, S., Hoog, J., Lu, C., et al. (2013). Endocrine-therapy-resistant ESR1 variants revealed by genomic characterization of breast-cancer-derived xenografts. Cell Rep. 4, 1116–1130. project funded by the European Community’s Seventh Framework Pro- gramme (FP7/2010-2014) under the grant agreement 242006. A.L. and A.-L.B.-D. are supported by Southern and Eastern Norway Regional Health Authority (2011079), Norwegian Cancer Society (0332), and the Norwegian Radium Hospital Research Foundation. 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https://openalex.org/W4309837635	https://link.springer.com/content/pdf/10.1007/s10278-022-00732-6.pdf	English	null	Deep Learning-based Non-rigid Image Registration for High-dose Rate Brachytherapy in Inter-fraction Cervical Cancer	Journal of digital imaging	2,022	cc-by	10,195	Abstract In this study, an inter-fraction organ deformation simulation framework for the locally advanced cervical cancer (LACC), which considers the anatomical flexibility, rigidity, and motion within an image deformation, was proposed. Data included 57 CT scans (7202 2D slices) of patients with LACC randomly divided into the train (n = 42) and test (n = 15) datasets. In addition to CT images and the corresponding RT structure (bladder, cervix, and rectum), the bone was segmented, and the coaches were eliminated. The correlated stochastic field was simulated using the same size as the target image (used for deformation) to produce the general random deformation. The deformation field was optimized to have a maximum amplitude in the rectum region, a moderate amplitude in the bladder region, and an amplitude as minimum as possible within bony structures. The DIRNet is a convolutional neural network that consists of convolutional regressors, spatial transformation, as well as resampling blocks. It was implemented by different parameters. Mean Dice indices of 0.89 ± 0.02, 0.96 ± 0.01, and 0.93 ± 0.02 were obtained for the cervix, bladder, and rectum (defined as at-risk organs), respectively. Furthermore, a mean average symmetric surface distance of 1.61 ± 0.46 mm for the cervix, 1.17 ± 0.15 mm for the bladder, and 1.06 ± 0.42 mm for the rectum were achieved. In addition, a mean Jaccard of 0.86 ± 0.04 for the cervix, 0.93 ± 0.01 for the bladder, and 0.88 ± 0.04 for the rectum were observed on the test dataset (15 subjects). Deep learning-based non-rigid image registration is, therefore, proposed for the high-dose-rate brachytherapy in inter-fraction cervical cancer since it outperformed conven- tional algorithms. Deep Learning‑based Non‑rigid Image Registration for High‑dose Rate Brachytherapy in Inter‑fraction Cervical Cancer Received: 29 September 2021 / Revised: 4 July 2022 / Accepted: 18 July 2022 © The Author(s) 2022 / Published online: 23 November 2022 Received: 29 September 2021 / Revised: 4 July 2022 / Accepted: 18 July 2022 © The Author(s) 2022 / Published online: 23 November 2022 https://doi.org/10.1007/s10278-022-00732-6 Journal of Digital Imaging (2023) 36:574–587 https://doi.org/10.1007/s10278-022-00732-6 Journal of Digital Imaging (2023) 36:574–587 ORIGINAL PAPER Introduction In other words, the output of the models would be a transformation matrix rather than transformed images. On the other hand, CNN models can be trained to directly perform image deforma- tion/alignment on the input image. In a study conducted by Miao et al. [37], a CNN model was used to estimate trans- formation parameters in a one-shot for rigid registration for 2D cone-beam CT to CT images. Similarly, Cao et al. [32] trained a CNN model to predict transformation parameters in a thin-palate spline for the DIR of brain MRI images. Eppenhof et al. [33] employed a 3D CNN model to learn thin-plate spline transformation parameters between pairs of 3D images. Sokooti et al. [41] proposed a CNN model which could directly predict a dense displacement vector field (DVF) from a pair of input images. Similar to other supervised deep learning tasks, the quality and quantity of the training dataset play a key role in the overall perfor- mance of the model. In this study, three major issues are addressed: (1) simu- lation of a realistic local deformation in the image domain that takes into account clinical considerations. As men- tioned above, defining a realistic deformation, especially locally and/or organ-wise, would be a challenging task for the creation of the registration ground-truth dataset. In this regard, a novel training data generation framework is proposed in this study, which enables the definition of any desirable deformations depending on the organ rigid- ity/mobility. (2) Creation of a dataset for DIR problems, which can be used by any machine learning approach. (3) Development of a multi-channel deep neural network to simultaneously process the original CT images together with the binary masks of the OARs. This study proposes a new method for the inter-fraction deformation simulation in the HDR brachytherapy to be used in a deep learning-based DIR algorithm. Further- more, a deep learning-based DIR algorithm is developed to directly perform the image alignment of the inter-fraction HDR sessions. A deep learning model consisting of five input channels was implemented and dedicated to the CT images of the bladder, cervix, rectum, and bone OARs in the form of binary masks to perform image alignment between two CT slices. Alternatively, unsupervised deep learning algorithm- based solutions are more appealing for this type of chal- lenge. For example, Wu et al. Introduction Cervical cancer is one the most common cause of can- cer-related female mortality in the world [1] and locally advanced cervical cancer (LACC) is the most common type of cervix cancer [2] which is normally external beam radia- tion therapy (EBRT) and brachytherapy (BT) and chemo- therapy were used to treat the patient. The combination of EBRT and chemotherapy is usually the first step in deliver- ing a prescribed dose to the planning target volume. BT is the second component of cervical cancer treatment, and it involves using low- or high-dose radioactive sources to irradiate the residual tumor locally. The applicators usu- ally set the sources near to the target in order to deliver the maximal dose while protecting normal/healthy tissues. The large inter-fraction organ deformations occur during the BT treatment (different treatment sessions) which causes noticeable uncertainties in the estimated cumulative dose * Isaac Shiri Isaac.shirilord@unige.ch * Reza Reiazi rreiazi@mdanderson.org 1 Department of Medical Physics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 2 Department of Theoretical Physics and Center for Astroparticle Physics, University of Geneva, Geneva, Switzerland 3 Institute of Pathology, RWTH Aachen University Hospital, Aachen, Germany 4 Division of Nuclear Medicine and Molecular Imaging, Geneva University Hospital, CH‑1211 Geneva 4, Switzerland 5 Division of Radiation Oncology, Department of Radiation Physics, University of Texas MD Anderson Cancer Center, Houston, USA 1 Department of Medical Physics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 1 Department of Medical Physics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 2 Department of Theoretical Physics and Center for Astroparticle Physics, University of Geneva, Geneva, Switzerland 3 Institute of Pathology, RWTH Aachen University Hospital, Aachen, Germany 4 Division of Nuclear Medicine and Molecular Imaging, Geneva University Hospital, CH‑1211 Geneva 4, Switzerland 5 Division of Radiation Oncology, Department of Radiation Physics, University of Texas MD Anderson Cancer Center, Houston, USA 1 Journal of Digital Imaging (2023) 36:574–587 575 method, it has been optimized for image reconstruction applications rather than dedicatedly for image registration, as such there is no guarantee that the extracted features are optimal for image registration. Dosovitskiy et al. [44] employed an unsupervised CNN model to estimate the optical flow across the video frames. This study addressed a 2D video sequencing, which contained relatively low levels of noise (compared to medical images), high con- trast, and a relatively small deformation between adjacent frames. Introduction Medial pictures, on the other hand, have a lot of noise, little tissue contrast across organs of interest, and may require a lot of 3D image modification. As mentioned earlier, in fully supervised transformation estimation, the creation of ground-truth data to train the deep learning models is the most challenging task in the deformable medical image registration problem. over the entire treatment course [3, 4]. For instance, target deformations of 20 mm and 48 mm have been reported for the cervix [5] and uterus [6], respectively. Moreover, some studies have reported an average of 63% tumor shrinkage during the treatment [7–10]. Dosimetry uncertainties for organs at risk (OARs) have been reported to be 20–25% in intra- and inter-fraction D2cc, indicating that organ defor- mation contributes significantly to ORAs dosage errors [11–13]. For toxicity prediction, some research advocated using a deformable image registration (DIR) approach to map the EBRT dose to the spatial coordinate of the initial BT percentage [14–16]. Intensity-based image alignment methods (e.g., Demons algorithms) are not an optimal solu- tion for DIR for cervical cancer BT due to large and low deformations in the different organs and tissue contrast, respectively [13, 17]. g g p However, for the DIR problem, a dense flow field ground-truth correspondence is rarely available. Some studies proposed specific frameworks to generate synthe- sized deformation parameters for the creation of a training dataset for the tasks of rigid registration [37], non-rigid image registration [33, 41], and manual annotation [43]. Unlike these methods, Uzunova et al. [45] used statistical appearance models to generate a ground-truth dataset in a 2D fashion for supervised training in the brain and cardiac imaging. Real time image registration could be achieved by using this approach; however, this method has several challenges, such as preparing the ground-truth or the train- ing dataset with clinical considerations. Recently, deep learning approaches, including supervised, unsupervised, and semi-supervised methods, are employed in different medical image analysis tasks [18–28], different BT tasks [29–31], and learn and carry out spatial alignment/ transformation between images [32–41]. These methods usu- ally used convolutional neural networks (CNNs) to extract informative features automatically to perform this task [32–41]. In these studies, different versions of CNN models are trained to learn small sets of transformation parameters, such as translation, rotation, scaling, and affine transforma- tions, in order to offer the optimal transformation parameters for a perfect alignment of the two images. Image Deformation Simulation Deformation Considerations Generally, bony structures do not have any significant deformation when the applicator is inserted into the vaginal cavity. On the other hand, flexible/ soft tissue organs may undergo noticeable deformation after the insertion of the applicator, usually very locally around the cervix region [3]. The cervix is a flexible organ which might be non-rigidly deformed depending on the patient’s posture. The bladder and rectum are also highly flexible organs which could be filled or empty within the course of therapy and may also cause deformation to the surround- ing organs. The bladder and rectum are regarded as OARs while the cervix is the target organ for dose delivery [46]. For a realistic simulation of the organ deformation in the pelvis regions, certain criteria should be met [3–5, 47, 48]. The uterus is a flexible organ which is prone to both rota- tional and translational motions [5]. Moreover, the uterine and vaginal deformation/motion also depend on the status Dataset Since there is no publicly available training dataset specific for the task of image alignment, a large dataset of the female pelvis was collected for the inter-fraction deformation simu- lation. Fifty-seven patients with cervical cancer diagnosed at the cancer institute of this study between 2017 and 2018 were included in this study. The collected data included planning CT images and the corresponding RT structure data. For each patient, a CT scan was acquired in the supine position from about L5 vertebrae to the femoral heads. All images were taken by the same CT scanner (SOMATOM Scope CT scan, Siemens Healthcare, Forchheim, Germany). All CT images were acquired with the same acquisition pro- tocol (KVp = 110 and mAs = 130). Image dimension was set at 512 × 512 for each trans-axial slice with about 80–150 slices with a pixel spacing of 0.84 mm and a slice thickness of 5 mm. In total, there were 7202 2D CT images. The RT structure data, in the form of manually defined contours for each organ, were delineated by a physician for the bladder, rectum, and cervix. The study protocol was approved by the ethical guidelines. The datasets of 57 patients were randomly divided into a 42-patient training set and a 15-patient test set. Couch Elimination One of the main steps in data prepara- tion for the deep learning models is to remove non-related components/regions from the input images to enhance the overall efficiency of the model. To this end, the couches were removed from the input CT images by applying an intensity-based thresholding method. To remove the couches from the entire slices, a CT intensity profile in the posterior- anterior direction was drawn to detect the coach intensity ranges (− 405 HU < couch intensity < − 340 HU) (Fig. 2). The couch removal was visually verified to avoid any mis- segmentation errors. Furthermore, the original CT images, stored in a 512 × 512 matrix, were cropped to a 256 × 512 matrix to fit the body contour in order to enhance memory and computa- tional efficiencies and image were checked to avoid remov- ing body regions in cropped images. Introduction [38] proposed a convolu- tional stacked auto-encoder (CAE) framework to extract discriminative features from pairs of fixed and moving images. They employed the Vercauteren et al. [42] and Shen et al. [43] methods to improve the registration on brain MR images. Though the CAE is an unsupervised 1 3 3 576 Journal of Digital Imaging (2023) 36:574–587 were obtained from CT images by an intensity-based seg- mentation on the CT number (HU > 150). The segmented bony structures were visually verified and manually cor- rected to avoid any mis-segmentation errors. were obtained from CT images by an intensity-based seg- mentation on the CT number (HU > 150). The segmented bony structures were visually verified and manually cor- rected to avoid any mis-segmentation errors. Fig. 1 Representative trans- axial CT slices of two different patients showing the differ- ent rectum volume/location between two sets of CT images Fig. 1 Representative trans- axial CT slices of two different patients showing the differ- ent rectum volume/location between two sets of CT images Methods Dataset were obtained from CT images by an intensity-based seg- mentation on the CT number (HU > 150). The segmented bony structures were visually verified and manually cor- rected to avoid any mis-segmentation errors. Fig. 3 A representative example of cross-organ deformation is shown wherein a rectum filling has greatly impacted the cervix. The trans-axial and sagittal CT views of two patients with dif- ferent rectum filling are shown. In A and C, the rectum with a normal filling has not affected the cervix. In B and D, the rectum filling has substantially deformed the cervix Binary Mask from RT Structures Due to the inter-patient anatomical structure variations and intra-patient anatomy motion, manual organ delineations were employed from the RT structure data to guide local image deformation. A representative sample of rectum deformation between two sets of CT images is presented in Fig. 1. Bone Extraction In the first step, the binary masks of the key organs in the pelvis region, including the rectum, bladder, and cervix, were obtained. It was also necessary to consider a binary mask of the bony structures for the simulation of image deformation. The binary masks of the bony structures 1 3 577 Journal of Digital Imaging (2023) 36:574–587 Fig. 2 Intensity-based thresholding approach was employed to remove couches from CT images. Left: an example of a 2D CT image with a line profile in the posterior-anterior direction. Right: CT inten- sity profile along the line depicted on the CT image shows the spe- cific intensity of the CT couch Fig. 2 Intensity-based thresholding approach was employed to remove couches from CT images. Left: an example of a 2D CT image with a line profile in the posterior-anterior direction. Right: CT inten- o e - sity profile along the line depicted on the CT image shows the spe- cific intensity of the CT couch sity profile along the line depicted on the CT image shows the spe- cific intensity of the CT couch Fig. 2 Intensity-based thresholding approach was employed to remove couches from CT images. Left: an example of a 2D CT image with a line profile in the posterior-anterior direction. Right: CT inten- stochastic deformation (YG) with an average of yc and a dis- persion of 휎 , the probability for YG = [y, y + dy] is given by Eq. (1): of the bladder and rectum (whether they are empty or not) [5]. The cross-organ deformation impacts vary greatly across different organs, as illustrated in Fig. 3 wherein the rectum filling has remarkably deformed the cervix, compared to the bladder filling which had less impact. Eq. (1): (1) p(y)dy = 1 √ 2휋휎2 exp − y −yc 2 2휎2 dy (1) Deformation Simulation To implement image deformation, the correlated stochastic field [49] was simulated using the same size as the target image (used for deformation) to produce general random deformation. Input images were patients’ CT scans and the target images were deformed images using the abovementioned method. Fig. 3 A representative example of cross-organ deformation is shown wherein a rectum filling has greatly impacted the cervix. The trans-axial and sagittal CT views of two patients with dif- ferent rectum filling are shown. In A and C, the rectum with a normal filling has not affected the cervix. In B and D, the rectum filling has substantially deformed the cervix Binary Mask from RT Structures For a Gaussian In such a Gaussian random field, the probability for the field at location × 1 would have a value of Y(× 1) = y1 at location × 2 and a value of Y(× 2) = y2, and so on for N points (i.e., for Y(xN) = yN at location xN). For a Gaussian field knowledge of all n points, the probability distributions would be: In such a Gaussian random field, the probability for the field at location × 1 would have a value of Y(× 1) = y1 at location × 2 and a value of Y(× 2) = y2, and so on for N points (i.e., for Y(xN) = yN at location xN). For a Gaussian field knowledge of all n points, the probability distributions would be: esentative example n deformation is in a rectum filling mpacted the cervix. al and sagittal CT patients with dif- filling are shown. he rectum with a has not affected B and D, the has substantially cervix 1 3 1 3 1 3 3 Journal of Digital Imaging (2023) 36:574–587 578 Fig. 4 Synthetic image deformation workflow. From left to right: input CT image was considered a fixed image, the binary masks of the key organs (created from the RT structure data) were defined on the fixed image, a non-linear deformation was applied on the binary mask, and subsequently, a deformed CT image was created from the deformed binary masks the fixed image, a non-linear deformation was applied on the binary mask, and subsequently, a deformed CT image was created from the deformed binary masks Fig. 4 Synthetic image deformation workflow. From left to right: input CT image was considered a fixed image, the binary masks of the key organs (created from the RT structure data) were defined on p(y1, … ., yn)dy1 … dyn = 1 2휋(detM)1∕2 exp{− y1, … ., yn M−1 ⎛ ⎜ ⎜⎝ y1, … yn, ⎞ ⎟ ⎟⎠ 2 }dy1 … dyn (b t h h i ht idth d h l ) Th f l In which M−1 is the inverse of the correlation matrix M: p(y1, … ., yn)dy1 … dyn = 1 2휋(detM)1∕2 exp{− y1, … ., yn M−1 ⎛ ⎜ ⎜⎝ y1, … yn, ⎞ ⎟ ⎟⎠ 2 }dy1 … dyn (batch, height, wi (2) (batch, height, width, and channels). Binary Mask from RT Structures Therefore, applying this deformation on either images or masks would allow the local deformation of an image considering anatomical condition/consideration (Fig. 4). In which M−1 is the inverse of the correlation matrix M: M = ( 휉(0) 휉(y1, y2) 휉(y1, y2) 휉(0) ) = ( 휎2 휉(0) 휉(0) 휎2 ) As shown in Fig. 5, bony structures did not change in the output deformed image after applying the image deforma- tion. This deformation field was optimized to have a maxi- mum amplitude in the rectum region, a moderate amplitude in the bladder region, and an amplitude as minimum as pos- sible within bony structures. In which 휉 is the covariance function.i In which 휉 is the covariance function.i The simulated deformation field moves all regions with the same strength. To add anatomical considerations, the field strength in bony structures and air (outer body) regions was decreased using regional information and a smoothed correction map. Afterward, the rectum expansion simulation was added, which is a random expansion modulated on the general deformation field. For the deformation offset field, a sigma ( 휎 ) of 10 was chosen while for the bony structure, 휎= 3 was used. Furthermore, to take into account the ana- tomical consideration in deformation offsets for the rectum field, the deformation amplitude was set within the range of 100 to 1000 and for the bladder, 100 to 500 were chosen. Next, a non-linear wrap was applied to the input image, in which the wrap field was specified by offset vectors which define the correspondence between pixel values in the source and the output images at the same original location. The pixel value was obtained by the bilinear interpolation of the 4 neighboring pixels. Additionally, for pixels residing out- side the considered ROI (i.e., the ROIs are binary masks specified for the OARs), the nearest pixel values at the cor- responding mask boundary were calculated using an image wrapping based on the per-pixel flow vectors. A dense image wrapping takes a 4D shape tensor (batch, height, width, and channels), as well as an offset 4D tensor (batch, height, width, channel) as inputs, and returns a 4D float tensor Deep Neural Network Architecture The DIRNet deep learning model proposed by de Vos et al. [50] was used as a baseline model. It is an end-to-end CNN that consists of convolutional regressors, spatial transfor- mation, and resampling block. The convolutional regressor receives concatenated pairs of fixed images (as input) and employs 4 alternating convolutional layers with a kernel size of 3 × 3 and 0-padding followed by 2 × 2 down sampling layers.i The spatial transformer creates a displacement vector field (DVF) that allows the resampler to wrap a moving image around a fixed image. The wrapped image is compared to the fixed image through a normalized cross-correlation (NCC) as a similarity metric which was employed as the loss function. The DIRNet is trained through optimizing a backpropagating dissimilarity between pairs of moving and fixed images using the mini-batch stochastic gradient descent (Adam) algorithm [51] (Fig. 6). 1 3 3 Journal of Digital Imaging (2023) 36:574–587 579 Fig. 5 First row: original and deformed images. Second row: original and deformed masks. Third row: original and deformed grids/fields 579 Journal of Digital Imaging (2023) 36:574 587 Fig. 5 First row: original and deformed images. Second row: original and deformed masks. Third row: original and deformed grids/fields ConvNet Regressor Second, to show the effect of the down- sampling layer, the DIRNet 3 was designed without an aver- age pooling. Third, to evaluate the effect of the number of Conv layers, the DIRNet 4 with 8 Conv layers were designed Fig. 6 Training workflow of the DIRNet architecture. The DIRNet model takes a concatenated pair of fixed and deformed images as inputs. The convolutional regressor generates a grid of control points by analyzing the spatial correspondence between the input images. The spatial transformer generates a displacement vector field that enables the ReSampler to wrap the moving image to the fixed image. Solid lines indicate the baseline DIRNet and the blue dash-line indi- cates the proposed modification for the loss calculation in the back- propagating flow Table 1 Key parameters of the baseline DIRNet and all its variants Models Number of layers Number of trainable parameters Kernel size Loss function Pooling layer Activation function Batch normalization DIRNet 1 (baseline) 4 446,722 3 × 3 Normalized cross-correlation Yes (2 × 2) ELU ✔ DIRNet 2 4 446,722 3 × 3 Mean square error Yes (2 × 2) ELU ✔ DIRNet 3 4 815,874 3 × 3 Normalized cross-correlation No ELU ✔ DIRNet 4 8 446,722 3 × 3 Normalized cross-correlation Yes (2 × 2) ELU ✔ point leading to a continuous/smooth and natural deforma fixed image Since a local deformation was used the bicubic Fig. 6 Training workflow of the DIRNet architecture. The DIRNet model takes a concatenated pair of fixed and deformed images as inputs. The convolutional regressor generates a grid of control points by analyzing the spatial correspondence between the input images. The spatial transformer generates a displacement vector field that enables the ReSampler to wrap the moving image to the fixed image. Solid lines indicate the baseline DIRNet and the blue dash-line indi- cates the proposed modification for the loss calculation in the back- propagating flow enables the ReSampler to wrap the moving image to the fixed image. Solid lines indicate the baseline DIRNet and the blue dash-line indi- cates the proposed modification for the loss calculation in the back- propagating flow Fig. 6 Training workflow of the DIRNet architecture. The DIRNet model takes a concatenated pair of fixed and deformed images as inputs. The convolutional regressor generates a grid of control points by analyzing the spatial correspondence between the input images. ConvNet Regressor The spatial transformer generates a displacement vector field that fixed image. Since a local deformation was used, the bicubic interpolation was employed to transform the moving images. point leading to a continuous/smooth and natural deforma- tion effect. According to Eq. (3), in the case of 2D, f(x) would depend on a couple of control points ( x to update the location of the target point ( ∅(x −i))). Network Training and Implementation Details (3) f(x) = ∑ i=1,2,3훼i∅(x −i) f(x) = ∑ i=1,2,3훼i∅(x −i) (3) Different types of DIRNet were trained to investigate the impact of various DIRNet parameters, such as the loss func- tion, as well as the number of Conv layers and the max- pooling layers. First, to evaluate the effect of the loss func- tion, the DIRNet 2 was designed with the mean square error (MSE) loss function. Second, to show the effect of the down- sampling layer, the DIRNet 3 was designed without an aver- age pooling. Third, to evaluate the effect of the number of Conv layers, the DIRNet 4 with 8 Conv layers were designed ELU Exponential Linear Units ConvNet Regressor The spatial transformer introduced by Jaderberg et al. [34], a differentiable attention to any spatial transforma- tion, can be trained using a backpropagation. The spatial transformer generates a dense DVF for local deformation parameters produced by ConvNet Regressor and allows a neural network to learn how to perform a spatial trans- formation on the input image to enhance the geometric invariance of the model. In this model, a cubic B-spline transformer was employed to its local support capability as a spatial transformer. It takes into account the data points in the neighborhood to modify the location of the target The baseline DIRNet model utilized a Convolutional Net Regressor which consists of 4 layers of 3 × 3 convolutional (Conv) layer with a 0-padding and average pooling layers with a size of 2 × 2 to retain the underlying information dur- ing the down-sampling. Sixteen kernels were used per Conv layer. All Conv layers used exponential linear units (ELU) [52] as an activation function, except for the last layer, which had a linear output. ConvNet Regressor expected a concat- enated pair of fixed and deformed images as input. In addi- tion, a batch normalization [52] with a momentum of 0.9 was applied for each layer. 1 3 Journal of Digital Imaging (2023) 36:574–587 580 point leading to a continuous/smooth and natural deforma- tion effect. According to Eq. (3), in the case of 2D, f(x) would depend on a couple of control points ( x to update the location of the target point ( ∅(x −i))). ReSampler The DVF generated by the spatial transformer needs to be applied to the moving image to transform it into the space of the fixed image. This process requires resampling the mov- ing image on the grid belonging to the pixels (voxels) in the (3) f(x) = ∑ i=1,2,3훼i∅(x −i) fixed image. Since a local deformation was used, the bicubic interpolation was employed to transform the moving images. Network Training and Implementation Details Different types of DIRNet were trained to investigate the impact of various DIRNet parameters, such as the loss func- tion, as well as the number of Conv layers and the max- pooling layers. First, to evaluate the effect of the loss func- tion, the DIRNet 2 was designed with the mean square error (MSE) loss function. 1 https://matplotlib.org/ Table 3 Jaccard coefficients calculated over deformed pelvic masks and the ground-truth masks for different variants of DIRNet models. Results are given for all DIRNet models for the cervix, bladder, and rectum Journal of Digital Imaging (2023) 36:574–587 Journal of Digital Imaging (2023) 36:574–587 581 Table 3 Jaccard coefficients calculated over deformed pelvic masks and the ground-truth masks for different variants of DIRNet models. Results are given for all DIRNet models for the cervix, bladder, and rectum Jaccard coefficient Cervix (mean ± SD) Bladder (mean ± SD) Rectum (mean ± SD) Before registration 0.62 ± 0.15 0.85 ± 0.06 0.51 ± 0.19 SimpleElastix 0.71 ± 0.08 0.83 ± 0.04 0.67 ± 0.11 DIRNet Base model 0.82 ± 0.06 0.91 ± 0.03 0.78 ± 0.07 2 0.76 ± 0.10 0.81 ± 0.04 0.63 ± 0.23 3 0.72 ± 0.08 0.85 ± 0.04 0.66 ± 0.10 4 0.86 ± 0.04 0.93 ± 0.01 0.88 ± 0.04 and visualization, the Scikit-Image2 (version 0.15.0) for image processing, and the Numpy3 package (version 1.16.4) for numerical computing. All models were trained on a 2 NVIDIA® GTX 1080 GPU and Intel Core i9 Xeon CPU with 128 GB RAM. and implemented. Table 1 summarizes the baseline DIRNet model and its variant properties. Furthermore, for the training of the DIRNet and its variants, the binary masks in the loss function were incorporated for the calculation of backpropagation flows. For this purpose, all DIRNet models take a pair of concatenated fixed and moving images as input. The extracted DVF was also applied to the moving binary mask to produce a wrapped binary mask. The loss function tries to minimize the image dissimilarity error between the wrapped and fixed binary masks to maximize image similarity between fixed and deformed images (Fig. 6). Table 2 Dice coefficients calculated over deformed pelvic masks and the ground-truth masks for the different variants of DIRNet model. Results are given for all DIRNet models for the cervix, bladder, and rectum 2 https://scikit-image.org/ 3 https://numpy.org/ ReSampler The DVF generated by the spatial transformer needs to be applied to the moving image to transform it into the space of the fixed image. This process requires resampling the mov- ing image on the grid belonging to the pixels (voxels) in the Table 1 Key parameters of the baseline DIRNet and all its variants ELU Exponential Linear Units Models Number of layers Number of trainable parameters Kernel size Loss function Pooling layer Activation function Batch normalization DIRNet 1 (baseline) 4 446,722 3 × 3 Normalized cross-correlation Yes (2 × 2) ELU ✔ DIRNet 2 4 446,722 3 × 3 Mean square error Yes (2 × 2) ELU ✔ DIRNet 3 4 815,874 3 × 3 Normalized cross-correlation No ELU ✔ DIRNet 4 8 446,722 3 × 3 Normalized cross-correlation Yes (2 × 2) ELU ✔ Table 1 Key parameters of the baseline DIRNet and all its variants 1 Table 2 Dice coefficients calculated over deformed pelvic masks and the ground-truth masks for the different variants of DIRNet model. Results are given for all DIRNet models for the cervix, bladder, and rectum Dice coefficient Cervix (mean ± SD) Bladder (mean ± SD) Rectum (mean ± SD) Before registration 0.75 ± 0.14 0.91 ± 0.05 0.65 ± 0.16 SimpleElastix 0.80 ± 0.06 0.90 ± 0.03 0.78 ± 0.09 DIRNet Base model 0.86 ± 0.08 0.95 ± 0.01 0.87 ± 0.20 2 0.83 ± 0.08 0.89 ± 0.02 0.74 ± 0.22 3 0.79 ± 0.06 0.91 ± 0.02 0.79 ± 0.85 4 0.89 ± 0.02 0.96 ± 0.01 0.93 ± 0.02 Table 3 Jaccard coefficients calculated over deformed pelvic masks and the ground-truth masks for different variants of DIRNet models. Results are given for all DIRNet models for the cervix, bladder, and rectum Jaccard coefficient Cervix (mean ± SD) Bladder (mean ± SD) Rectum (mean ± SD) Before registration 0.62 ± 0.15 0.85 ± 0.06 0.51 ± 0.19 SimpleElastix 0.71 ± 0.08 0.83 ± 0.04 0.67 ± 0.11 DIRNet Base model 0.82 ± 0.06 0.91 ± 0.03 0.78 ± 0.07 2 0.76 ± 0.10 0.81 ± 0.04 0.63 ± 0.23 3 0.72 ± 0.08 0.85 ± 0.04 0.66 ± 0.10 4 0.86 ± 0.04 0.93 ± 0.01 0.88 ± 0.04 581 Journal of Digital Imaging (2023) 36:574–587 Journal of Digital Imaging (2023) 36:574–587 Performance Measurement After training, the accuracy of each DIRNet model was evaluated by three different similarity metrics. The trained models were applied to the concatenated pair of fixed and moving images to extract the DVF. Next, the extracted DVF was applied to the moving binary mask to wrap it into the fixed binary mask. As such, each organ could be evalu- ated separately with pixel-level precision. Each similarity measurement score was averaged over the entire patient slices. For the entire OARs (bladder, cervix, and rectum), three different similarity metrics, including two boundary- based and one surface-based metric, were calculated for each slice separately. Afterward, its average was calculated over the entire 2D slices. The mean ± SD calculated over the 2D slices are reported for each of the abovementioned metrics. In the present experiments, the input image was of 256 × 512 voxel size. The 2D Convs were applied followed by the ELU activation functions, except for the final layer which had a linear output with a kernel size of 3 × 3. The down sampling layer had an average pooling layer of 2 × 2 and the batch normalization was applied to every layer. A mini batch of size 4 was utilized since the image size was large. Each model was trained using 10,000 iterations through optimizing (employing mini-batch stochastic gradi- ent descent) image similarity metrics (MSE for the DIRNet 2 and the normalized cross-correlation for the others) as the loss function (comparing the model outputs and fixed images). The learning rate was set at 1e − 4 for all models.f Different packages and libraries were employed, such as the TensorFlow (version 1.13.2) for implementing deep learning models, the Matplotlib1 (version 3.1.2) for plotting 2 https://scikit-image.org/ 3 https://numpy.org/ 1 3 3 Journal of Digital Imaging (2023) 36:574–587 582 Table 4 Average symmetric surface distance (ASSD) calculated over deformed pelvic masks and the ground-truth masks for the different variants of DIRNet models. Performance Measurement Results are given for the entire DIRNet models for the cervix, bladder, and rectum Average symmetric surface distance (ASSD) Cervix (mean ± SD) Bladder (mean ± SD) Rectum (mean ± SD) Before registration 4.00 ± 1.82 mm 2.68 ± 1.09 mm 7.70 ± 3.90 mm SimpleElastix 2.94 ± 0.78 3.26 ± 0.74 3.04 ± 1.50 DIRNet Base model 1.85 ± 0.45 1.52 ± 0.86 1.97 ± 0.78 2 2.63 ± 1.01 3.46 ± 0.90 3.70 ± 2.75 3 2.86 ± 0.65 2.87 ± 2.37 2.92 ± 0.94 4 1.61 ± 0.46 1.17 ± 0.15 1.06 ± 0.42 Table 4 Average symmetric surface distance (ASSD) calculated over deformed pelvic masks and the ground-truth masks for the different variants of DIRNet models. Results are given for the entire DIRNet models for the cervix, bladder, and rectum Average symmetric surface distance (ASSD) Jaccard Coefficient patients in the external validation dataset. For the Dice and Jaccard, a higher value indicates a better deformation accuracy. For average symmetric distance, lower values indicate better agreement between the contours of the two corresponding images. Registration performance of the basic DIRNet model and its variants were compared to the con- ventional iterative intensity-based image registration imple- mented in the Elastix [54]. To compare the conventional registration algorithm with the DIRNet, a similar grid spac- ing setting and NCC were chosen. Furthermore, stochastic gradient descent was used for the iterative optimization. Registration was performed in 500 iterations. Moreover, the Gaussian smoothing image Pyramid and multiresolu- tion approach were chosen. Average Symmetric Surface Distance (ASSD) Average symmetric surface distance (ASSD) is a surface distance-based measure. According to Eq. (6), the ASSD measures the average of all the distances from the bound- ary between two surfaces of the reference and predicted segmentations: (6) ASSD = 1 \|\|\|Bp + \|\|BG\|\| \|\|\| × ( ∑ x휖BP d(x, BG) + ∑ y∈BG d(y, Bp) ) (6) where Bp and BG represent a set of voxels belonging to boundary contours of the predicted mask and ground-truth, respectively. d(x, BG) and d(y, Bp) are the Euclidean distances between distinct voxels (y and x) from the ground-truth and predicted contours, respectively [53]. Among other models, the basic DIRNet model (DIRNet 1) achieved a higher score in terms of the Dice, Jaccard, and ASSD for the cervix. Furthermore, the DIRNet 3 exhibited a higher score, compared to the SimpleElastix based on ASSD and Jaccard scores. However, based on the Dice score, DIRNet 3 and SimpleElastix had the same performance on the cervix. More detailed results are summarized in Tables 2, 3, and 4 and illustrated in Fig. 7. Representative image slices from the input moving and fixed CT image pairs, together with the registered CT images, are provided in Fig. 7. Dice Coefficient Similarity Metric (DC) The Dice coefficient is very similar to the Jaccard index Eq. (5). (5) DC = 2\|A ∩B\| \|A\| + \|B\| (5) Performance on Cervix The DIRNet 4 model was able to achieve a higher Dice, Jaccard, and lower average symmetric surface distance among all other DIRNet variants evaluated across 15 patients. On average, the DIRNet 4 model achieved closer contours (a lower ASSD with a mean of 1.61 ± 0.46 mm) than all other models. The DIRNet 4 achieved mean scores of 0.89 ± 0.02, 0.86 ± 0.04, and 1.61 ± 0.46 mm for the Dice, Jaccard, and ASSD, respectively, which are higher than other models (Tables 2, 3, and 4). To illustrate the model’s performance on each organ, the Marching Square algorithm [55] was used to generate contours from the 2D predicted binary masks for each DIRNet model (Fig. 7, blue contours). Jaccard Coefficient The intersection-over-union, also known as the Jaccard index Eq. (4), was employed as follows: (4) Jaccard = \|A ∩B\| \|A ∪B\| (4) where A and B are the binary masks of the OARs in the fixed and deformed images, respectively. Dice Coefficient Similarity Metric (DC) Performance on Bladder The DIRNet 4 network, trained with more Conv layers, showed a better performance with respect to the basic DIR- Net model in terms of the Dice, ASSD, and Jaccard indices. This indicates that adding more Conv layers would signifi- cantly improve the performance of the model for all three organs. Furthermore, the basic DIRNet and DIRNet 4 mod- els exhibited improved performance in image registration for the bladder. However, the remaining models failed to improve bladder deformation. Based on the Dice, Jaccard, and average symmetric surface distance metrics, the DIRNet 4 model achieved a higher score for the rectum. Mean Dice, ASSD, and Jaccard coefficients of 0.93 ± 0.02, 1.06 ± 0.42, and 0.88 ± 0.04, respectively, were obtained from the DIRNet 4 model for the rectum. More detailed results are presented in Tables 2, 3, and 4. Additionally, a radar plot of the results was generated to show the models’ performance based on multiple quantitative metrics (Fig. 8). Representative Fig. 8 Radar plots of the quantitative metrics. Results are shown for three metrics, five DIRNet models, and three organs averaged over 15 patients. The colored polygon represents the organs (blue: cervix, red: bladder, and green: rectum). Each corner corresponds to a vari- ant of DIRNet models. Each model has three scores (at the corners) for three organs. For the Dice measure (left plot), the DIRNet 4 has a greater dice score relative to all other DIRNet variants, except for the DIRNet 3 in the bladder. Similarly, for the Jaccard coefficient (mid- dle plot), the DIRNet 4 has a greater score for all organs, in compari- son with all other DIRNet variants. Regarding the average symmetric surface distance (ASSD) (right plot), the DIRNet 4 also has a lower score (best match) for all organs, and the DIRNet 3 has a similar ASSD score as the DIRNet 4 for the cervix DIRNet 3 in the bladder. Similarly, for the Jaccard coefficient (mid- dle plot), the DIRNet 4 has a greater score for all organs, in compari- son with all other DIRNet variants. Regarding the average symmetric surface distance (ASSD) (right plot), the DIRNet 4 also has a lower score (best match) for all organs, and the DIRNet 3 has a similar ASSD score as the DIRNet 4 for the cervix Fig. 8 Radar plots of the quantitative metrics. Results Tables 1, 2, and 3 present the mean ± SD of the Dice, Jac- card, and the average symmetric distance for the differ- ent DIRNet models, respectively, calculated across the 15 3 Journal of Digital Imaging (2023) 36:574–587 583 Fig. 7 Representative slices of the registered image by the different DIRNet models, in comparison with the conventional intensity-based image registration (SimpleElastix). Red, blue, and yellow contours correspond to the bladder, cervix, and rectum, respectively Fig. 7 Representative slices of the registered image by the different DIRNet models, in comparison with the conventional intensity-based image registration (SimpleElastix). Red, blue, and yellow contours correspond to the bladder, cervix, and rectum, respectively Discussion In comparison with similar studies on the rigid-registration problem, Miao et al. [37] used a CNN model to predict rigid transformation parameters for 2D/3D X-ray attenuation maps and 2D X-ray images of the volar plate for virtual implant planning systems. They proposed a hierarchical regression in which the six transformation parameters were categorized into three groups. By transforming the aligned data, synthetic ground-truth data was created. Their proposed methodol- ogy beat registration algorithms (by using mean target reg- istration error (TRE)) based on gradient correlation (TRE: 0.315 mm), mutual information (TRE: 0.285 mm), and an optimization-based (TRE: 0.282 mm) method. In a study conducted by Eppenhof et al. [33], a 3D CNN was used to estimate deformable image transformation directly from two input images. To automatically annotate the ground-truth data for supervised training, they applied a random modification of aligned inhale-exhale 3D lung CT picture pairings. Synthetic deformations generated by a random transformation estima- tor were applied to images. They compared their approach against other state-of-the-art lung registration by measuring the registration error across 10 subjects with 300 landmarks. A mean TRE of 4.02 ± 3.08 mm across 10 subjects was achieved by this model. Fast registration time and automatic annotation are the most important aspects of their study. To enhance the diversity of the registration dataset, Sokooti et al. [41] used random DVFs to augment their dataset. They used a multi-scale CNN on intra-subject 3D chest CT images to estimate the DVF. They proposed a late fusion approach for the input data to the model. For single resolution, B-spline RegNet has a better TRE (4.39 ± 7.54 mm), in comparison with a single resolution with a TRE of 5.48 ± 7.56 mm. How- ever, multi-resolution B-spline exhibited an improved TRE of 2.19 ± 6.22 mm. This study demonstrated the feasibility of deep learning- based DIR to account for inter-fraction organ motion in the HDR brachytherapy. This method exploits image similar- ity features between fixed and moving images to train a CNN model for the DIR. The CNN model was evaluated for pelvis CT images. Since there is no specific dataset (publicly available) for such an application, a deforma- tion simulation was introduced considering realistic image deformation within the bladder and rectum organs. In the proposed method, DIRNet architecture, introduced by de Vos et al. [50], was considered the basic model. Discussion To evalu- ate the effect of the loss functions, Conv layer, and pooling layers, different types of the DIRNet model were trained and tested. All models were separately evaluated for all three organs (i.e., bladder, cervix, and bladder) using the binary masks obtained from the models’ output and the ground-truth data. For this purpose, the Dice, Jaccard, and average symmetric surface distance metrics were employed to assess the performance of deep learning- based image deformation within the specified organs. Fur- thermore, the synthetic deformation based on correlated stochastics field was applied to simulate an inter-fraction organ deformation. g To compare the proposed method to conventional inten- sity-based DIR algorithms, SimpleElastix was implemented on the dataset with tuned parameters. Results showed that the DIRNet 4 model that benefits from more Conv layers (eight layers, in comparison with the basic DIRNet model) achieved better results on all three organs; nevertheless, slightly increased errors were observed for the bladder due to the large deformation of this organ. It is suggested that the number of Conv layers has a significant effect on the perfor- mance of the model, in comparison with the average pool- ing layer. The reason is as Conv layers increase, the model can extract more discriminative features from the input images that help the transformation function to estimate better deformation parameters. On the other hand, the aver- age pooling layer may decrease the image dimension which results in poor resolution in the deformation estimation field. However, for large image deformation within the rectum, pooling layers had a greater effect on the performance of the model, compared to the loss function. For medium deforma- tion relative to the rectum, such as within the cervix, the loss function was important, in comparison with the pool- ing layer. The mean squared loss function, which magnifies Hu et al. [56] proposed an end-to-end CNN approach to predict the displacement field for multimodal image regis- tration between multiple labeled structures. Furthermore, they addressed the challenges of the ground-truth genera- tion for supervised learning by higher-level correspondence information for voxel-level labeling. They used 108 pairs of T2-weighted MRI and 3D transrectal US images for several network architectures. The mean TRE of 3.6 mm on land- mark centroids and the median Dice of 0.87 on the pros- tate gland were achieved. In a study conducted by de Vos et al. Performance on Bladder Results are shown for three metrics, five DIRNet models, and three organs averaged over 15 patients. The colored polygon represents the organs (blue: cervix, red: bladder, and green: rectum). Each corner corresponds to a vari- ant of DIRNet models. Each model has three scores (at the corners) for three organs. For the Dice measure (left plot), the DIRNet 4 has a greater dice score relative to all other DIRNet variants, except for the 1 3 Journal of Digital Imaging (2023) 36:574–587 584 the estimated errors, would show more sensitivity to small errors between the predicted and the ground-truth images. Therefore, the MSE loss function could not be a good choice for small deformations. Furthermore, for low deformation, such as bladder, most of the DIRNet models did not improve registration accuracy. However, DIRNet 1 model (the basic DIRNet model) improved bladder registration. slices from the input fixed and moving images, together with the registered image, are provided in Fig. 7 for quali- tative visual assessment of the registration results based on the test data. Conclusion 4. Kobayashi K, et al.: Dosimetric Variations due to Interfraction Organ Deformation in Cervical Cancer Brachytherapy. 117:555- 558, 2015 In summary, a deep learning-based DIR was introduced in this study to take into account inter-fraction deformation in high-dose-rate cervical cancer BT. The trained network ena- bles a fast and fully-automatic DIR algorithm using a pair of fixed and deformed images. The trained models were applied to 15 cervical cancer patients with manually defined labels for the bladder, cervix, and rectum. The deep learning-based registration results were compared to the SimpleElastix, which is a conventional intensity-based DIR algorithm. The proposed method outperformed the SimpleElastix in all three organs based on the Dice, Jaccard, and ASSD metrics. Finally, it could be concluded that DIRNet 4 model is able to consider large and low deformation for the rectum and bladder, respectively. 5. 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Al-Mansour Z, Verschraegen CJCoio: Locally Advanced Cervical Cancer: What Is the Standard of Care? 22:503–512, 2010 3. Hellebust TP, Dale E, Skjonsberg A, Olsen DR: Inter fraction variations in rectum and bladder volumes and dose distributions during high dose rate brachytherapy treatment of the uterine cer- vix investigated by repetitive CT-examinations. Radiother Oncol 60:273-280, 2001 Conflict of Interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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/. In the present experiment, it was found that the main advan- tage of this study was conducting deformation simulation based on anatomical changes by inserting applicators inside the vaginal cavity. This simulation also improved the proposed models through the use of binary masks which resulted in pixel-level accuracy for model evaluation. Future studies will aim to further improve the proposed method by adding more Conv layers and testing different loss functions. Although a dataset of 57 cervical patients was used in this study, increas- ing the training dataset would result in a model with improved accuracy and generalizability by using data augmentation [57] and decentralize federated learning [58] approaches. Another suggestion would be the use of more structure contours to take into account more anatomical considerations. Defor- mation simulation has a great effect on results; therefore, in future studies more sophisticated deformation models would be applied, and a variety of more patients may be investigated from different treatment strategies in terms of the type of appli- cators and the relevant anatomical deformation. Discussion [50], an end-to-end deep learning DIR called DIRNet was proposed. 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https://openalex.org/W4309224255	https://hal.science/hal-04286073/document	English	null	Optic Flow Processing in Patients With Macular Degeneration	Investigative ophthalmology & visual science	2,022	cc-by	9,115	Optic Flow Processing in Patients With Macular Degeneration Jade Guénot, Yves Trotter, Paul Fricker, Marta Cherubini, Vincent Soler, Benoit R Cottereau To cite this version: Jade Guénot, Yves Trotter, Paul Fricker, Marta Cherubini, Vincent Soler, et al.. Optic Flow Processing in Patients With Macular Degeneration. Investigative Ophthalmology & Visual Science, 2022, 63 (12), pp.21. 10.1167/iovs.63.12.21. hal-04286073 Optic Flow Processing in Patients With Macular Degeneration Jade Guénot, Yves Trotter, Paul Fricker, Marta Cherubini, Vincent Soler, Benoit R Cottereau Guénot, Yves Trotter, Paul Fricker, Marta Cherubini, Vincent Soler, Benoit R Cottereau To cite this version: Jade Guénot, Yves Trotter, Paul Fricker, Marta Cherubini, Vincent Soler, et al.. Optic Flow Processing in Patients With Macular Degeneration. Investigative Ophthalmology & Visual Science, 2022, 63 (12), pp.21. 10.1167/iovs.63.12.21. hal-04286073 Optic Flow Processing in Patients With Macular Degeneration 1Centre de Recherche Cerveau et Cognition, Université Toulouse III–Paul Sabatier, Toulouse, France 2Centre National de la Recherche Scientifique, Toulouse Cedex–CNRS: UMR5549, Toulouse, France 3Unité de rétine, consultation d’ophtalmologie, hôpital Pierre-Paul-Riquet, CHU Toulouse, Toulouse, France PURPOSE. Optic flow processing was characterized in patients with macular degeneration (MD). PURPOSE. Optic flow processing was characterized in patients with macular degeneration (MD). Correspondence: Jade Guénot and Benoit R. Cottereau, CNRS CERCO UMR 5549, Pavillon Baudot, CHU Purpan, BP 25202, Toulouse Cedex 31052, France; METHODS. Twelve patients with dense bilateral scotomas and 12 age- and gender-matched control participants performed psychophysical experiments. Stimuli were dynamic random-dot kinematograms projected on a large screen. For each component of optic flow (translational, radial, and rotational), we estimated motion coherence discrimina- tion thresholds in our participants using an adaptive Bayesian procedure. jade.guenot@cnrs.fr, benoit.cottereau@cnrs.fr. jade.guenot@cnrs.fr, jade.guenot@cnrs.fr, benoit.cottereau@cnrs.fr. Received: July 19, 2022 Accepted: October 24, 2022 Published: November 15, 2022 RESULTS. Thresholds for translational, rotational, and radial patterns were comparable between patients and their matched control participants. A negative correlation was observed in patients between the time since MD diagnosis and coherence thresholds for translational patterns. Citation: Guénot J, Trotter Y, Fricker P, Cherubini M, Soler V, Cottereau BR. Optic flow processing in patients with macular degeneration. Invest Ophthalmol Vis Sci. 2022;63(12):21. CONCLUSIONS. Our results suggest that in patients with MD, selectivity to optic flow patterns is preserved. Keywords: optic flow, macular degeneration, AMD, Stargardt disease, motion processing, aging https://doi.org/10.1167/iovs.63.12.21 tional. In line with this hypothesis, Tarita-Nistor et al.19 found that rotational and translational optic flow patterns elicited strong perception of motion in patients with MD. The correlation between their perceptual reports and objec- tive measures of head tilt was actually more significant than that measured in a group of age-matched controls. Another study found that the sensation of vection elicited by a radial pattern was stronger in patients with MD than in controls with normal visual fields.20 Altogether, these two studies suggest that optic flow processing could even be enhanced in patients, possibly through functional reorgani- zations occurring after the onset of the scotoma and improv- ing perception in peripheral vision. However, they only char- acterized the sensation of self-motion and did not investigate whether patients with MD could also outperform control participants in other visual tasks based on optic flow (e.g., in fine motion discrimination tasks). HAL Id: hal-04286073 https://hal.science/hal-04286073v1 Submitted on 15 Nov 2023 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. Low Vision Copyright 2022 The Authors iovs.arvojournals.org \| ISSN: 1552-5783 METHODS Participants The main clinical data of these patients (sex, age, time since MD diagnosis, diagno- sis, visual acuity, tested eye, scotoma area, PRL eccentricity, and polar angle) are provided in the Table. The psychophys- ical tasks were performed monocularly. The patients were recruited from the Retina Center of the Purpan Hospital in Toulouse, France, where they underwent ophthalmologic examinations. Only patients who met all the inclusion crite- ria were included in the study. Inclusion criteria were as follows: absolute central binoc- ular scotoma of less than 20° in diameter, measured by 30° and 12° central visual fields analyzed with the Octo- pus 300 perimeter (Haag-Streit, Köniz, Switzerland) and with spectral-domain optical coherence tomography (Spec- tralis Optical Coherence Tomography (OCT); Heidelberg Engineering, Heidelberg, Germany; see the “Scotoma Size” section); age between 18 and 90 years; residual vision in both eyes (i.e., visual acuity between 1.3 and 0.5 logMAR); and stable ocular fixation (i.e., presence of a single PRL as detected with OCT measurements; see the “PRL Localization” section). To date, no study has assessed how patients with MD process the three components of optic flow (trans- lational, rotational, and radial). Here, we used a motion direction discrimination task combined with an adaptive Bayesian psychophysical procedure to robustly estimate motion coherence thresholds for each of these components in a group of patients with MD and in a group of gender- and age-matched controls. The experiments in the control group were performed using simulated scotomas of the same dimensions as those of their corresponding patients so that the two populations receive comparable visual inputs. Measurements in the control group were also reproduced without a simulated scotoma to evaluate the contribution of central vision in our task. Patients with a concomitant presence of other visual diseases (cataract, retinal detachment, glaucoma), an inabil- ity to understand the task, or unilateral MD were excluded. TABLE. METHODS Participants stimuli (and therefore focused more on local motion processing), Eisenbarth et al.22 found that patients had higher contrast thresholds than age-matched controls when discriminating the motion direction of a moving plaid presented at eccentricities up to 20°. Even though it is diffi- cult to know exactly which part of the retina was stim- ulated in this case because the authors did not measure the preferred retinal locus (PRL) of patients (defined by Crossland et al.23 as a region of the functioning retina, repeatedly aligned with a visual target for a specific task, that may also be used for attentional deployment and as the oculomotor reference), this result nonetheless suggests that their processing of dynamic stimuli could be impaired beyond the border of the scotoma. Altogether, these studies support the idea that when considering fine discrimination tasks, performances of patients with MD are not enhanced as in the studies described above19,20 and could even be impaired, even though stimuli are presented in peripheral vision. stimuli (and therefore focused more on local motion processing), Eisenbarth et al.22 found that patients had higher contrast thresholds than age-matched controls when discriminating the motion direction of a moving plaid presented at eccentricities up to 20°. Even though it is diffi- cult to know exactly which part of the retina was stim- ulated in this case because the authors did not measure the preferred retinal locus (PRL) of patients (defined by Crossland et al.23 as a region of the functioning retina, repeatedly aligned with a visual target for a specific task, that may also be used for attentional deployment and as the oculomotor reference), this result nonetheless suggests that their processing of dynamic stimuli could be impaired beyond the border of the scotoma. Altogether, these studies support the idea that when considering fine discrimination tasks, performances of patients with MD are not enhanced as in the studies described above19,20 and could even be impaired, even though stimuli are presented in peripheral vision. Thirteen patients were initially included in the study. One of them (87 years old) was not able to perform the task correctly and was excluded. We therefore collected the data from 12 patients (six females, mean age, 64.67 ± 14.72 years) with central vision loss. Six of them had a confirmed diag- nosis of age-related MD (AMD), three of them with atrophic MD and three with Stargardt disease. Optic Flow Processing in Patients With Macular Degeneration M M acular degeneration (MD) is the leading cause of legal blindness in countries with an aging population. A recent meta-analysis estimated that 67 million people are affected in Europe, and this number is expected to increase by 15% by 2050.1 Patients with MD experience a gradual loss of central vision that strongly impairs their ability to perform tasks such as reading2,3 or face recognition.4,5 The evolution of visual functions that mostly rely on periph- eral vision has been much less explored in patients. In particular, very little is known about their ability to process motion and more particularly optic flow—the projection of the visual scene on the retinas during self-displacement.6 Optic flow can be decomposed into different visual patterns (translational and rotational, both due to eye and/or head rotation and radial due to observer forward/backward displacements) that are processed by distinct neural popu- lations in the brain.7,8 This is crucial for heading percep- tion,9,10 path integration,11 and estimating object move- ment during navigation (flow parsing).12 Its processing is even more important in aged populations, who more strongly rely on vision than on other senses during their displacements.13,14 One previous study21 measured motion direction and speed discrimination thresholds in patients with MD using wide-field translations (random-dot kinematograms moving along one direction) presented in peripheral vision. Thresh- olds did not significantly differ between patients with MD and their age-matched controls. Odom et al. (Odom JV, et al. IOVS 2010;51:ARVO E-Abstract 3619) asked their partic- ipants to determine the location of the focus of expansion (FoE) of a radial optic flow pattern and found that head- ing precision was impaired in patients with MD when stim- uli were noisy. Finally, although they did not use full-field Optic flow processing is believed to mostly rely on peripheral vision,15,16 although some studies supported the idea that central vision might also contribute.17 Peripheral stimulation can notably determine the sensation of vection.18 Optic flow processing could therefore be preserved in patients with MD as their peripheral vision remains func- 1 1 Downloaded from iovs.arvojournals.org on 11/15/2022 Downloaded from iovs.arvojournals.org on 11/15/2022 IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 2 Optic Flow Processing in Patients With MD METHODS Participants Summary of Characteristics of Patients and Age-Matched Controls Included in the Study Visual Acuity (logMAR) Subject Sex Age, y Time Since MD Diagnosis, y Diagnosis OD OS Tested Eye Scotoma Area, deg2 PRL Eccentricity, deg PRL Polar Angle, deg MD1 F 57 20 Stargardt 1.17 0.71 OS 15.36 10.58 265.93 MD2 M 63 8 Atrophic MD 0.66 0.56 OD 6.6 2.49 322.19 MD3 M 26 10 Stargardt 1.23 1.16 OD 12.96 14.97 329.19 MD4 F 58 23 Atrophic MD 0.76 ≥1.72 OD 12.18 3.10 187.43 MD5 F 62 7 Atrophic MD 0.68 0.54 OS 14.94 8.94 189.73 MD6 M 59 8 Stargardt 0.63 0.5 OS 7.8 4.75 268.43 MD7 M 80 8 AMD 0.42 ≥1.72 OD 8.52 2.21 302.25 MD8 F 71 9 AMD 0.8 0.75 OD 20 15.56 315 MD9 M 71 5 AMD ≥1.72 0.47 OS 20 4.17 342.56 MD10 F 74 6 AMD 0.95 0.88 OS 20 12.40 204.29 MD11 M 75 2 AMD 0.82 1.61 OD 7.72 3.99 101.56 MD12 F 80 5 AMD 0.49 0.55 OD 14.48 4.10 255.89 C1 F 56 — — –0.09 –0.1 OS — — — C2 M 62 — — –0.01 –0.16 OD — — — C3 M 27 — — –0.21 –0.17 OD — — — C4 F 61 — — 0.02 –0.15 OD — — — C5 F 62 — — 0.01 0.02 OS — — — C6 M 57 — — 0.12 0.11 OS — — — C7 M 80 — — 0.08 –0.05 OD — — — C8 F 71 — — 0.15 0.25 OD — — — C9 M 71 — — 0.14 –0.08 OS — — — C10 F 76 — — –0.01 0.09 OS — — — C11 M 75 — — 0.11 0.38 OD — — — C12 F 76 — — 0.15 0.23 OD — — — Scotoma area and PRL polar coordinates (eccentricity and angle) correspond to the tested eye. OD, right eye; OS, left eye. TABLE. Summary of Characteristics of Patients and Age-Matched Controls Included in the Study a and PRL polar coordinates (eccentricity and angle) correspond to the tested eye. OD, right eye; OS, left eye. Scotoma Size For each patient, an ophthalmologist delineated the border of the scotoma from the fundus of the eye captured by stan- dard and autofluorescence imaging (see Fig. 1). The area of the scotoma (in deg2 of visual angles) was extracted using MATLAB (MathWorks, Natick, MA, USA). This area was used to compute the simulated scotoma of the age- and gender-matched controls (see more details in “Experimental Design” section). Motion Stimuli Visual stimuli were created in MATLAB (R2017a) using the Psychophysics toolbox28,29 and consisted of random-dot kinematograms projected on a large convex screen (58.1° × 43.7° of visual angle, refresh rate: 60 Hz, resolution: 1400 × 1050 pixels) in a dark room and at a viewing distance of 180 cm. Dots were bright disks (0.2° in diameter moving on a dark background, contrast: 100%). They had a density of 0.3945 dots per degree of visual angle. We adapted the protocol described in a previous electrophysiologic study30 to define motion patterns. Each dot had a lifetime of 200 ms (12 frames), during which it moved along a straight line at a constant speed. At the end of this lifetime, it was randomly reassigned to a new location within the screen and given a trajectory and speed corresponding to this new location. To avoid a coherent flickering of the stimulus every 200 ms, each dot initial age was randomly picked between 0 and 166 ms (11 frames) at the beginning of each trial. Dots reaching the border of the screen were immediately relocated at a new position. These processes permit equalizing dot density and mean luminance across the screen during the experiment. We used three different types of optic flow patterns: transla- tional, radial, and rotational (see Fig. 2A). Speed in the trans- lation condition was equal to 7°/s, a value that corresponds to the average preferred speed of MT neurons measured in macaques.31 The radial and rotational optic flow patterns had identical speed distributions. In these conditions, the speed of a dot was a function of its eccentricity (Ecc) and was given by S × Ecc. S was chosen to equalize the average speed in the radial and rotational conditions with the speed in the translational condition, in order to obtain compara- ble thresholds between the three optic flow components. Because the trajectories contained no curvature or accelera- tion and because dot size, density, and speed were equalized between the translational, radial, and rotational optic flow patterns, the only difference between these three conditions was the global motion. Optic Flow Processing in Patients With MD The control group consisted of 12 age- and gender- matched participants (six females; mean age, 64.5 ± 14.33 years; see the Table). All the control participants had a corrected visual acuity over 6/10 on the tested eye. They were recruited via advertisements in local journals and on social media. Test.27 In the subsequent psychophysical tasks, each patient was tested monocularly in the eye with the better acuity (the other eye was patched) except for MD2 and MD3, whose ocular fixation (see the previous paragraph) was much more stable with the other eye, and for MD8, who had a macular hemorrhage on her best eye when she performed the study. Age-matched controls were tested on the same eye as their matched patient. Test.27 In the subsequent psychophysical tasks, each patient was tested monocularly in the eye with the better acuity (the other eye was patched) except for MD2 and MD3, whose ocular fixation (see the previous paragraph) was much more stable with the other eye, and for MD8, who had a macular hemorrhage on her best eye when she performed the study. Age-matched controls were tested on the same eye as their matched patient. Both patients and controls gave written informed consent. The research was conducted at the Centre de Recherche et Cognition (Toulouse, France), and the experimental proto- col was approved by a national ethics committee before the beginning of the study (CPP, Comité de Protection des Personnes, protocols 13018–14/04/2014 and 2020-A02441- 38). PRL Localization For each patient with MD and for each eye, we estimated the position of the PRL with respect to the fovea (see Fig. 1) from high-resolution scans of the retinal fundus acquired with spectral-domain optical coherence tomog- raphy (Spectralis OCT; Heidelberg Engineering) using a method previously described by Maniglia et al.24 (see Supplementary Text 1 for a detailed explanation). Measures of the PRL coordinates were repeated three times to make sure that patients had a stable ocular fixation (i.e., less than 2° of variability across measurements, based on the ocular fixation classification proposed by previ- ous studies).25,26 The final PRL coordinates were obtained by averaging these three measurements together (see the Table) and were subsequently converted into polar coordinates. Downloaded from iovs.arvojournals.org on 11/15/2022 IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 3 Determination of the Tested Eye The visual acuity of each participant was determined for each eye using the Sloan letters of the Freiburg Visual Acuity FIGURE 1. Standard (A) and autofluorescence (B) imaging of the eye fundus of one typical patient (MD5). The macular atrophy (circled in yellow in B) was delineated by an ophthalmologist. The PRL and fovea of this patient are respectively given by the red and green dots. Note that the eye fundus in these two panels has been flipped relative to the horizontal meridian to be aligned with the visual field. (C) Positions of the PRLs for the 12 patients with MD. The PRL of patient MD5 (used in A and B) is marked in red. FIGURE 1. Standard (A) and autofluorescence (B) imaging of the eye fundus of one typical patient (MD5). The macular atrophy (circled in yellow in B) was delineated by an ophthalmologist. The PRL and fovea of this patient are respectively given by the red and green dots. Note that the eye fundus in these two panels has been flipped relative to the horizontal meridian to be aligned with the visual field. (C) Positions of the PRLs for the 12 patients with MD. The PRL of patient MD5 (used in A and B) is marked in red. Downloaded from iovs.arvojournals.org on 11/15/2022 Experimental Design given the short duration and relatively low speed of our stim- uli (see above), it is unlikely that they elicited vection (as a comparison, Tarita-Nistor et al.19 used translational stimuli moving at 60°/s for 2 minutes in their study on vection). Our participants therefore reported the motion direction of the stimuli rather than their self-motion perception. During each block, we manipulated motion coherency (i.e., the percent- age of dots moving in the same direction while the other dots had random directions) and estimated the thresholds corresponding to 80% of correct detection using a Bayesian adaptive psychometric method (QUEST, see below). The three first blocks (one for each optic flow pattern) were considered a training and were thus discarded from further analyses. During the experiments, participants sat in a chair whose height was adapted so that their eyes were at the same level as the center of the screen. Their head was placed on a head-support device clamped on top of a table and equipped with both chin and forehead supports. The chair and head-support devices were positioned so as to ensure a fine alignment between the participants’ head and trunk axes. Participants were instructed to keep this position as constant as possible. Stimuli were presented in blocks of 64 trials and about 3 minutes using the Psychophysics toolbox.28 Only one optic flow pattern (translation, rotation, or expansion/contraction) was used during a block. Participants were involved in a two-alternative forced-choice task. For each trial, they had to report the motion direction of the stimuli (leftward versus rightward for translational, clockwise versus anti- clockwise for rotational patterns, and inward versus outward for radial). Stimuli were presented for 200 ms and partici- pants had up to 2 seconds to respond. Responses reported after this time limit were considered incorrect. Importantly, During the psychophysical measurements, patients with MD were instructed to gaze monocularly on a central fixation cross with their PRL (see Fig. 3A). This central position was also the FoE of optic flow and coincided with the simulated heading for radial patterns. Control participants performed the experiments in two viewing conditions. In the main one, they had to gaze on a fixation cross presented at the posi- tion of the fovea of their paired patient, and their central FIGURE 3. Schematic representation of visual inputs during the task. (A) Patients with MD gazed at a central fixation red cross with their PRL. Downloaded from iovs.arvojournals.org on 11/15/2022 IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 4 Optic Flow Processing in Patients With MD FIGURE 2. Stimuli and experimental protocol. (A) Stimuli were random-dot kinematograms (RDKs) moving in different directions. We used three components of optic flow: translational (upper), rotational (central), and radial (lower panel). (B) RDKs were projected on a large convex screen at a viewing distance of 180 cm. Participants were instructed to fixate on a red cross and to report the motion direction of the stimuli (leftward versus rightward for translational motion, clockwise versus counterclockwise for rotational motion, and inward versus outward for radial motion). We manipulated motion coherence parametrically and estimated the thresholds corresponding to 80% of correct detection. FIGURE 2. Stimuli and experimental protocol. (A) Stimuli were random-dot kinematograms (RDKs) moving in different directions. We used three components of optic flow: translational (upper), rotational (central), and radial (lower panel). (B) RDKs were projected on a large convex screen at a viewing distance of 180 cm. Participants were instructed to fixate on a red cross and to report the motion direction of the stimuli (leftward versus rightward for translational motion, clockwise versus counterclockwise for rotational motion, and inward versus outward for radial motion). We manipulated motion coherence parametrically and estimated the thresholds corresponding to 80% of correct detection. Experimental Design Their scotoma (circled in yellow) masked one portion of the visual field. (B) In the main viewing condition, controls gazed at a red fixation point presented at the position of the fovea of their paired patient, and their central vision was masked with a simulated scotoma. The size and the position of this simulated scotoma corresponded to the one measured in their paired patient. Illustrations in A and B are based on the clinical data (PRL and scotoma) of MD5 (as in Fig. 1). (C) In the second viewing condition, control participants gazed centrally and their visual field was unmasked. FIGURE 3. Schematic representation of visual inputs during the task. (A) Patients with MD gazed at a central fixation red cross with their PRL. Their scotoma (circled in yellow) masked one portion of the visual field. (B) In the main viewing condition, controls gazed at a red fixation point presented at the position of the fovea of their paired patient, and their central vision was masked with a simulated scotoma. The size and the position of this simulated scotoma corresponded to the one measured in their paired patient. Illustrations in A and B are based on the clinical data (PRL and scotoma) of MD5 (as in Fig. 1). (C) In the second viewing condition, control participants gazed centrally and their visual field was unmasked. Downloaded from iovs.arvojournals.org on 11/15/2022 Impact of Central Vision on Optic Flow Processing for Control Participants Before analyzing data in patients, we determined the influ- ence of central vision on optic flow processing in our control participants. Figure 4 shows the distributions of thresh- olds measured in this group with and without a simulated scotoma for translational (in red), rotational (in green), and radial (in blue) patterns. In this case, statistical analyses consisted of a two-way ANOVA with the viewing condition (with or without a simu- lated scotoma) and the optic flow pattern (translational, rota- tional, or radial) as within-subject factors. This ANOVA did not lead to a significant effect of the viewing condition, F(1, 11) = 2.25, P = 0.162, or to a significant interaction between the viewing condition and the optic flow pattern, F(2, 22) = 1.44, P = 0.258 (see Fig. 4). It suggests that in control partic- ipants, optic flow processing is performed by peripheral vision. The ANOVA led to a significant effect of the optic flow pattern, F(2, 22) = 6.87, P = 0.004, driven by higher thresh- olds for translational (mean ± SD, 36.1 ± 19.67) than for rotational (25.03 ± 16.72, P = 0.036, for the paired post hoc t-test with a Bonferroni correction for multiple comparisons) and radial (24.43 ± 12.72, P = 0.015) patterns. Coherence thresholds did not significantly differ between rotational and radial patterns (P = 1.0). RESULTS The aim of this study was to characterize the impact of macu- lar degeneration on optic flow processing. We estimated psychophysical thresholds during a motion discrimination task performed by patients and age-matched control partic- ipants with and without a simulated scotoma. We ran three blocks for each optic flow pattern (trans- lation, rotation, or expansion/contraction) in both patients with MD and controls. We included breaks (3 to 4 minutes on average) between blocks to reduce fatigue. Blocks and condition sequences were randomized to minimize possi- ble learning effects on the results. The whole experiment was completed in one session of about an hour in patients. Two sessions of about the same duration were necessary in controls because measurements in this group included two viewing conditions (see above). Robust Estimation of the Motion Coherence Thresholds Using a Bayesian Adaptive Psychometric Method Because it is difficult to perform long psychophysical measurements in aged populations, we estimated motion coherence thresholds using the QUEST adaptive procedure, which permits rapid and robust estimations. QUEST is a Bayesian method that assumes that the psychophysics func- tion underlying the performance of the participants follows a Weibull distribution. During a block, the estimated param- eters of this function were updated after each trial on the basis of the participant’s performance. Coherence values corresponded to the current maximum likelihood estimate of the threshold. We fixed the maximum number of trials at 64 as it was previously shown that this value leads to robust thresholds in most circumstances.34 We used an initial threshold value of 58% based on pilot recordings performed in patients and age-matched controls. The robust- ness and reproducibility of our psychophysical protocol were also validated from tests–retests performed in two patients and four controls. Results showed that the esti- mated thresholds were very stable across both blocks and sessions. Statistical Analysis For each participant and each optic flow pattern (trans- lational, rotational, and radial), we averaged together the thresholds estimated in the three recording blocks. To compare the performances of the control participants with and without a simulated scotoma, we used a two-way ANOVA with the optic flow pattern (translational, rotational, or radial) and the viewing condition (with or without an artificial scotoma) as within-subject factors. To compare performances between the groups of patients and controls, we used a two-way ANOVA with the optic flow patterns (translational, rotational, or radial) as within-subject factor and the group (patient or control) as the between-subject factor. Sphericity was always respected, and thus, we did not apply any corrections. Significant effects of the ANOVA were explored with post hoc paired t-tests, corrected for multiple comparisons (Bonferroni). The strength of the relationships between the measured thresholds and age, visual acuity, scotoma size, and time since MD diagnosis was assessed with Pearson r correlations. IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 5 Because in this case, control participants did not gaze straight ahead, we measured their eye position with an eye-tracker to control that our measures were not affected by fixation instability. These analyses demonstrated that eye fixation was actually very stable in this case and similar to that measured under straight-ahead fixation. They are presented in supplemen- tary materials (see Supplementary Text 2 and Supplementary Fig. S1). We did not measure eye position in patients with MD because calibration is very time-consuming in this popu- lation and would have significantly reduced the duration and hence the robustness of our psychophysical measures. We nonetheless controlled that they had a stable fixation with their PRL (see the “PRL Localization” section). In the second viewing condition, control participants gazed centrally and stimuli were presented without a simulated scotoma (see Fig. 3C). We used this control condition to determine the contribution of central vision in our task. IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 5 IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 5 vision was masked with a simulated scotoma, as in previous studies32,33 (see Fig. 3B). The size of this simulated scotoma corresponded to the one measured in their paired patient (see the “Scotoma Size” section). In this case, the center of the screen (and therefore the FoE for radial patterns) was projected on their peripheral vision, at the PRL loca- tion of their paired patient. This protocol permits patients and controls to receive comparable visual inputs (it notably equalizes the position and surface of the occluded part of the visual field) and thereby provides a very good frame- work to characterize the effect of macular degeneration on visual processing under wide-field stimulation. Because in this case, control participants did not gaze straight ahead, we measured their eye position with an eye-tracker to control that our measures were not affected by fixation instability. These analyses demonstrated that eye fixation was actually very stable in this case and similar to that measured under straight-ahead fixation. They are presented in supplemen- tary materials (see Supplementary Text 2 and Supplementary Fig. S1). We did not measure eye position in patients with MD because calibration is very time-consuming in this popu- lation and would have significantly reduced the duration and hence the robustness of our psychophysical measures. We nonetheless controlled that they had a stable fixation with their PRL (see the “PRL Localization” section). In the second viewing condition, control participants gazed centrally and stimuli were presented without a simulated scotoma (see Fig. 3C). We used this control condition to determine the contribution of central vision in our task.l vision was masked with a simulated scotoma, as in previous studies32,33 (see Fig. 3B). The size of this simulated scotoma corresponded to the one measured in their paired patient (see the “Scotoma Size” section). In this case, the center of the screen (and therefore the FoE for radial patterns) was projected on their peripheral vision, at the PRL loca- tion of their paired patient. This protocol permits patients and controls to receive comparable visual inputs (it notably equalizes the position and surface of the occluded part of the visual field) and thereby provides a very good frame- work to characterize the effect of macular degeneration on visual processing under wide-field stimulation. Downloaded from iovs.arvojournals.org on 11/15/2022 IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 5 Optic Flow Processing in Patients With MD Comparison Between Motion Coherence Thresholds in Patients and in Age-Matched Controls With a Simulated Scotoma We compared performances in patients and controls. Because we did not observe significant differences between the thresholds measured in the control group for the two Downloaded from iovs.arvojournals.org on 11/15/2022 Downloaded from iovs.arvojournals.org on 11/15/2022 IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 6 Optic Flow Processing in Patients With MD FIGURE 4. Motion coherence thresholds estimated for translational (red), rotational (green), and radial (blue) patterns. The boxplots show the different values for the first quartile, median, last quartile, and the extreme data points. Light and dark boxes respectively represent data from age-matched controls in the conditions without scotoma (light colors) and with scotoma (dark colors). Triangles provide the individual data points of the distributions. Note that they were slightly offset horizontally to improve their visibility. FIGURE 4. Motion coherence thresholds estimated for translational (red), rotational (green), and radial (blue) patterns. The boxplots show the different values for the first quartile, median, last quartile, and the extreme data points. Light and dark boxes respectively represent data from age-matched controls in the conditions without scotoma (light colors) and with scotoma (dark colors). Triangles provide the individual data points of the distributions. Note that they were slightly offset horizontally to improve their visibility. FIGURE 5. Motion coherence thresholds estimated for translational (red), rotational (green), and radial (blue) patterns. The boxplots show the different values for the first quartile, median, last quartile, and the extreme data points. Dark and light boxes respectively represent data from patients with MD and from their age-matched controls. Individual datapoints are respectively given by triangles (for controls) and circles (for patients with MD). Note that they were slightly offset horizontally to improve their visibility. Stars provide significant differences in the post hoc paired t-tests after a Bonferroni correction for multiple comparisons (see details in the text). P < 0.05. P < 0.01. FIGURE 5. Motion coherence thresholds estimated for translational (red), rotational (green), and radial (blue) patterns. The boxplots show the different values for the first quartile, median, last quartile, and the extreme data points. Dark and light boxes respectively represent data from patients with MD and from their age-matched controls. Individual datapoints are respectively given by triangles (for controls) and circles (for patients with MD). Note that they were slightly offset horizontally to improve their visibility. Stars provide significant differences in the post hoc paired t-tests after a Bonferroni correction for multiple comparisons (see details in the text). P < 0.05. **P < 0.01. Downloaded from iovs.arvojournals.org on 11/15/2022 viewing conditions (with and without a simulated scotoma; see above), here, we used only the data collected with a simulated scotoma. for the translational pattern (mean ± SD, 48.05 ± 27.14) were significantly higher than those for the rotational (22.84 ± 14.03, P < 0.001) and radial (29.23 ± 20.3, P = 0.009) patterns. No differences were observed between rotational and radial patterns (P = 0.897). The ANOVA did not lead to a group effect, F(1, 22) = 1.19, P = 0.287, but revealed a signif- icant interaction between groups and patterns, F(2, 44) = 3.28, P = 0.047, although this effect was statistically smaller than the pattern effect. Post hoc pairwise t-tests corrected for multiple comparisons (Bonferroni) indicated that there were no significant differences between thresholds measured in the two groups for translational (P = 0.21), rotational (P = 0.887), and radial (P = 0.964) patterns. Paired t-tests also Figure 5 shows the distributions of these thresholds for translational (in red), rotational (in green), and radial (in blue) patterns. Statistical analyses consisted of a two-way ANOVA with the optic flow pattern (translational, rotational, or radial) as the within-subject factor and the group (patient or control) as the between-subject factor. In agreement with the results observed in the previous section, this ANOVA led to a significant effect of the optic flow pattern, F(2, 44) = 14.29, P < 0.001. Here as well, post hoc t-tests indicated that motion coherence thresholds IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 7 IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 7 Optic Flow Processing in Patients With MD Optic Flow Processing in Patients With MD IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 7 FIGURE 6. (A) Correlations between motion coherence thresholds and scotoma sizes (in degrees), estimated for translational (red, leftward), rotational (green, middle), and radial (blue, rightward) patterns. Data points and associated regression lines are provided for patients (dark colors) and also for their age-matched controls (light colors). Pearson correlation coefficients (R) and associated P values are provided on the upper parts of each panel. (B) Correlations between motion coherence thresholds and durations (in years) since the MD diagnosis. FIGURE 6. (A) Correlations between motion coherence thresholds and scotoma sizes (in degrees), estimated for translational (red, leftward), rotational (green, middle), and radial (blue, rightward) patterns. Data points and associated regression lines are provided for patients (dark colors) and also for their age-matched controls (light colors). Pearson correlation coefficients (R) and associated P values are provided on the upper parts of each panel. (B) Correlations between motion coherence thresholds and durations (in years) since the MD diagnosis. the three optic flow patterns were related to these data, we computed Pearson correlation coefficients. revealed that threshold differences between translational patterns, on the one hand, and the rotational and radial patterns, on the other hand, were significant in patients but not in controls (patients: respectively, P = 0.001 and P = 0.014; mean ± SD: translational, 58.7 ± 30.05; rotational, 22.24 ± 15.76; radial, 29.43 ± 25.5; controls: respectively, P = 1.0 and P = 1.0; mean ± SD: translational, 37.39 ± 20.39; rotational, 23.44 ± 12.74; radial, 29.04 ± 14.54). The correlations between motion coherence thresholds and the two main clinical characteristics (i.e., scotoma size and time since the MD diagnosis) are presented in Figure 6. Other correlations are provided in Supplementary Figure S3. Scotoma size did not correlate with motion coher- ence thresholds measured for the three optic flow patterns (P > 0.05; see Fig. 6A). This result is in agreement with the idea that optic flow is mostly processed by peripheral vision in our experiment (see above). However, in patients, a signif- icant negative correlation was found between time since the MD diagnosis and coherence thresholds for translational patterns, r(10) = –0.64, P = 0.026 (see Fig. 6B). IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 \| 7 No correla- tions were found between visual acuity and motion coher- ence thresholds (P > 0.05; see Supplementary Fig. S3B) or between age and motion coherence thresholds measured for the three optic flow patterns (P > 0.05; see Supplementary Fig. S3A) in controls or patients. However, in patients, a trend was observed for the correlation between age and threshold for translations, r(10) = 0.56, P = 0.058. This trend should be taken with care as the distribution of ages was skewed (one patient and his control were much younger than the other participants) but could indicate an effect of age on transla- tion perception, in agreement with a previous study.35 This effect was not observed in the control group, r(10) = 0.27, P = 0.369. In order to control whether the extreme data points measured in our experiment (i.e., the thresholds measured in two patients for rotational and radial optic flow patterns in Fig. 5) had an impact on our results, we reproduced the statistical analyses described above without these patients (and their associated controls) and found the same effects. Altogether, these results suggest that in patients with MD, the processing of rotational, radial, and translational patterns of optic flow is preserved. Their robustness was controlled by reproducing the analyses using the threshold measured in control participants without a simulated scotoma. Effects remain unchanged in this case (see Supplementary Text 3 for more details). Correlations Between Motion Coherence Thresholds Estimated in Patients With MD and Clinical Data Patients with MD are very different from one another, and there was a wide variety of clinical characteristics in our group of participants (i.e., scotoma size, time since the MD diagnosis, age, visual acuity, sex, etc.; see the Table). To test whether the motion coherence thresholds estimated for Finally, for each optic flow pattern, we examined whether behavioral responses were biased toward one direction or the other (e.g., toward leftward or rightward motions for translational patterns). Our analyses of the correct Downloaded from iovs.arvojournals.org on 11/15/2022 Downloaded from iovs.arvojournals.org on 11/15/2022 DISCUSSION The aim of this study was to characterize the impact of MD and notably AMD on optic flow processing. This is important because the elderly mostly rely on visual cues during loco- motion.36,37 We used an adaptive psychophysical approach to estimate motion coherence thresholds for translational, rotational, and radial patterns in patients with MD and in a group of gender- and age-matched controls with a simulated scotoma. Altogether, we found that despite their disease and notably their very low visual acuity, patients with central vision loss are still able to discriminate the motion direction of optic flow patterns. Motion coherence thresholds for radial and rotational patterns had never been estimated in patients with MD. We found that they did not significantly differ between our two groups, which suggests that motion direction percep- tion is preserved for these patterns in patients. One previ- ous study based on preliminary data found that heading judgments could be impaired in patients when noise is added to radial stimuli (Odom JV, et al. IOVS 2010;51:ARVO E-Abstract 3619). Patients might therefore be affected for some visual functions (heading) but not for others (motion direction discrimination), and further explorations will be needed to get a full picture of the consequences of MD on the processing of radial and rotational optic flow patterns. Our results also suggest that thresholds for translational patterns are preserved in patients. In a previous study,21 patients with MD and age-matched controls performed motion direction and speed discrimination tasks with trans- lational patterns presented in peripheral vision. In agree- ment with our measurements, thresholds did not signif- icantly differ between the two groups. In another study based on local rather than full-field stimuli, Eisenbarth et al.22 reported that contrast thresholds were significantly higher in patients with MD than in a group of age-matched controls when discriminating the motion direction of a plaid presented at 10° and 20° of eccentricity. Our results are not in direct agreement with this study but used a different task based on local motion, which suggests that impairment in local motion processing does not necessarily imply a deficit in optic flow processing. Recordings from a larger popula- tion may clarify whether or not patients might be impaired in this case. DISCUSSION Beyond the comparison between performances in patients with MD and in controls, one of the main results of our study is that thresholds for translational patterns were significantly higher than thresholds for rotational and radial patterns in both groups. This effect is unlikely to be caused by differences in our stimuli because all three patterns were based on identical parameters (dot density, average speed, luminance, etc.; see the Methods section). One possibility is that it reflects an effect of age, as suggested by Billino et al.35 or Kuba et al.,42 who showed that aging impairs the perception of translational but not radial patterns. In our study, we had only two young participants (one 26- year-old patient and his age-matched control), and it is thus difficult to conclude on this point. Future studies should explore the validity of this hypothesis through psychophys- ical measurements in a large population of young and old participants. Another important result of our study is that in the control group, motion coherence thresholds remained unchanged for visual stimulations with and without a simulated scotoma defined from clinical measurements in the paired patients. We used these simulated scotomas because previous studies reported that central vision might contribute to optic flow processing17 and because we wanted to make the signals received by each patient and his or her control as compa- rable as possible. It appears that under our experimen- tal protocol, only peripheral vision was useful to perform the task, in line with the results of previous studies15,16 and with the idea that optic flow is processed within high-level visual areas whose neurons have large receptive fields.43,44 In a previous study performed by Tarita-Nistor et al.,19 patients with bilateral AMD and age-matched controls were exposed to translational and rotational optic flow patterns and had to report their sensation of self-motion (vection). Results showed that patients experienced vection sooner than controls, which suggests that self-induced motion might be improved in this population. In line with this study, Luu et al.20 reported higher vection strength and enhanced spatial presence in patients with AMD compared to healthy subjects when exposed to radial optic flow patterns. In our case, participants were instructed to report the motion direction of different optic flow patterns. Downloaded from iovs.arvojournals.org on 11/15/2022 IOVS \| November 2022 \| Vol. 63 \| No. 12 \| Article 21 Optic Flow Processing in Patients With MD \| 8 ated coherence thresholds and not vection. Moreover, it is unlikely that our visual stimuli induced self-motion percep- tion because of their short duration (200 ms) and relatively slow speed (7°/s on average). Our results therefore cannot be directly compared to those of Tarita-Nistor et al.19 and Luu et al.20 The fact that vection is enhanced in patients with central vision loss19,20 while motion perception is not indi- cates that the cortical networks underlying these two cogni- tive functions might substantially differ. responses did not lead to any significant bias in both patients and controls (see Supplementary Fig. S4). For translational patterns, we reproduced these analyses by taking into account the tested eye (to check for a bias toward temporal or nasal motions) and did not observe significant effects (see Supplementary Fig. S4D). We also did not observe differ- ences between the motion coherence threshold measured in men and women (see Supplementary Fig. S5). We found a significant negative correlation between motion coherence thresholds for translational patterns and time since the MD diagnosis in our group of patients (no other significant correlations were found between thresh- olds and data in the two groups). If this correlation should be interpreted with care and does not imply causality, it raises the interesting possibility that despite their disease and their age, patients with central visual loss might partially improve their ability to perceive translational motion over time, potentially through neural plasticity, as observed in animal models (e.g., Burnat et al.38 showed reorganizations of area MT/V5 and improved motion perception in adult cats a few months after a central retinal lesion). This idea opens promising perspectives for the development of future and innovative therapeutic approaches based on perceptual learning24,39,40 or oculomotor training.41 DISCUSSION We measured the associ- In our study, participants were instructed to gaze on a cross and not to move their head because it permitted to better control their retinal projections. During natural loco- motion, the eyes and the head can move, which greatly complexify retinal projections. 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https://openalex.org/W2995747970	https://mhealth.jmir.org/2020/3/e13461/PDF	English	null	Long-Term Weight Management Using Wearable Technology in Overweight and Obese Adults: Systematic Review	JMIR mhealth and uhealth	2,020	cc-by	7,296	Long-Term Weight Management Using Wearable Technology in Overweight and Obese Adults: Systematic Review Emily Fawcett1; Michelle Helena Van Velthoven1, BSc, MSc, PhD; Edward Meinert1,2, MA, MSc, MBA, MPA, PhD, CEng FBCS, EUR ING 1Department of Paediatrics, University of Oxford, Oxford, United Kingdom 2Department of Primary Care and Public Health, Imperial College London, London, United Kingdom Department of Paediatrics, University of Oxford, Oxford, United Kingdom 2Department of Primary Care and Public Health, Imperial College London, London, United Kingdom Corresponding Author: Edward Meinert, MA, MSc, MBA, MPA, PhD, CEng FBCS, EUR ING Department of Paediatrics University of Oxford John Radcliffe Hospital Children's Hospital Oxford, OX3 9DU United Kingdom Phone: 44 7824446808 Email: e.meinert14@imperial.ac.uk Abstract Background: Although there are many wearable devices available to help people lose weight and decrease the rising prevalence of obesity, the effectiveness of these devices in long-term weight management has not been established. Objective: This study aimed to systematically review the literature on using wearable technology for long-term weight loss in overweight and obese adults. Methods: We searched the following databases: Medical Literature Analysis and Retrieval System Online, EMBASE, Compendex, ScienceDirect, Cochrane Central, and Scopus. The inclusion criteria were studies that took measurements for a period of ≥1 year (long-term) and had adult participants with a BMI >24. A total of 2 reviewers screened titles and abstracts and assessed the selected full-text papers for eligibility. The risk of bias assessment was performed using the following tools appropriate for different study types: the Cochrane risk of bias tool, Risk Of Bias In Nonrandomized Studies-of Interventions, A MeaSurement Tool to Assess systematic Reviews, and 6 questions to trigger critical thinking. The results of the studies have been provided in a narrative summary. Results: We included five intervention studies: four randomized controlled trials and one nonrandomized study. In addition, we used insights from six systematic reviews, four commentary papers, and a dissertation. The interventions delivered by wearable devices did not show a benefit over comparator interventions, but overweight and obese participants still lost weight over time. The included intervention studies were likely to suffer from bias. Significant variances in objectives, methods, and results of included studies prevented meta-analysis. Conclusions: This review showed some evidence that wearable devices can improve long-term physical activity and weight loss outcomes, but there was not enough evidence to show a benefit over the comparator methods. A major issue is the challenge of separating the effect of decreasing use of wearable devices over time from the effect of the wearable devices on the outcomes. Consistency in study methods is needed in future long-term studies on the use of wearable devices for weight loss. (JMIR Mhealth Uhealth 2020;8(3):e13461) doi: 10.2196/13461 (JMIR Mhealth Uhealth 2020;8(3):e13461) doi: 10.2196/13461 Review Long-Term Weight Management Using Wearable Technology in Overweight and Obese Adults: Systematic Review Emily Fawcett1; Michelle Helena Van Velthoven1, BSc, MSc, PhD; Edward Meinert1,2, MA, MSc, MBA, MPA, PhD, CEng FBCS, EUR ING 1Department of Paediatrics, University of Oxford, Oxford, United Kingdom 2Department of Primary Care and Public Health, Imperial College London, London, United Kingdom Fawcett et al JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Fawcett et al JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 1 (page number not for citation purposes) Eligibility Criteria Textbox 1 summarizes the inclusion and exclusion criteria for the participant, intervention, comparators, outcomes, and study types of this systematic review. Textbox 1. Inclusion and exclusion criteria for study selection. • Population: We included studies with obese or overweight adult participants. Overweight was defined as having a BMI ranging between 25 kg/m2 and 29.99 kg/m2 or as defined by the study. Obese was defined as having a BMI of 30 kg/m2 or more. Participants in hospital settings were excluded as these studies were unlikely to focus on long-term effects and as they do not represent the real-world use of wearable devices. • Intervention: Interventions included digital wearable technologies used for monitoring or managing weight. Studies that only included a mobile phone app were excluded. • Intervention: Interventions included digital wearable technologies used for monitoring or managing weight. Studies that only included a mobile phone app were excluded. • Comparators: Comparators included traditional behavioral weight loss approaches, usual care, or another intervention. Studies that did not have a comparator were also included if they met the other inclusion criteria. • Outcomes: The primary outcome was change in physical activity and weight after using digital wearable technology for at least a year. Secondary outcomes were the duration of wearable technology use, the accuracy of wearable technology vs self-reporting, and the use of wearable technology by people with specific medical conditions. • Study types: All types of studies were included. Owing to the rapid advances in technology, studies from only the past 10 years were used—from 2008 onward. reflective of the rapid change in the use of mobile phones and wearables. Keywords related to participant, intervention, comparators, and outcome items were used to search for relevant papers. A librarian was consulted for advice on the searches. The search was adjusted and modified for each database. KEYWORDS telemedicine; mHealth; eHealth; mobile health; obesity; wearable electronic devices; wearable technology; wearable device; digital technology; weight loss; overweight; fitness trackers JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 1 (page number not for citation purposes) https://mhealth.jmir.org/2020/3/e13461 XSL•FO RenderX JMIR MHEALTH AND UHEALTH Fawcett et al these devices [7]. Digital wearables could be a novelty that wears off over time, rather than being part of a sustained lifestyle change [9]. Protocol A protocol was registered with the International Prospective Register for Systematic Reviews (CRD42018096932), with the review structure following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Multimedia Appendix 1). We narrowed down the review question of the protocol, focusing on long-term weight management. In combination with an effective weight management intervention based on a behavior change model, wearable technologies can help people lose weight through various means, eg, by promoting physical exercise, by monitoring food consumption, or by encouraging interuser communication and support [6]. Research on the effectiveness of interventions delivered by wearable devices suggests that these interventions can help lose weight [7]. However, long-term weight loss (>1 year) is often unsuccessful [8]. Wearable devices have only demonstrated a statistically significant weight loss lasting for a few weeks, which greatly reduces the potential usefulness of Introduction Obesity is a rising concern worldwide [1]. By 2030, obesity prevalence in the United States is predicted to be 50% to 51% in men and 45% to 52% in women, and it is estimated that in the United Kingdom, 41% to 48% of men and 35% to 43% of women will be obese [2]. Obesity is well known to be a risk factor for different medical conditions, leading to increased morbidity and mortality [2,3]. Various interacting factors influence the prevalence of obesity, including people’s upbringing, lifestyle, environment, and genetics [4]. Over the past decades, numerous strategies for losing weight have been developed that mainly focus on reducing calorie intake and increasing energy expenditure [1]. It is important to tackle obesity early on, as the ability of a person to increase his or her activity levels decreases as his or her weight increases (particularly BMI >40) [4]. Previous research on weight loss interventions without wearable technology has shown that over a 5-year period, only 20% of individuals maintained a weight loss of more than 5 kg (after an initial loss of around 10 kg) [10,11]. Therefore, this review focused on studies that can aid long-term weight loss. Evidence on the long-term effects of wearables to manage or prevent obesity could be relevant for people seeking to reach a healthy weight and for their medical practitioners [12]. This study systematically reviewed the use of wearable devices for long-term weight loss in overweight and obese adults. This review had four objectives: (1) to investigate the effects of using wearable devices on physical activity and weight outcomes, (2) to examine the duration of wearable technology use, (3) to assess the accuracy of wearable technology vs self-reporting, and (4) to explore the use of wearable technology by people with specific medical conditions. The rapid development of technology has led to a growing market of wearable devices claiming to help people lose weight. Over 100 million wearable devices were sold in 2016, and sales were expected to continue to rise over the next years [5]. Wearable technology refers to any electronic device that is worn on the body, commonly being fitness trackers containing some form of an activity monitor. https://mhealth.jmir.org/2020/3/e13461 JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 2 (page number not for citation purposes) • Population: We included studies with obese or overweight adult participants. Overweight was defined as having a BMI ranging between 25 kg/m2 and 29.99 kg/m2 or as defined by the study. Obese was defined as having a BMI of 30 kg/m2 or more. Participants in hospital settings were excluded as these studies were unlikely to focus on long-term effects and as they do not represent the real-world use of wearable devices. JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Fawcett et al Reviews was used for systematic reviews, and 6 questions to trigger critical thinking were used for qualitative papers [13-16]. Results A standardized data extraction sheet was used to extract data. The extracted data included the title, the research question, the data sources, how the data were analyzed, the main findings, and the conclusions. Study Selection and Characteristics We found 1116 references, and after removing duplicates and adding six references identified through searching reference lists of included studies, 684 titles and abstracts were screened (Figure 1). Furthermore, 44 full texts were assessed for inclusion, of which 28 were excluded (Multimedia Appendix 3). We included five intervention studies: four RCTs and one nonrandomized study of an intervention as shown in Table 1. In addition, we used insights from six systematic reviews, four commentary papers, and a dissertation for additional insights (Table 2). Study Selection The references found were imported into EndNote X9 (Clarivate, Pennsylvania), and duplicates were removed. Overall, 2 reviewers conducted title and abstract screening. Any differences in the chosen studies were discussed until a consensus was reached. The full texts of potentially eligible studies were retrieved and analyzed for eligibility by 2 reviewers. Synthesis of Results We have provided a narrative overview and tabular summary of the findings. A meta-analysis of the studies could not be conducted because of the heterogeneity in their interventions, participants, and outcomes. Information Sources and Search We searched the following databases: Medical Literature Analysis and Retrieval System Online, Compendex, ScienceDirect, Cochrane Central, Scopus, and EMBASE through Ovid. Multimedia Appendix 2 outlines the search terms. Data published before 2008 were not included as these data are not JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 2 (page number not for citation purposes) https://mhealth.jmir.org/2020/3/e13461 XSL•FO RenderX JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 3 (page number not for citation purposes) Quality Appraisal of Individual Studies All included studies underwent a methodological quality appraisal. Relevant appraisal tools were used for different study designs. The Cochrane risk of bias tool was used for randomized controlled trials (RCTs), Risk Of Bias In Nonrandomized Studies-of Interventions was used for nonrandomized studies of interventions, A MeaSurement Tool to Assess systematic Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram showing the selection of included studies. JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 3 (page number not for citation purposes) https://mhealth.jmir.org/2020/3/e13461 https://mhealth.jmir.org/2020/3/e13461 JMIR MHEALTH AND UHEALTH Fawcett et al Table 1. Included intervention studies. Type of paper Title Author (year) Study Randomized con- trolled trial Change in physical activity during a weight management intervention for breast cancer survivors: Association with weight outcomes Fazzino et al (2017) [17] 1 Randomized con- trolled trial Potential impact of wearable technology as part of a multidisciplinary treatment strategy for weight regain following RYGBa Chiang et al (2017) [18] 2 Randomized con- trolled trial Effect of wearable technology combined with a lifestyle intervention on long-term weight loss: The IDEA randomized clinical trial Jakicic et al (2016) [19] 3 Randomized con- trolled trial Effect of a stepped-care intervention approach on weight loss in adults: A randomized clinical trial Jakicic et al (2012) [20] 4 Nonrandomized study of an intervention Long-term outcomes of a Web-based diabetes prevention program: 2-year results of a single-arm longitudinal study Sepah et al (2015) [21] 5 aRYGB: Roux-en-Y gastric bypass. Table 1. Included intervention studies. Table 2. Secondary research and other papers. Quality Appraisal of Individual Studies Type of paper Title Author (year) Study Systematic review Critical review and meta-analysis of multicomponent behavioural e-health interventions for weight loss Podina and Fodor (2018) [16] 6 Systematic review A systematic review and meta-analysis of mobile devices and weight loss with an inter- vention content analysis Lyzwinski (2014) [22] 7 Systematic review Technology-assisted weight management interventions: Systematic review of clinical trials Allen et al (2014) [23] 8 Systematic review Persuasive system design does matter: A systematic review of adherence to Web-based interventions Kelders et al (2012) [24] 9 Systematic review The impact of interventions that integrate accelerometers on physical activity and weight loss: A systematic review Goode et al (2017) [25] 10 Systematic review Technology interventions to curb obesity: A systematic review of the current literature Coons et al (2012) [26] 11 Comment Wearable technology and long-term weight loss Kulick (2017) [27] 12 Comment Wearable technology and long-term weight loss-Reply Jakicic and Belle (2017) [28] 13 Comment Advancing models and theories for digital behavior change interventions [6] Hekler et al (2016) 14 Comment Wearable fitness device does not help maintain weight loss, study finds: Fitness device doesn’t maintain weight loss Dyer (2016) [29] 15 Dissertation Evidence-based psychotherapeutic interventions and mHealth for weight management in overweight: A biopsychosocial framework Assar (2018) [30] 16 JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 4 (page number not for citation purposes) Risk of Bias Within Studies Table 3 outlines the studies that contributed findings to the fulfillment of the objectives of this review: physical activity and weight outcomes, duration of wearable technology use, accuracy of wearable technology over self-reporting, and use by people with specific medical conditions. There was a large variation in bias in all the included papers (Multimedia Appendix 4). In most of the RCTs, there was a high risk of bias because of the lack of blinding of participants, as a blinded version of a wearable device intervention is not possible. All the included RCTs used random sequence generation to generate groups, although information about allocation concealment was missing in half of them. https://mhealth.jmir.org/2020/3/e13461 JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 4 (page number not for citation purposes) https://mhealth.jmir.org/2020/3/e13461 XSL•FO RenderX JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Table 3. Included intervention studies and findings. Population with a spe- cific medical condi- tion Accuracy com- pared with self- reporting Long-term use Physical activ- ity and weight outcomes Description Author (year) Study Breast cancer preven- tion Yes Yes Yes An RCTa assessing the effects of mobile health weight management on physical activity, weight loss, and weight mainte- nance Fazzino et al (2017) [17] 1 After repair of failed Roux-en-Y gastric by- pass No No Yes An RCT on the weight loss after repaired RYGBb surgery, with and without wear- able devices Chiang et al (2017) [18] 2 No Yes Yes Yes An RCT comparing outcomes of technol- ogy-enhanced interventions with standard behavioral interventions Jakicic et al (2016) [19] 3 No No Yes No An RCT comparing a standard and stepped-care intervention in weight loss Jakicic et al (2012) [20] 4 Prediabetes No Yes No A diabetes prevention study measuring the outcomes of weight and hemoglobin A1c Sepah et al (2015) [21] 5 aRCT: randomized controlled trial Table 3. Included intervention studies and findings. bRYGB: Roux-en-Y gastric bypass. group, but it is not noted whether the comparator group were given similar recommendations. A significant benefit was only found at 2 years (P=.03). Association Between Weight Outcomes and Change in Physical Activity The three studies reporting on weight loss and physical activity outcomes concluded that using wearable devices had a benefit on these outcomes, but not compared with the comparator groups. Study 1 showed a significant rise in moderate-to-vigorous physical activity over 18 months and divided participants into high or low original weight loss and high or low weight regain groups. At 6 months, the high weight loss groups had significantly higher level of moderate-to-vigorous physical activity than the low weight loss group. However, at 12 and 18 months, the high loss and high regain groups’ level of moderate-to-vigorous physical activity fell, leaving the high loss and low regain group with a significantly higher level of moderate-to-vigorous physical activity than all other groups. In both the standard and wearable device groups of study 3, there was an increase in the duration of moderate-to-vigorous physical activity sessions ≥10 min over a 12-month period, but there was no statistically significant difference between the two groups. The percentage of weight lost did differ, with significantly greater weight loss in the comparator group compared with the wearable devices group from 12 months onward. Study 3 did not find an association between physical activity and weight loss within its groups. aRCT: randomized controlled trial. Principal Findings Accuracy was reported by two studies. Study 1 found that self-reported moderate-to-vigorous physical activity was significantly higher than that recorded by the wearable device. The groups with poorer outcomes (low loss or high regain) had larger discrepancies between the two methods. The self-reported and accelerometer-derived moderate-to-vigorous physical activities were most similar to the high loss and low regain group. the moderate-to-vigorous physical activity data from self-reporting and the accelerometer were, however, collected on different weeks. However, the high loss and low regain group still overestimated moderate-to-vigorous physical activity, and the overestimation did not reduce over time. Study 1 suggested “social desirability to report physical activity adherence,” with participants inflating self-reported moderate-to-vigorous physical activity. This review showed some evidence that wearable devices can improve long-term physical activity and weight loss outcomes, but there was not enough evidence to show a benefit over the comparator methods. The comparator interventions differed among studies, which adds to the difficultly in determining the impact on outcomes. Although the term standard was used, there was no standardization in the comparators’ intervention, with different levels of support and procedures. Overall physical activity levels increased from baseline, but there was no difference between wearable and comparator interventions. Study 1 found that those who sustained higher physical activity levels were more likely to maintain weight loss. Retention was fairly high in the included intervention studies. The mechanism through which wearable devices have an effect compared with other methods was not known as diet and physical activity were not different. The accuracy of wearable devices varied, which could be explained by the different features and technology of wearables. A total of three included studies focused on populations with a specific medical condition. The difference in populations added a challenge to comparing the studies as the results of a study on the weight management of patients with one medical condition may not apply to patients with another medical condition or the general population. Use of Wearable Technology by People With Specific Medical Conditions Overall, three out of the five studies focused on populations with a specific medical condition (Table 4). This included a history of breast cancer (study 1), repair of failed Roux-en-Y gastric bypass (study 2), or prediabetes (study 5). Study 1 analyzed those who attended the visits but had invalid or missing data and found no significant difference in cancer treatment–related variables. Study 5 measured blood glucose (hemoglobin A1c) levels and showed a significant beneficial reduction over 24 months. Maintenance of Wearable Technology Use Retention was fairly high in four studies, but study 2 did not provide data (Table 4). Study 4 was the only study mentioning to offer a monetary incentive for assessments (US $10-$25). Study 4 compared a standard intervention with a stepped-care intervention, where the intensity of support (such as telephone intervention and additional individual sessions) increased if certain goals were not met. Study 2 compared 27 individuals using wearable devices with 260 individuals who were not using a wearable device. A total of 8000 steps per day was recommended for the intervention JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 5 (page number not for citation purposes) JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 5 (page number not for citation purposes) https://mhealth.jmir.org/2020/3/e13461 https://mhealth.jmir.org/2020/3/e13461 XSL•FO RenderX JMIR MHEALTH AND UHEALTH JMIR MHEALTH AND UHEALTH Table 4. Retention rate across included intervention studies. Notes Retention at 24 months, % Retention at 18 months, % Retention at 6 months, % Description Author (year) Study 80% maintained intervention use at 18 months but without valid accelerometer data. N/A 68 N/Ab An RCTa assessing the effects of mobile health weight manage- ment on physical activity, weight loss, and weight maintenance Fazzino et al (2017) [17] 1 N/A N/A N/A N/A An RCT on the weight loss after repaired RYGBc surgery, with and without wearable devices Chiang et al (2017) [18] 2 N/A 75 N/A N/A An RCT comparing outcomes of technology-enhanced interven- tions with standard behavioral interventions Jakicic et al (2016 [19] 3 N/A N/A 72 N/A An RCT comparing a standard and stepped-care intervention in weight loss Jakicic et al (2012) [20] 4 N/A 70.1 N/A 79.1 A diabetes prevention study measuring the outcomes of weight and hemoglobin A1c Sepah et al (2015) [21] 5 able 4. Retention rate across included intervention studies. aRCT: randomized controlled trial. bN/A: not applicable. cRYGB: Roux-en-Y gastric bypass. JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 6 (page number not for citation purposes) bN/A: not applicable. aRCT: randomized controlled trial. Authors' Contributions EM conceived the study objectives and oversaw the original study protocol. EF reviewed the initial study protocol, made amendments as per this manuscript’s methods, executed the review independently (with peer review on study inclusion), and drafted the final manuscript on her own. EM gave feedback to EF, and EF incorporated all feedback. DB also provided feedback on iterations. MV rewrote the paper and made major revisions based on peer-review feedback. All authors approved the final manuscript. EM is the guarantor. Acknowledgments The authors would like to thank Karine Barker at the Radcliffe Science Library. Research issues identified and prioritized by the members of the public in a workshop at the European Scientific Institute in July 2017 were used to guide the focus of this study. The authors declare that the manuscript is an honest, accurate, and transparent account of the study being reported, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. This study was a systematic review of publicly available literature. This work was supported by the Sir David Cooksey Fellowship in Healthcare Translation and the Final Honour School of Medical Sciences, Cell and Systems Biology and Neuroscience at the University of Oxford. Limitations There were only five studies with a relatively small sample size assessing the long-term use of wearable devices. It was not possible to undertake a meta-analysis because of the heterogeneity among participants, wearables, methods, and https://mhealth.jmir.org/2020/3/e13461 XSL•FO RenderX JMIR MHEALTH AND UHEALTH Fawcett et al medical conditions or risk factors could suffer from higher dropout because of the higher risk of a medical event. However, having a specific condition or medical event could be a stronger motivation than having a vaguer risk factor such as being overweight or obese [37]. outcomes. The included studies were likely to suffer from bias. Wearable device interventions cannot be blinded to the user. Only outcome assessors could have been blinded, which most studies did not attempt to do. The use of wearables in these studies may not be applicable to real-world scenarios as the companies selling these wearable devices do not offer the support that was offered by researchers in the studies. A limitation of this review is that we only conducted a basic search limited to a few keywords and phrases. In addition, databases such as Institute of Electrical and Electronics Engineers Xplore and Cumulative Index of Nursing and Allied Health Literature for clinical and behavioral science research were not searched. Recommendations for Future Work Different aspects of weight loss maintenance and wearable devices have been studied, but large areas are still unknown. These include the mechanisms through which using wearable devices can lead to weight loss and studies into the usefulness of wearable devices for long-term weight management. Those who managed to sustain raised physical activity levels had a weight maintenance benefit. Not all groups managed to sustain increased activity levels, and it would be valuable to understand why. Individuals who sustained exercise could have been more likely to commit to other lifestyle changes around weight management. Comparison With Prior Work It is important to retain participants in studies to separate the effect of the study design and intervention [31]. Study 5 compared a standard intervention with a stepped-care intervention where the intensity of support (such as telephone intervention and additional individual sessions) increased if certain goals were not met. Interventions of this kind have been shown to reduce attrition [32]. Other strategies for improving long-term data collection are offering incentives, reducing barriers by offering alternative data collection modes, and reminder calls [32]. Improving adoption and retention through methods such as monetary incentives could be counterproductive as this is not possible in real-life settings. Investigating the reasons for dropout could help to understand to what extent this is caused by study design and/or flaws in wearable devices. Discovering how wearable devices are being used, and whether their use is improved through outside support, would give valuable information for designing more effective wearable devices. It could also help health care practitioners to advice and support people who are trying to lose weight and are interested in using wearable devices. Consciously or subconsciously, self-reported physical activity levels are often overestimated [33]. Wearable devices are more accurate at estimating physical activity levels than self-reporting, though a truly objective method is currently not available for everyday purposes. Accelerometers, which were used in the wearable devices in the included studies, can lead to different estimates, even when using the same device [34]. The accuracy of heart rate monitors has been reported to be higher but still insufficient [35]. Conclusions We found a small number of long-term studies showing some evidence that wearable devices can improve long-term physical activity and weight loss outcomes, but there was not enough evidence to show a benefit over the comparator methods. A major issue is the challenge to separate the effect of the decreasing use of wearable devices over time from the effect of wearable devices on the outcomes. Consistency in study methods is needed in future long-term studies on the use of wearable devices for weight loss. Wearable devices have shown benefit in managing medical conditions, eg, diabetes [36]. Studies with populations having JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 7 (page number not for citation purposes) Multimedia Appendix 4 Quality appraisal of randomized controlled trials. [DOCX File , 41 KB-Multimedia Appendix 4] Multimedia Appendix 2 Results found in each database and the search strings use [DOCX File , 13 KB-Multimedia Appendix 2] Results found in each database and the search strings used. Multimedia Appendix 3 Excluded studies. [DOCX File , 18 KB-Multimedia Appendix 3] Multimedia Appendix 1 Identify the report as a systematic review, meta-analysis, or both. [DOCX File 19 KB Multimedia Appendix 1] Identify the report as a systematic review, meta-analysis, or both. Identify the report as a systematic review, meta- [DOCX File , 19 KB-Multimedia Appendix 1] References 1. Caballero B. The global epidemic of obesity: an overview. Epidemiol Rev 2007;29:1-5. [doi: 10.1093/epirev/mxm012] [Medline: 17569676] 2. Wang YC, McPherson K, Marsh T, Gortmaker SL, Brown M. Health and economic burden of the projected obesity trends in the USA and the UK. Lancet 2011 Aug 27;378(9793):815-825. [doi: 10.1016/S0140-6736(11)60814-3] [Medline: 21872750] ] 3. Pi-Sunyer X. 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JMIR MHEALTH AND UHEALTH A systematic review and meta-analysis of mobile devices and weight loss with an intervention content analysis. J Pers Med 2014 Jun 30;4(3):311-385 [FREE Full text] [doi: 10.3390/jpm4030311] [Medline: 25563356] 22. Lyzwinski LN. A systematic review and meta-analysis of mobile devices and weight loss with an intervention content analysis. J Pers Med 2014 Jun 30;4(3):311-385 [FREE Full text] [doi: 10.3390/jpm4030311] [Medline: 25563356] analysis. J Pers Med 2014 Jun 30;4(3):311 385 [FREE Full text] [doi: 10.3390/jpm4030311] [Medline: 25563356] 23. Allen JK, Stephens J, Patel A. Technology-assisted weight management interventions: systematic review of clinical trials. Telemed J E Health 2014 Dec;20(12):1103-1120 [FREE Full text] [doi: 10.1089/tmj.2014.0030] [Medline: 25409001] 24. Kelders SM, Kok RN, Ossebaard HC, van Gemert-Pijnen JE. 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BMJ Open 2018 Mar 12;8(3):e017466 [FREE Full text] [doi: 10.1136/bmjopen-2017-017466] [Medline: 29530904] JMIR Mhealth Uhealth 2020 \| vol. 8 \| iss. 3 \| e13461 \| p. 9 (page number not for citation purposes) https://mhealth.jmir.org/2020/3/e13461 XSL•FO RenderX JMIR MHEALTH AND UHEALTH Abbreviations PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses RCT: randomized controlled trial Edited by G Eysenbach; submitted 22.01.19; peer-reviewed by D Baines, K Ng, D Gratzer, M Bardus, T Fazzino, M Stuc to author 22.02.19; revised version received 06.11.19; accepted 16.12.19; published 10.03.20 Please cite as: Fawcett E, Van Velthoven MH, Meinert E Long-Term Weight Management Using Wearable Technology in Overweight and Obese Adults: Systematic Review JMIR Mhealth Uhealth 2020;8(3):e13461 URL: https://mhealth.jmir.org/2020/3/e13461 doi: 10.2196/13461 PMID: 32154788 ©Emily Fawcett, Michelle Helena Van Velthoven, Edward Meinert. Originally published in JMIR mHealth and uHealth (http://mhealth.jmir.org), 10.03.2020. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR mHealth and uHealth, is properly cited. The complete bibliographic information, a link to the original publication on http://mhealth.jmir.org/, as well as this copyright and license information must be included. https://mhealth.jmir.org/2020/3/e13461 https://mhealth.jmir.org/2020/3/e13461 XSL•FO RenderX
https://openalex.org/W4214595902	https://jesr.ub.ro/1/article/download/297/278	English	null	INFLUENCE OF THE CRANK MECHANISM POSITION IN THE MOTION OF AN OSCILLATING SIEVE	Journal of Engineering Studies and Research	2,021	cc-by	4,846	* Corresponding author, email: claudia.tomozei@ub.ro © 2021 Alma Mater Publishing House 1“Vasile Alecsandri” University of Bacau, Calea Marasesti 157, Bacau, 600115, Romania Abstract: This article presents a study that aims to visualize the influence exerted by the position of the crank mechanism on the motion performed by the sieve supports respectively, on the displacement of an oscillating sieve. The study was oriented for a specific constructive case and involves the connecting rod position changing, the drive mechanism in relation to the horizontal, respectively at the angles 0 - 75 and -45 - 0. Also, were identifying the variation of the angle described by the motion of the sieve support. From the obtained results analysis, it was found that in the case of 12.71 of the connecting rod angle, in relation to the horizontal, the lowest value of the angle described by the motion of the tie rod between the extreme points was obtained. Also, for the value of the connecting rod angle of approximately 6.5 equal angles described by the extreme positions of the sieve support in relation to the vertical were obtained. It is found that the variation of the connecting rod angle directly influences the variation of the angles described by the connecting rod in relation to the horizontal, the measured value being corresponding to the extreme points P1 and P2. Keywords: oscillating motion, crank mechanism, connecting rod angle variation Journal of Engineering Studies and Research – Volume 27 (2021) No. 4 Journal of Engineering Studies and Research – Volume 27 (2021) No. 4 36 36 1. INTRODUCTION In this article, a theoretical study was performed to visualize the influence exerted by the position of a crank mechanism used for the movement mechanism of a sieve. 2. MODELING THE DRIVE MECHANISM For the analysis of the influence exerted by the position of the driving mechanism of the sieve on its movement, a sieve was choose performs an oscillating movement (Fig. 1). This system is a classic drive of a sieve block and is composed of [21, 22]: sieve; tie rods; Sieve drive mechanism; connecting crank mechanism; fastening element of the sieve connecting rod-crank mechanism. Fig. 1. Sieves block drive system. The location of the connecting crank mechanism [23], the one that drives, in relation to the gripping joint, varies in a range of 60. The study was performed for a variation of the work position from 15to 15(Fig. 2). Fig. 1. Sieves block drive system. Fig. 1. Sieves block drive system. The location of the connecting crank mechanism [23], the one that drives, in relation to the gripping joint, varies in a range of 60. The study was performed for a variation of the work position from 15 to 15 (Fig. 2). Figure 3 shows the variation of the angle generated by the drive device in relation to the gripping joint. The study was performed for values: -45, -30, -15, 0, 13, 30, 45, 60, 75. Fig. 2. Variation of the position of the drive system. Fig. 3. Variation of the connecting rod position. For positions -60 and -75, the study could not be performed because from a physical point of view the movement cannot be performed, respecting the positioning of the crank. It is known that the entire drive mechanism of the seat block is composed of the two components: tie rods and the drive mechanism (connecting rod-crank mechanism). Thus, it was concluded that this assembly can be done in a simplified way. Figure 4 shows the model used in this paper [24, 25]. To determine in value terms the influence exerted by the position of the drive device a model was generated Fig. 3. Variation of the connecting rod position. Fig. 2. Variation of the position of the drive system. Fig. 3. Variation of the connecting rod position. Fig. 2. Variation of the position of the drive system. For positions -60 and -75, the study could not be performed because from a physical point of view the movement cannot be performed, respecting the positioning of the crank. 1. INTRODUCTION A mixture of solid particles, in order to be used in different industrial processes, is subjected to different separation processes, through which the most used is the separation on sieve. Different types of separators are used in the process of solid particle’s heterogeneous mixture sieve separation [1-14]. In order, to increase the efficiency of this process, the sieve motions can be ensured by using different devices [15-20]: y p , y g [ ] - Elliptical motion - used for the purpose of retaining by sieving some coarse particles from a mixtur - Elliptical motion - used for the purpose of retaining by sieving some coarse particles from a mixture. - Linear motion - the eccentric is induced on the work surface and a vertical movement, to increase the action of sieving and preventing compaction. - Linear motion - the eccentric is induced on the work surface and a vertical movement, to increas action of sieving and preventing compaction. g p g p - Circular motion - used for sorting particles in several fractions, depending on thickness and granulation, respectively, or for sifting ground products, with separation according to particle size. C if l i h d i h f i l h h f fl b i d f Centrifugal motion - the most used in the case of particles that have a free flow being used f calibration operations. - Gyroscopic movement - high efficiency equipment used for long filtration processes of light partic From all these methods of sieves entrainment, the most used system is the one with oscillating motion, being found in a series of equipment that performs the process of separating mixtures of solid particles according to their length and width. It is also used in other industrial processes such as packing, grinding or transporting solid particles. From all these methods of sieves entrainment, the most used system is the one with oscillating motion, being found in a series of equipment that performs the process of separating mixtures of solid particles according to their length and width. It is also used in other industrial processes such as packing, grinding or transporting solid particles. Journal of Engineering Studies and Research – Volume 27 (2021) No. 4 37 In this article, a theoretical study was performed to visualize the influence exerted by the position of a crank mechanism used for the movement mechanism of a sieve. To determine in value terms, the influence exerted by the position of the drive device, a model was generated whose dimensions are (Fig. 5): tie rods (2): 0.587 m; crank: 0.065 m; connecting rod: 0.27 m. 2. MODELING THE DRIVE MECHANISM It is known that the entire drive mechanism of the seat block is composed of the two components: tie rods and the drive mechanism (connecting rod-crank mechanism). Thus, it was concluded that this assembly can be done in a simplified way. Figure 4 shows the model used in this paper [24, 25]. To determine in value terms, the influence exerted by the position of the drive device, a model was generated whose dimensions are (Fig. 5): tie rods (2): 0.587 m; crank: 0.065 m; connecting rod: 0.27 m. To determine in value terms, the influence exerted by the position of the drive device, a model was generated whose dimensions are (Fig. 5): tie rods (2): 0.587 m; crank: 0.065 m; connecting rod: 0.27 m. Journal of Engineering Studies and Research – Volume 27 (2021) No. 4 38 38 The following programs were used to conduct this study: The following programs were used to conduct this study: - The simulation program of mechanisms, Linkage, was used to identify the influence exerted by the position of the connecting rod-crank mechanism on the movements exerted by the free end of the tie rod; - The simulation program of mechanisms, Linkage, was used to identify the influence exerted by the position of the connecting rod-crank mechanism on the movements exerted by the free end of the tie rod; od; - The Mathcad program used to process the values obtained. od; The Mathcad program used to process the values obtained. Fig. 4. Simplified model of the analyzed mechanism. Fig. 5. Dimensional representation of the drive system studied. Fig. 6. The movements of the tie rod. Fig. 4. Simplified model of the analyzed mechanism. Fig. 5. Dimensional representation of the drive system studied. Fig. 6. The movements of the tie rod. f the Fig. 5. Dimensional representation of the drive system studied. Fig. 6. The movements of the tie rod. As a result of the processing of the obtained data, the following parameters were identified: As a result of the processing of the obtained data, the following parameters were identified: - The angle achieved by the movement of the free end of the tie rod, angle obtained by summing the angles α and β (Figure 6). - The angle achieved by the movement of the free end of the tie rod, angle obtained by summing the angles α and β (Figure 6). 2. MODELING THE DRIVE MECHANISM - The angles made by the connecting rod corresponding to the two extremes of the movement made by the free end of the tie rod. For each position of the drive device, the angle made by the tie rod was determined. For this purpose, the extreme positions of the tie rod were identified and the values of the angles α and β were determined (Figure 6). For each position of the drive device, the angle made by the tie rod was determined. For this purpose, the extreme positions of the tie rod were identified and the values of the angles α and β were determined (Figure 6). gure 7 shows the working mode for processing the data obtained in order to determine the parameters stud Fig. 7. Working methodology. Fig. 7. Working methodology. Journal of Engineering Studies and Research – Volume 27 (2021) No. 4 39 3. RESULTS AND DISCUSSION Following the simulations with the help of the Linkage program, a series of variations of the angles given by the support system of the sieve, respectively of the tie rod were obtained (Figure 8). The graphical representation of these variations was made taking into account the position that the tie rod, the support system of the site, have in the starting position. Compared to these working positions, the movement made by the tie rod was performed for a complete stroke of the crank. The physical value of the extreme points of the free end displacement of the tie rod (P1 and P2) were used for the graphical representations in Figure 8. a) 75 b) 60 c) 45 d) 30 e) 13 f) 0 a) 75 b) 60 d) 30 f) 0 b) 60 d) 30 a) 75 a) 75 c) 45 e) 13 c) 45 e) 13 d) 30 d) 30 d) 30 f) 0 e) 13 f) 0 Journal of Engineering Studies and Research – Volume 27 (2021) No. 4 40 40 g) -15; h) -30; i) -45; Fig. 8. Variation of the angle described by the tie rod according to the position of the drive device: a) 75; b) 60; c) 45; d) 30; e) 13s; f) 0; g) -15; h) -30; i) -45. h) -30; g) 15; g) -15; h) i) 45 i) 45 ; Fig. 8. Variation of the angle described by the tie rod according to the position of the drive device: a) 75; b) 60; c) 45; d) 30; e) 13s; f) 0; g) -15; h) -30; i) -45. ) ; Fig. 8. Variation of the angle described by the tie rod according to the position of the drive device: a) 75; b) 60; c) 45; d) 30; e) 13s; f) 0; g) -15; h) -30; i) -45. From the analysis of the variation of the angle described by the tie rods, depending on the position of the drive device, the following aspects can be evidenced: From the analysis of the variation of the angle described by the tie rods, depending on the position of the drive device, the following aspects can be evidenced: g p - The angle described by the two extreme positions of the free end of the tie rod was analyzed according to the starting position of the study, respectively the position of the tie rod, being 90. 3. RESULTS AND DISCUSSION 9) it is found that: Analyzing the angular values presented (Fig. 9) it is found that: y g g p ( g ) The smallest angular opening, of 12.71, corresponds to the connecting rod angle of 13; - The smallest angular opening, of 12.71, corresponds to the connecting rod angle of 13; - For the variation of the connecting rod angle between 30 and 75, an exponential increase of the value of the studied angle was obtained, reaching the maximum value of 17.78; - For the variation of the connecting rod angle between 30 and 75, an exponential increase of the value of the studied angle was obtained, reaching the maximum value of 17.78; - For the variation of the connecting rod angle between 0 and -30, the same exponential increase of the value of the studied angle was found, obtaining for the last value an angle of 17.49; - A specific analysis can be performed for the connecting rod angle of -45 where it is found that the calculated angle has a value of 30.5. This value is due to the positioning of the crank in relation to the driven assembly. - A specific analysis can be performed for the connecting rod angle of -45 where it is found that the calculated angle has a value of 30.5. This value is due to the positioning of the crank in relation to the driven assembly. Fig. 9. Variation of the angle described by the free end of the tie rod. Fig. 10. Variation of angles α and β. Fig. 10. Variation of angles α and β. Fig. 9. Variation of the angle described by the free end of the tie rod. Fig. 9. Variation of the angle described by the free end of the tie rod. Fig. 10. Variation of angles α and β. From the study of the results presented in figure 10 it can be concluded: - The two angles analyzed, α and β, have equal values, of approximately 6.4, for a connecting rod angle in relation to the horizontal of approximately 6.5. This value of the angle is closely related to the dimensional values of the components that make up the analyzed mechanism; - The two angles analyzed, α and β, have equal values, of approximately 6.4, for a connecting rod angle in relation to the horizontal of approximately 6.5. 3. RESULTS AND DISCUSSION g - The angle described by the two extreme positions of the free end of the tie rod was analyzed according to the starting position of the study, respectively the position of the tie rod, being 90. - In relation to this position, it is found that the angle described by the tie rod varies in value. This is highlighted by the variations of the angles on the right and left side of the tie rod, respectively the angle α and the angle β (Figure 8). g β ( g - In relation to the position of the drive device it is observed that the position of the angle varies, as follows: o For the value of 13 the values of the angles α and β are approximately equal, this value being corresponding for a symmetrical displacement of the free end of the tie rod in relation to the starting position of the displacement. o When changing the position of the drive device from the value of the angle of 30 to the value of the angle of 75 it is observed that the spring described by the free end of the tie rod moves to the right. g o For values of the connecting rod angle c between 0 and -45 it is found that the free end of the tie rod generates an angle that moves to the left. Analyzing the positions of the extreme points, obtained from the displacement of the free end of the tie rod, P1 and P2, and of the fixed end of the tie rod, it was possible to determine the angular value between the three points. The variation of this angle is shown in Figure 9. Analyzing the positions of the extreme points, obtained from the displacement of the free end of the tie rod, P1 and P2, and of the fixed end of the tie rod, it was possible to determine the angular value between the three points. The variation of this angle is shown in Figure 9. The analysis of the individual values of the two angles, α and β, is given in graphical representation from Figure 10. The analysis of the individual values of the two angles, α and β, is given in graphical representation from Figure 10 Journal of Engineering Studies and Research – Volume 27 (2021) No. 4 41 Analyzing the angular values presented (Fig. 4. CONCLUSIONS Following the study performed on the influence exerted by the position of the drive device of an oscillating sieve, the following conclusions could be drawn: Following the study performed on the influence exerted by the position of the drive device of an oscillating sieve, the following conclusions could be drawn: - A number of methods can be used to carry out the process of mechanical separation of a heterogeneous mixture of solid particles; - One of the most used methods of mechanical separation is the separation on sieves, respectively the dimensional separation of the mixture of solid particles; In order to streamline the separation process on sieves, they must perform a series of movements th can be simple or complex; - The simplest method of driving a sieves block is the oscillating movement, a movement performed by a crank connecting rod mechanism; g - The solid particles movement on the surface of the oscillating sieve is influenced by the angle of inclination of the sieve but also by the movement made by the support system of the sieve block; - Study of the movement performed by the oscillating sieve, a series of simulations were performed through the Linkage program; - In order to carry out this study, the initial angle of the connecting rod was modified and in relation to it the following parameters were studied: o The angle achieved by the movement of the free end of the tie rod, angle obtained by summing the angles α and β; o The angles made by the connecting rod corresponding to the two extreme points of th movement made by the free end of the tie rod. Following the study, it was found that: o The initial angle variation of the connecting rod in relation to the horizontal directly influences both the value of the angle made by the free end of the tie rod and the angles made by the connecting rod in relation to the horizontal, for the two extreme points P1 and P2; o For an angle value made by the connecting rod with a horizontal of 12.71, the smallest angular opening made by the tie rod was obtained in the case under study. 3. RESULTS AND DISCUSSION This value of the angle is closely related to the dimensional values of the components that make up the analyzed mechanism; - The increase of the connecting rod angle in relation to the horizontal, it is found that the values of the angle α decrease, and the values of the angle β increase; - The increase of the connecting rod angle in relation to the horizontal, it is found that the values of the angle α decrease, and the values of the angle β increase; - In case of reduction of the connecting rod angle in relation to the horizontal (for negative values of the angle) the value variation of the analyzed angles is reversed. - In case of reduction of the connecting rod angle in relation to the horizontal (for negative values of the angle) the value variation of the analyzed angles is reversed. It was also determined the value of the angle made by the connecting rod in relation to the horizontal, for the two extreme points, P1 and P2. The variation of these angles is shown in Figure 11. Fig. 11. Angle variation of the connecting rod in relation to the horizontal for the extreme points. Fig. 11. Angle variation of the connecting rod in relation to the horizontal for the extreme points. Journal of Engineering Studies and Research – Volume 27 (2021) No. 4 42 ollowing conclusions can be drawn from the analysis of the variation of the two studied angles: - The value of the angle corresponding to point P1 is greater than the value of the angle P2 regardless of the position of the connecting rod; - The minimum value of the two angles, of 0, corresponds to an initial angle, of inclination of the connecting rod, of 13; - As the value of the inclination angle of the connecting rod increases, it is found that the values o two angles also increase, this increase being a linear increase. 4. CONCLUSIONS Starting from this point, the value of the angle described by the free end of the tie rod increases in direct proportion to the value of the connecting rod angle; o The two angles analyzed, α and β, have equal values, of approximately 6.4, for a connecting rod angle in relation to the horizontal of approximately 6.5; o A minimum value of the two angles described by the connecting rod in relation to the horizontal corresponding to the extreme points P1 and P2, respectively 0, corresponds to an initial angle of inclination of the connecting rod in relation to the horizontal of 13; o The variation of the angles described by the connecting rod in relation to the horizontal, corresponding to the extreme points P1 and P2, is directly proportional to the variation of the angle of the connecting rod in relation to the horizontal. This study can be continued by identifying the value of the entrainment speed of a solid particle positioned on the oscillating surface, respectively determining the inertial force generated by the working surface on the solid particle, and then identifying the working regime of the oscillating surface. This study can be continued by identifying the value of the entrainment speed of a solid particle positioned on the oscillating surface, respectively determining the inertial force generated by the working surface on the solid particle, and then identifying the working regime of the oscillating surface. [1] Bernotat, S., Gregor, W., Some aspects about separation in a cross-flow air-classifier, Chemical Engineering Science, vol. 33, no. 6, 1978, p. 751-757. REFERENCES [1] Bernotat, S., Gregor, W., Some aspects about separation in a cross-flow air-classifier, Chemical Engineering Science, vol. 33, no. 6, 1978, p. 751-757. 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[21] Mosnegutu, E., Nedeff, V., Narcis, B., Chitimus, D., Rusu, D.I., Influence of screening block supporting h b h i f lid i l ill i f J l f E i i S di d R h [20] Voicu, G., Stoica, D., Constantin, G., Carp-Ciocardia, D.-C., Stefan, E.-M., Influence of the granular mixture particles sizes and oscillation frequency on the separation process at the conical sieves with oscillating movement, Journal of Engineering Studies and Research, vol. 20, 2014. [21] Mosnegutu, E., Nedeff, V., Narcis, B., Chitimus, D., Rusu, D.I., Influence of screening block supporting way on the behaviour of a solid particle on an oscillating surface, Journal of Engineering Studies and Research, vol. 21, 2016. 2] Hartenberg R., Denavit J., Kinematic synthesis of Linkages. McGraw Hill Book Company, N.Y, 1964. [23] Goessner, S., Crank-rocker-Design and analysis, https://github.com/goessner/crocker#readme (12.01.2021). 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https://openalex.org/W2795569320	https://darchive.mblwhoilibrary.org/bitstream/1912/10249/1/genes-09-00197.pdf	English	null	Transgenic Xenopus laevis Line for In Vivo Labeling of Nephrons within the Kidney	Genes	2,018	cc-by	7,185	genes G C A T T A C G G C A T genes G C A T T A C G G C A T genes Transgenic Xenopus laevis Line for In Vivo Labeling of Nephrons within the Kidney Mark E. Corkins 1,† ID , Hannah L. Hanania 1,2,† ID , Vanja Krneta-Stankic 1,3, Bridget D. DeLay 1, Esther J. Pearl 4,‡, Moonsup Lee 3,5,§, Hong Ji 5, Alan J. Davidson 6, Marko E. Horb 4 and Rachel K. Miller 1,5,7,8,* 1 Department of Pediatrics, Pediatric Research Center, UTHealth McGovern Medical School, Houston, TX 77030, USA; Mark.E.Corkins@uth.tmc.edu (M.E.C.); hlh2@rice.edu (H.L.H.); Vanja.Stankic@uth.tmc.edu (V.K.-S.); Bridget.D.Delay@uth.tmc.edu (B.D.D.) j g y 2 Program in Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA 3 Program in Genes and Development, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA; moonsup.lee@nih.gov 4 National Xenopus Resource and Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543, USA; esther.pearl@kcl.ac.uk (E.J.P.); mhorb@mbl.edu (M.E.H.) partment of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; ngji@mdanderson.org 6 Department of Molecular Medicine and Pathology, University of Auckland, Auckland 1010, New Zealand; a.davidson@auckland.ac.nz 7 Program in Genetics and Epigenetics, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA Program in Biochemistry and Cell Biology, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA Correspondence: Rachel.K.Miller@uth.tmc.edu; Tel.: +1-713-500-6537 * Correspondence: Rachel.K.Miller@uth.tmc.edu; Tel.: +1-713-500-6537 These authors contributed equally to this work. † These authors contributed equally to this work. ‡ Current address: Centre for Craniofacial and Regenerative Biology, Dental Institute, King’s College London, London WC2R 2LS, UK. Current address: Cancer and Developmental Biology Laboratory, Center for Cancer Research, National § Current address: Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. § Current address: Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. ceived: 28 February 2018; Accepted: 4 April 2018; Published: 6 April 2018 1. Introduction Xenopus laevis is an established vertebrate model for studying developmental processes. X. laevis produce free-living, relatively transparent embryos, enabling direct visualization of development during chemical and genetic screens. This, combined with the numerous embryos produced in a single clutch, allows for hundreds of embryos to be manipulated and observed in a single experiment [1–3]. Additionally, the large size of the embryos and established fate maps allow for targeted microinjection into a selected blastomere to manipulate gene expression in a tissue of interest [4–9]. This method of targeted injections directs knockdown or overexpression of genes to a selected subset of tissues in F0 generation embryos. Targeted injections allow for a delivery of constructs to organs of interest, while avoiding tissues that affect early development and prevent assessment of subsequent phenotypes. This technique generates tissue-targeted knockdown or knockout mutant embryos similar to the Cre–LoxP systems commonly used in mouse and zebraﬁsh studies. Mammalian kidneys go through three developmental stages: the pronephros, mesonephros and metanephros [10]. Although the mammalian pronephros is non-functional, it is essential for the subsequent development of the mesonephric and metanephric kidney forms [11]. Each X. laevis pronephric kidney consists of one functional nephron which runs down the side of the tadpole. Like nephrons in the mammalian metanephros, the X. laevis pronephros consists of a glomus (functionally similar to the mammalian glomerulus), as well as proximal, intermediate, distal and connecting tubules, which together ﬁlter waste products and resorb nutrients from the blood [10,12–14]. Additionally, many of the markers that label each region of the kidney are conserved between the Xenopus pronephros and the mammalian metanephros [15,16]. X. laevis is a useful model for studying processes involved in vertebrate kidney development and disease [17]. The X. laevis pronephros undergoes rapid development and becomes functional within 2–3 days after fertilization [18,19]. A number of assays to assess kidney function, such as development of edema and visualization of the passage of ﬂuorescent molecules through the kidney, have been developed for use in X. laevis [13,20]. Modulating the function or expression of putative disease-causing genes through the injection of morpholinos, messenger RNA (mRNA) or clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing constructs results in kidney developmental phenotypes in X. laevis [21,22]. This allows for further study of the role that these genes play in human kidney pathologies. X. Received: 28 February 2018; Accepted: 4 April 2018; Published: 6 April 2018 Abstract: Xenopus laevis embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between Xenopus and humans, allowing for the study of human disease-causing genes. Xenopus embryos are also amenable to large-scale screening, but studies of kidney disease-related genes have been impeded because assessment of kidney development has largely been limited to examining ﬁxed embryos. To overcome this problem, we have generated a transgenic line that labels the kidney. We characterize this cdh17:eGFP line, showing green ﬂuorescent protein (GFP) expression in the pronephric and mesonephric kidneys and colocalization with known kidney markers. We also demonstrate the feasibility of live imaging of embryonic kidney development and the use of cdh17:eGFP as a kidney marker for secretion assays. Additionally, we develop a new methodology to isolate and identify kidney cells for primary culture. We also use morpholino knockdown of essential kidney development genes to establish that GFP expression enables observation of phenotypes, previously only described in ﬁxed embryos. Taken together, this transgenic line will enable primary kidney cell culture and live imaging of pronephric and mesonephric kidney development. It will also provide a simple means for high-throughput screening of putative human kidney disease-causing genes. Keywords: cdh17; pronephros; mesonephros; kidney; nephron; live imaging; primary culture; Xenopus; transgenic Genes 2018, 9, 197; doi:10.3390/genes9040197 www.mdpi.com/journal/genes 2 of 14 Genes 2018, 9, 197 1. Introduction laevis is also a useful model for use in stem cell and primary cell culture research. In early embryos, cells are weakly held together and can be easily dissociated in calcium- and magnesium-free saline. An established example of this is the animal cap [23]. Additionally, the cells of X. laevis embryos contain yolk, allowing them to survive in simple saline solutions for days. X. laevis cells can also survive at temperatures from 15 to 25 ◦C under normal atmosphere, allowing for easy growth without the need for a special incubator. Given that there are no clear live markers for kidney cells, one limitation of primary tissue culture of kidney cells in X. laevis is the inability in live cells to identify cells of interest. Although the external development of X. laevis embryos allows for live imaging of many tissues, [24] live imaging of the kidney is difﬁcult because there are currently no live markers. To facilitate the study of the dynamic cellular movements that occur during nephrogenesis, we generated a X. laevis transgenic line that labels the kidney cells. Here, we characterized this cdh17:eGFP line and demonstrate its usefulness in examining kidney development in vivo over a wide range of developmental stages. We also show that GFP expression within the labeled kidney is useful for monitoring the effects of targeted microinjection of morpholinos. Additionally, we develop a method for primary culture of X. laevis kidney cells. This transgenic line allows for live embryonic 3 of 14 Genes 2018, 9, 197 kidney labeling without the need to ﬁx and stain embryos, enabling live observation of X. laevis kidneys and kidney function during development. kidney labeling without the need to ﬁx and stain embryos, enabling live observation of X. laevis kidneys and kidney function during development. 2.1. Transgenic Animal Generation and Embryo Culture The cdh17:eGFP [Xla.Tg(Dre.cdh17:eGFP)NXR; RRID:NXR_0.0102] transgenic frog line was generated by I-SceI mediated integration [25,26]. Approximately a 4.3-kb genomic fragment upstream of the Danio rerio cdh17 gene was ampliﬁed from pTol2(cdh17::eGFP) [27,28] and cloned into the I-SceIpBSIISK+ vector. GFP-positive transgenic embryos were generated and maintained until sexual maturity at the National Xenopus Resource (Woods Hole, MA, USA). Transgenic F0 males were crossed to wild type X. laevis females to generate backcrossed F1 generation animals (Figure 1). Figure 1. Schematic of cdh17:eGFP transgenic line creation. (A) cdh17:eGFP construct ﬂanked by I-SceI sites was co-injected with I-SceI meganuclease into one-cell embryos. (B) Eight female and 11 male Xenopus laevis adults were grown from these F0 embryos. (C) Upon mating of an F0 transgenic male to a wild type female, 14 F1 females were generated. Figure 1. Schematic of cdh17:eGFP transgenic line creation. (A) cdh17:eGFP construct ﬂanked by I-SceI sites was co-injected with I-SceI meganuclease into one-cell embryos. (B) Eight female and 11 male Xenopus laevis adults were grown from these F0 embryos. (C) Upon mating of an F0 transgenic male to a wild type female, 14 F1 females were generated. Wild type X. laevis adult males were purchased from Nasco (LM00713M, Fort Atkinson, WI, USA). Embryos were obtained from cdh17:eGFP adult females and reared as previously described [29]. All embryos were grown in 1/3× Marc’s Modiﬁed Ringer’s (MMR) (33 mM NaCl, 0.66 mM KCl, 0.33 mM MgSO4, 0.66 mM CaCl2, 1.66 mM HEPES pH 7.4) [29]. This protocol was approved by the UTHealth’s Center for Laboratory Animal Medicine Animal Welfare Committee, which serves as the Institutional Animal Care and Use Committee (protocol #: AWC-16-0111). 2. Materials and Methods 2.1. Transgenic Animal Generation and Embryo Culture 2.3. Imaging Embryos were scored and imaged using an Olympus SZX16 ﬂuorescent stereomicroscope (Olympus, Tokyo, Japan) equipped with an Olympus DP71 camera. Confocal kidney images were taken using a Zeiss LSM800 confocal microscope (Zeiss, Oberkochen, Germany). Fixed embryos were cleared in a solution of BABB/Murray’s clearing solution (1:2 volume of benzyl alcohol to benzyl benzoate). For live imaging, animals were anesthetized with 10 µg/mL Benzocaine in 1/3× MMR [32,33]. For time-lapse movies, embryos were immobilized using coverslips anchored down using vacuum grease [34]. An image was taken every 30 min over a period of 15 h. Images were processed with Zen blue (Zeiss), Adobe Photoshop, and Microsoft PowerPoint. 2.2. Immunostaining Embryos were staged [19], ﬁxed [9], and immunostained using established protocols [30]. Proximal tubule lumens were labeled with antibody 3G8 (1:30, European Xenopus Resource Centre, Portsmouth, United Kingdom), while cell membranes of the intermediate, distal and connecting tubules were labeled with antibody 4A6 (1:5, European Xenopus Resource Centre) [31]. Proximal tubule lumens were also detected using Fluorescein isothiocyanate (FITC)-conjugated lectin from Erythrina cristagalli (1:1000, FL-1141 Vector labs, Burlingame, CA, USA). Rabbit anti green ﬂuorescent 4 of 14 Genes 2018, 9, 197 protein (anti-GFP) (1:500, iclLab, Portland, OR, USA) antibody was used to detect GFP expression. Goat anti-mouse or anti-rabbit conjugated to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (1:2000, Invitrogen, Carlsbad, CA, USA) secondary antibodies were used to visualize antibody staining. protein (anti-GFP) (1:500, iclLab, Portland, OR, USA) antibody was used to detect GFP expression. Goat anti-mouse or anti-rabbit conjugated to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (1:2000, Invitrogen, Carlsbad, CA, USA) secondary antibodies were used to visualize antibody staining. protein (anti-GFP) (1:500, iclLab, Portland, OR, USA) antibody was used to detect GFP expression. Goat anti-mouse or anti-rabbit conjugated to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (1:2000, Invitrogen, Carlsbad, CA, USA) secondary antibodies were used to visualize antibody staining. 2.6. Microinjection Microinjections were performed as previously described [9]. 10 nL of injection mix (described below) was injected into the indicated blastomere. 20 ng of Daam1 morpholino 5′-GCCGCAGGTCTG TCAGTTGCTTCTA-3′ or Standard morpholino 5′-CCTCTTACCTCAGTTACAATTTATA-3′ were injected along with 25 ng rhodamine-dextran as a tracer, to mark targeted cells, into the V2 blastomere at the 8-cell stage to target the kidney [6,35]. Error is given as Standard Error of the Mean (SEM) across at least three trials. Microinjections were performed as previously described [9]. 10 nL of injection mix (des below) was injected into the indicated blastomere. 20 ng of Daam1 morpholino 5′-GCCGCAGG 2.4. Secretion Assay Two–4 pulses of 5 nL of 1 mg/mL rhodamine-dextran 10,000 MW (10–20 nL total) was injected into the coelomic cavity above the heart of benzocaine-anesthetized stage 40–43 embryos [13]. Embryos were either directly visualized with a dissection microscope, or placed on a confocal microscope for high resolution imaging. 2.5. Kidney Cell Primary Culture Animals expressing GFP within the kidney were identiﬁed by ﬂuorescent microscopy. The pronephric primordium was identiﬁed by anatomical position and explanted using sharp forceps from stage 30–32 embryos under white light. All explants were carried out on 2% agar-coated plates. Approximately 10–20 kidney explants were disassociated in Calcium Magnesium Free Media (CMFM) [1/3× MMR without MgSO4 and CaCl] for 1 h. Once dissociated cells were washed in Danilchik’s for Amy (DFA) [53 mM NaCl, 5 mM Na2CO3, 4.5 mM potassium gluconate, 32 mM sodium gluconate, 1 mM CaCl2, 1 mM MgSO4, 0.1% (w/v) BSA, buffered to pH 8.3 with 1 M bicine] and allowed to attach. Dissociation and attachment procedures were carried out in Attoﬂuor™Cell Chamber (Thermo Fisher A7816, Waltham, MA, USA) with ﬁbronectin-coated coverslips (Roche 10838039001, Basel, Switzerland). Alternatively, to increase the yields of individualized cells, explants were treated with Trypsin-EDTA (Ethylenediaminetetraacetic acid) (Sigma T4049, St. Louis, MO, USA) for 5 min instead of CMFM, followed by inactivation of Trypsin-EDTA with 10% fetal calf serum in DFA. 3.1. Generation of Xenopus laevis cdh17:eGFP Line In zebraﬁsh (Danio rerio), Cadherin-17 (cdh17) is a gene that functions in maintaining pronephric integrity [36]. Previously, the cdh17 promoter was used to generate a zebraﬁsh transgenic line to label the pronephric and the mesonephric kidney [27,28]. Given that this promoter was previously characterized in zebraﬁsh, we conﬁrmed pTol2(cdh17::eGFP) expression in the X. laevis pronephros in F0 embryos using the Tol2 system [37]. To improve the transgene integration rate into the X. laevis genome, we cloned the zebraﬁsh cdh17::eGFP fragment into the I-SceIpBSIISK+ vector to generate 5 of 14 Genes 2018, 9, 197 Genes 2018, 9, 197 a X. laevis transgenic line [27]. This vector allows for I-SceI-mediated rather than Tol2-mediated integration. Eight F0 GFP-positive female animals and 11 F0 males were grown until adulthood. A GFP-positive male was mated to a wild type female to generate 14 F1 generation female animals (Figure 1). Of the eight F0 females, six animals produced cdh17:eGFP-positive offspring, although three of the F0 females produced offspring with weak GFP expression. The remaining two F0 animals produced embryos with undetectable levels of GFP in the kidney. Thirteen of the F1 animals produced uniformly strong kidney GFP expression, while one F1 female was sterile and did not produce any eggs. The consistency of GFP expression observed in the embryos of the fertile F1 females was anticipated given that these females were produced from the same transgenic father and wild type mother. In contrast, the F0 females produced progeny with a range of GFP intensity, likely due to the different integration events in each animal. 3.2. cdh17:eGFP Colocalizes with Kidney Markers To assess the spatial expression of GFP in F2 embryos, we grew tadpoles to stage 40, ﬁxed, and then stained them with markers of the pronephros. The 3G8 antibody labels the lumen of the proximal tubules, while the 4A6 antibody labels the cell membranes of the intermediate, distal and collecting tubules (Figures 2–4) [31]. Additionally, we used Lectin as a marker to further verify proximal tubules (Video S1) [35]. GFP is expressed in the same cells as these markers, indicating that the kidney cells are expressing GFP. Additionally, a few tissues other than the kidney weakly expressed GFP, including the myotome and a population of cells dispersed within the cloaca (Figure 2). However, in tadpole stages, this expression is weak in comparison with the kidney expression. As predicted, GFP expression appears to be cytoplasmic and nuclear, since a signal peptide was not included in the construct (Figure 3). We ﬁnd that the cdh17:eGFP transgene is not deleterious to the embryos. The 3G8/4A6 staining in the transgenic embryos looks similar to their non-transgenic siblings as well as wild type embryos grown in parallel, suggesting that the transgene does not alter kidney morphology. Additionally, the transgenic animals do not develop edema, indicating that they have functional kidneys. We also see that 52% ± 7% (n = 488 embryos) of the embryos had GFP expression in their kidneys. This is about the predicted rate, given that the transgenic mothers were heterozygous for the transgene, and the testes used to fertilize the eggs were from wild type X. laevis males. We also ﬁnd that the transgenic embryos grow at the same rate as their wild type siblings. Together, our data suggest that the cdh17:eGFP transgene is not being selected against and is likely not affecting embryo survival or kidney morphology. 6 of 14 Genes 2018, 9, 197 Figure 2. cdh17:eGFP expression co-localizes with the kidney markers 3G8 and 4A6. Stage 42 cdh17:eGFP transgenic X. laevis embryos were co-immunostained with antibodies for green ﬂuorescent protein (GFP) and the kidney markers 3G8 and 4A6. (A) GFP immunostained cdh17:eGFP embryo. (B) 3G8/4A6 immunostained embryo. (C) Merged differential interference contrast (DIC) and ﬂuorescent image of cdh17:eGFP embryo. Embryo is surrounded in a bubble of 1:2 volume benzyl alcohol to benzyl benzoate (BABB). Figure 2. cdh17:eGFP expression co-localizes with the kidney markers 3G8 and 4A6. Stage 42 cdh17:eGFP transgenic X. 3.2. cdh17:eGFP Colocalizes with Kidney Markers laevis embryos were co-immunostained with antibodies for green ﬂuorescent protein (GFP) and the kidney markers 3G8 and 4A6. (A) GFP immunostained cdh17:eGFP embryo. (B) 3G8/4A6 immunostained embryo. (C) Merged differential interference contrast (DIC) and ﬂuorescent image of cdh17:eGFP embryo. Embryo is surrounded in a bubble of 1:2 volume benzyl alcohol to benzyl benzoate (BABB). Figure 3. Subcellular GFP expression in the cdh17:eGFP line is largely cytoplasmic and nuclear. Fixed stage 42 cdh17:eGFP embryos were stained with (A,E) anti-GFP, (B,F) 3G8/4A6, and (C,G) DAPI (4′,6-diamidino-2-phenylindole). (E–H) Magniﬁed image of proximal tubules. Figure 3. Subcellular GFP expression in the cdh17:eGFP line is largely cytoplasmic and nuclear. Fixed stage 42 cdh17:eGFP embryos were stained with (A,E) anti-GFP, (B,F) 3G8/4A6, and (C,G) DAPI (4′,6-diamidino-2-phenylindole). (E–H) Magniﬁed image of proximal tubules. 7 of 14 Genes 2018, 9, 197 Figure 4. cdh17:eGFP labels the kidney throughout pronephric development. Stage 32 through stage 44, cdh17:eGFP transgenic X. laevis embryos were immunostained with antibodies for GFP and the kidney markers 3G8 and 4A6. GFP expression is present throughout pronephric kidney development. (Left) GFP expression, (right) GFP (Green) and 3G8/4A6 (red) kidney marker expression. Figure 4. cdh17:eGFP labels the kidney throughout pronephric development. Stage 32 through stage 44, cdh17:eGFP transgenic X. laevis embryos were immunostained with antibodies for GFP and the kidney markers 3G8 and 4A6. GFP expression is present throughout pronephric kidney development. (Left) GFP expression, (right) GFP (Green) and 3G8/4A6 (red) kidney marker expression. 3.4. Assessment of Kidney Function in cdh17:eGFP Embryos Various dyes, such as 10,000 MW dextrans, are secreted into the urine by the embryonic Xenopus kidney, allowing for a simple visual readout of kidney function. This is performed by injecting labeled substances into the coelomic cavity. Uptake of the dye into the kidney can be directly visualized, followed by excretion of the dye in the urine. It takes approximately ﬁve minutes for the dyes to be secreted and visualized in real time in live embryos, allowing for easy phenotypic determination of kidney functionality (Video S3). However, coelomic dye injection can result in a strong signal from the coelomic cavity, making the tubules of the kidney difﬁcult to visualize. The contrast between the GFP-labeled kidney tubules and the red rhodamine-dextran dye in the cdh17:eGFP line, facilitates the visualization of the tubules and the localization of the dextran passing through them (Figure 6). Figure 6. cdh17:eGFP transgenic line labels the kidney for tracking of rhodamine secretion. Rhodamine-dextran was injected into the coelomic cavity of stage 42 X. laevis embryos and tracked using GFP as a marker for the kidney. Figure 6. cdh17:eGFP transgenic line labels the kidney for tracking of rhodamine secretion. Rhodamine-dextran was injected into the coelomic cavity of stage 42 X. laevis embryos and tracked using GFP as a marker for the kidney. 3.3. In Vivo Characterization of cdh17:eGFP Transgenic Line As there is still much to be learned about how the kidney tubules normally develop, we wished to observe nephrogenesis in real time. To test the feasibility of live imaging of the cdh17:eGFP line, we anesthetized embryos with benzocaine, immobilized them using coverslips, and then directly imaged them (Figure 5). cdh17:eGFP is expressed in the developing pronephros between tadpole stages 30 through 44. We also imaged embryos starting at stage 35 over a period of 15 h (Video S2). We were able to revive animals by washing them following benzocaine treatment and imaging, indicating that embryos were not severely harmed in the process. 8 of 14 Genes 2018, 9, 197 Figure 5. Live imaging of kidney development using cdh17:eGFP reporter line. Kidney development within a transgenic embryo was visualized by GFP expression from stages 30 to 44. Figure 5. Live imaging of kidney development using cdh17:eGFP reporter line. Kidney development within a transgenic embryo was visualized by GFP expression from stages 30 to 44. 3.4. Assessment of Kidney Function in cdh17:eGFP Embryos 3.5. Primary Culture of Xenopus laevis Kidney Cells X. laevis embryos are amenable to dissection and primary cell culture. However, primary culture of X. laevis kidney cells has not been previously performed. Given that the cdh17:eGFP expression in the kidney allows for easy tracking of the dissociated cells, we developed a protocol for culturing kidney cells on coverslips. Similar to animal caps, the cells of the kidney disassociate in calcium- and magnesium-free media. After disassociation, cells were plated on ﬁbronectin coated coverslips. The cells attached to the coverslips in a few hours, and were viable for at least 24 h. Kidney cells were easily detectable by their GFP expression (Figure 7). This 24 h point was chosen as it would be long enough to assess if our treatment of the cells was acutely toxic, or if the GFP expression reduced 9 of 14 Genes 2018, 9, 197 following explant of the cells without having to use media containing growth factors. GFP expression did not appear to reduce in strength over the 24 h culture period. The vast majority of explanted cells appeared healthy by GFP expression. As single cell suspensions were difﬁcult to obtain with this method, we also utilized Trypsin-EDTA to completely disassociate the cells. We found that the cells were still viable after treatment, though many cells were lost during the washing steps. Figure 7. X. laevis cdh17:eGFP transgenic line is useful for primary culture of kidney tubules. (A) Diagram showing region of embryo that was explanted. (B) Schematic of methodology used to culture cells. Media used for each step is as indicated DFA (Danilchik’s for Amy) and CMFM (Calcium Magnesium Free Media). (C) Image of GFP-positive cells cultured for 24 h in DFA. Cells are outlined using dashed lines. Figure 7. X. laevis cdh17:eGFP transgenic line is useful for primary culture of kidney tubules. (A) Diagram showing region of embryo that was explanted. (B) Schematic of methodology used to culture cells. Media used for each step is as indicated DFA (Danilchik’s for Amy) and CMFM (Calcium Magnesium Free Media). (C) Image of GFP-positive cells cultured for 24 h in DFA. Cells are outlined using dashed lines. 3.7. Utility of cdh17:eGFP for Identifying Genes Involved in Kidney Diseases and Development We next tested the utility of the cdh17:eGFP line to determine genes that result in kidney malformations. In X. laevis, antisense morpholinos are commonly used to alter gene activity [38]. As many genes that play a role in kidney development also play a role in the development of other tissues, we microinjected the V2 blastomere to target the kidney progenitors while avoiding most other tissues [9]. To test the potential of utilizing the cdh17:eGFP transgenic line to identify genes involved in kidney development, we chose to assess the expression of the cdh17:eGFP reporter upon disruption of Daam1. Daam1 is a component of the planar cell polarity pathway, and loss of Daam1 is known to disrupt tubule morphology [35]. We injected Daam1 or a standard control morpholino into the V2 cell of 8-cell stage cdh17:eGFP transgenic embryos to target the kidney (Figure 9). The embryos were then ﬁxed and immunostained with αGFP and 3G8/4A6 antibodies, allowing us to compare the effectiveness of monitoring GFP expression versus validated kidney markers to assess kidney development [29]. By monitoring the phenotype using GFP expression as a marker for kidney tubules, we were able to clearly visualize the effects of the morpholino injections on kidney development on the injected side of the embryos as compared with the uninjected side. Of the 62 transgenic embryos injected with Daam1 morpholino, 62% showed disrupted kidney tubule development, while only 1.4% of the 94 embryos injected with standard morpholino showed disrupted kidney development. In order to verify that our Daam1 morpholino was affecting kidney function as well as morphology, we assayed embryos for the development of edema after knocking down Daam1 in both kidneys: we found that they do develop edema. The major advantage of the cdh17:eGFP animals is that kidney development can be directly visualized in living embryos. This reduces the time, effort, and cost needed to screen for disease-causing genes. In order to directly compare the efﬁcacy of using living embryos, we injected Daam1 or standard morpholino and then assayed for abnormal GFP expression (Figure 9). Although the signal intensity is weaker as compared to the immunostained embryos, we ﬁnd that immunostained kidneys show a similar number of abnormal kidneys when compared to living embryos. 3.6. Expression of Green Fluorescent Protein in Adult Animals In zebraﬁsh, the cdh17::eGFP reporter line labels the pronephric as well as the mesonephric kidneys [28]. To test weather this holds true in X. laevis, we euthanized two F0 and one F1 generation adult female animals. All of these frogs tested positive for GFP expression within the kidney as tadpoles, though the two F0 generation animals did not produce GFP-positive offspring, and the F1 generation animal was sterile. We imaged the mesonephric kidneys and found that the cdh17:eGFP reporter is expressed in tubules within the mesonephric kidney (Figure 8). To ensure that the signal we observed was not due to background signal, an adult wild type animal kidney was imaged and no detectable green ﬂuorescent signal was observed. 10 of 14 Genes 2018, 9, 197 Figure 8. X. laevis adult mesonephric tubules express cdh17:eGFP. Adult kidneys were dissected, then imaged from an F1 generation cdh17:eGFP animal. (A) Brightﬁeld image of X. laevis cdh17:eGFP kidney. (B,C) GFP ﬂuorescence imaging of cdh17:eGFP kidney. Dashed boxes indicate regions taken for ﬂuorescence imaging. Figure 8. X. laevis adult mesonephric tubules express cdh17:eGFP. Adult kidneys were dissected, then imaged from an F1 generation cdh17:eGFP animal. (A) Brightﬁeld image of X. laevis cdh17:eGFP kidney. (B,C) GFP ﬂuorescence imaging of cdh17:eGFP kidney. Dashed boxes indicate regions taken for ﬂuorescence imaging. 3.7. Utility of cdh17:eGFP for Identifying Genes Involved in Kidney Diseases and Development 3.7. Utility of cdh17:eGFP for Identifying Genes Involved in Kidney Diseases and Development The added beneﬁt of not needing to stain the embryos prior to imaging saves approximately three days of processing time, allowing for faster screening of embryos for phenotypes, and making this transgenic line suitable for high throughput screening of human kidney development genes. 11 of 14 Genes 2018, 9, 197 Figure 9. Disruption of kidney development using Daam1 morpholino (MO) can be tracked by cdh17:eGFP. cdh17:eGFP transgenic embryos were injected with Standard or Daam1 morpholino, then stage 42 embryos were assayed for kidney abnormalities. (A) Representative images of kidneys under indicated conditions. (B) Percentage of abnormal kidneys under indicated conditions. (C) Daam1 morpholino was injected into both V2 blastomeres, resulting in the development of edema, which indicates kidney functional defects. Arrow indicates site of edema. Figure 9. Disruption of kidney development using Daam1 morpholino (MO) can be tracked by cdh17:eGFP. cdh17:eGFP transgenic embryos were injected with Standard or Daam1 morpholino, then stage 42 embryos were assayed for kidney abnormalities. (A) Representative images of kidneys under indicated conditions. (B) Percentage of abnormal kidneys under indicated conditions. (C) Daam1 morpholino was injected into both V2 blastomeres, resulting in the development of edema, which indicates kidney functional defects. Arrow indicates site of edema. 4. Discussion X. laevis is an attractive model for identifying genes responsible for human diseases [39]. Approximately 79% of genes implicated in human diseases have X. laevis orthologues, including genes that are involved in human kidney disorders [40]. Many of these disease-causing genes display phenotypes in the X. laevis pronephric kidney [21], although the mechanisms by which these disease-causing genes affect kidneys remain unknown. The cdh17:eGFP line allows us to follow the developmental processes underlying kidney formation as they are occurring, in order to further understand how kidney defects arise during embryonic development. The rapid development and large numbers of embryos allows for in vivo and real-time phenotypic assessment of large numbers of genes in a considerably shorter time than would be possible using mammalian systems. There is still much to be learned about normal kidney development. This line will allow us to follow morphogenetic movements within the pronephric and mesonephric kidney throughout development. The cdh17 promoter drives GFP expression beginning around embryonic stage 30 (early tadpole stage). This is before the 3G8 and 4A6 antibodies label the pronephros and during a time when the proximal tubules undergo large developmental changes. Additionally, the cdh17:eGFP reporter line labels the pronephric as well as the mesonephric kidneys of X. laevis. Little is known about the pronephric to mesonephric transition, and with this line, we have the potential to monitor these developmental changes as they occur. Examining cells in culture provides some advantages when performing certain experiments. The primary advantage is that it is easier to visualize the staining of subcellular components in cells in culture. In the whole embryo, visualization of through other tissues may decrease image quality by blocking and refracting light. Additionally, getting antibodies to penetrate deeply into the embryo requires chemicals such as methanol or Proteinase K, which may be incompatible with some antibodies or stains. Additionally, primary cell culture has the advantage that cells can be isolated from external sources of growth factors. This allows for easier studying of factors that function in specializing cells into tissues of interest. Genes 2018, 9, 197 12 of 14 Previously, an immortal kidney cell line (A6) was generated from spontaneous kidney tumor in X. laevis [41]. Although stable transgenic cell lines have been generated using A6 cells, this process is frequently difﬁcult, because A6 cells grow slowly and transfection rates are low [42–44]. 4. Discussion Additionally, these cells are a cancerous line that requires growth factors to survive [43]. Here we describe a novel method of isolating, culturing and identifying primary cell lines using X. laevis. Primary culture of these kidney cells has advantages over traditional cell culture. Primarily, these non-cancerous cells allow for better modeling of normal kidney functions. As these cells contain their own yolk, they can be grown in a saline solution without animal serum. This eliminates many factors that may induce these cells to take on abnormal characteristics. Additionally, as injections are routine, transfection of these cells are not necessary. Instead, embryos can be injected with morpholinos, RNAs or CRISPR constructs, and then the affected kidney cells can be cultured and assayed. GFP expression allows for conﬁrmation that the cells are kidney cells, and also allows for Fluorescence activated cell sorting (FACS) analysis of GFP-positive cells, a technique that would not have been feasible before this line was developed. We show that GFP is expressed under faithful control of the cdh17 promoter in the kidneys during organogenesis, and that this expression co-localizes with known kidney markers. We have used morpholino knockdown of Daam1 to conﬁrm that the GFP expression in the kidney recapitulates phenotypes described previously in ﬁxed embryos. We also demonstrate that live imaging of kidney development is feasible with this line, and that GFP expression functions as a reliable marker of kidney cells in secretion assays. Additionally, we have also developed a novel method for culturing primary cell lines from X. laevis kidneys. Taken together, this transgenic X. laevis line will be a useful tool, enabling high throughput screening of putative human kidney disease-causing genes. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/9/4/197/s1, Video S1: 3D structure of stage 40 cdh17:eGFP X. laevis. Transgene expression labeled using anti-GFP antibody (green), and Erythrina cristagalli lectin labeling the proximal tubules (red), Video S2: Live imaging of kidney development starting at stage 38 over a period of 15 h, Video S3: Movie of secretion assay in stage 42 X. laevis embryo. Acknowledgments: We are grateful to P.D. McCrea for facilitating the initiation of this project. We appreciate the helpful suggestions and advice throughout this project from the members of the laboratories of R.K.M. and P.D. McCrea, as well as M. Kloc. We thank the animal care technicians and veterinarians, including J.C. Whitney and T.H. 4. Discussion Gomez who took care of the animals even during hurricane Harvey. These studies were supported by a postdoctoral fellowship from the National Kidney Foundation (FLB1628 to R.K.M), a National Institutes of Health (NIH) KO1 grant (K01DK092320 to R.K.M.), and startup funding from UTHealth McGovern Medical School’s Department of Pediatrics (to R.K.M.). Author Contributions: M.E.C. designed the experiments upon creation of the transgenic line, performed injections, immunostained embryos, conducted secretion assays and wrote the manuscript; H.L.H. performed injections, immunostained and scored all embryos and wrote the manuscript; V.K.S. isolated cells for primary culture; B.D.D. performed injections; E.J.P. cloned the cdh17 promoter from Tol2 vector to I-SceIpBSIISK+ and generated transgenic animals; M.L. performed preliminary injections with the Tol2 vector and tested for cdh17 promoter expression prior to generation of lines; H.J. cloned alternative cdh17:eGFP variants related to Tol2; A.J.D. provided zebraﬁsh Tol2 construct; M.E.H. oversaw creation of cdh17:eGFP animals; R.K.M. conceived of the project, helped to design the strategy to generate cdh17:eGFP animals, and oversaw the experiments and article preparation. All authors edited and approved the ﬁnal manuscript. 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https://openalex.org/W4285418277	https://zenodo.org/record/5054799/files/Preprint.%20Investigation%20of%20High%20Sensitivity%20Piezoresistive%20Pressure%20Sensors%20for%20-0.5%E2%80%A6%2B0.5%20kPa.pdf	English	null	Development of High-Sensitivity Piezoresistive Pressure Sensors for -0.5…+0.5 kPa	null	2,021	cc-by	5,679	II. MODELING II. MODELING Abstract. The investigation of the pressure sensor chip’s design developed for operation in ultralow differential pressure ranges has been conducted. The optimum geometry of a membrane has been defined using available technological resources. The pres- sure sensor chip with an area of 6.15х6.15 mm has an average sensitivity S of 34.5 mV/кPa/V at nonlinearity 2KNL = 0.81 %FS and thermal hysteresis up to 0.6 %FS was created. Owing to the chip connection with stop elements, the burst pressure reaches 450 кPa. A mathematical model of a pressure sensor chip operating in a range of 0.5 to 0.5 кPa is based on analytical and comput- er-aided calculation for trade-off relationship between sensi- tivity (required: S > 30.0 mV/kPa/V) and a nonlinearity error (required: 2KNL < 1.5 %FS). The mechanical part of the sens- ing element includes a membrane with three separate and two bonded with the chip frame concentrators of mechanical stresses (MS), or rigid islands (RIs), where the thickness of RIs is equal to the initial thickness of a wafer. The selected structure of a membrane with RIs allows concentrating high MS at small values of deflection, which reduces a nonlinearity error [4, 14]. The geometrical shape of the membrane depends on the method of anisotropic wet etching that requires the ex- pansion of the chip area due to silicon etching at an angle of 54.7°. For these types of ultrahigh sensitivity pressure sensor chips reactive ion etching (RIE) is frequently used, which sig- nificantly saves the area of a chip [15-20]. Figure 1 shows the geometrical parameters of the selected structure of a chip: L – length of a chip side, W – thickness of the membrane thinned part, H – chip thickness, A – area of the membrane thinned part, D – width of a gap between RIs, Z – length of a RI edge, Y – width of the chip frame, G – width of the membrane etch taper projection, Q1 – distance between meddle piezoresistors (PRs), Q2 – distance between end PRs. PRs of p-type, covering an area of 20х400 µm, are made on a silicon substrate of n- type (NC = 9∙1014 cm-3) with a crystallographic plane (100) along the crystallographic direction [110] and have the follow- ing parameters: surface concentration NSp- = 5.5∙1018 cm-3, surface resistance RSp- = 200 Ohm/cm-2, depth of a p-n junc- tion xjp- = 2.5 µm. This work was supported by the Dukhov All-Russia Research Institute of Automatics (VNIIA). (Corresponding author: Mikhail Basov.) M. Basov and D. Prigodskiy are with VNIIA, Moscow 127055, Russian Federation (e-mail: engineerbasovm@gmail.com). M. BASOV, D. PRIGODSKIY: INVESTIGATION OF HIGH SENSITIVITY PIEZORESISTIVE PRESSURE SENSORS FOR -0.5…+0.5 KPA M. BASOV, D. PRIGODSKIY: INVESTIGATION OF HIGH SENSITIVITY PIEZORESISTIVE PRESSURE SENSORS FOR -0.5…+0.5 KPA II. MODELING The main piezoresistive coefficient is π44 = 1.26∙10-9∙Pa-1 at room temperature [21, 22]. The calculation of a change in PR rating and, consequently, output sensitivity and nonlinearity is performed using the theory of piezoresistive effect from the following formulas: Index Terms—piezoresistive pressure sensor, high sensitivity, temperature error, high mechanical strength, technology upgrad- ing. Investigation of High Sensitivity Piezoresistive Pressure Sensors for -0.5…+0.5 kPa Mikhail Basov, R&D Engineer Denis Prigodskiy, R&D Engineer 11. Wafer dicing. Technological processes of formation chip’s top side guar- anteed a spread in surface resistance RS and, consequently, resistor rating (Rbridge = 4.5 кOhm) within 7% error and the presence of leakage current Ileak on p-n junctions of no more than 0.5 µА at room temperature at a reverse bias Usample = 70 V. At anisotropic wet etching without self-hardening a spread in the membrane thickness was ∆W = ± 3 µm over 5 plates 100 mm in diameter. Besides large deviations of the mem- brane thickness from the required rated value W = 8 µm, we also observe asymmetry in the layout of RI edges due to a possible error of alignment at photolithography relative to a primary flat of the wafer and the use of silicon wafers with significantly misorientation crystallographic plane and prima- ry flat (Figure 6a) [27, 28]. This deficiency substantially in- creases a nonlinearity error as the resistor body (p--type) asymmetrically protrudes beyond the length of a RI edge. The further analysis of the obtained assemblies of pressure sensor chips, presented in the section below, showed that there was a difference in the rated values of sensitivity and nonlinearity, when applying pressure from the chip topside and backside, that was quite noticeable at W < 7 µm. Residual MS (com- pressive stresses) in chip structures are caused by the presence on the thinned part of the membrane of a step structure of SiO2 (WSiO2 = 0.2…0.6 µm) resulted from diffusion and oxidation processes for different types of doped regions. The elimination of the deficiencies detected for these types of ultrahigh sensi- tivity chips [29-38] will allow in the future both to increase the percent yield and improve output characteristics within the stated requirements. For the purpose of testing, ultrahigh sen- sitivity chips of pressure sensors were first combined in a sili- con assembly, comprising top and bottom stops and a base with low-temperature bonding glass between them (Figure 4). Connection with the selected geometry makes it possible not only to increase burst pressure of the sensor but also to reduce thermo-mechanical effect of the package [39-41]. The silicon assembly is connected to the package using silicon glue, the chip is wire to the package pins with the help of an aluminum wire (Figure 6b). I. INTRODUCTION n s О ne of the most important directions of MEMS pressure sensors development is realization of silicon sensing el- ements able to work in ultralow pressure ranges of 0.1 to 1.0 kPa. Such pressure sensors may find application in many large-scale production fields (medicine, automotive industry, HVAC) and highly specialized scientific developments (seis- mology, biophysics, robotics) [1]. The opposite side of the task of achieving high piezosensitivity has been and remains an intent to reduce dimensions of a chip and, as a conse- quence, the package size of MEMS pressure sensors [2,3]. We can minimize the area of a chip by reducing sensitivity, while maintaining the principle of its mechanical design [4]. There are alternative methods for enhancing piezosensitivity or re- ducing chip dimensions that are associated with the applica- tion of a novel electric circuit PDA [5-8] using BJT. The de- velopment of such pressure sensors is still at the beginning of the road, but they have already demonstrated an obvious ad- vantage over those with a classical Wheatstone bridge. О Simulated and experimental data on the proposed type of an ultrahigh sensitivity chip of a pressure sensor with a piezore- sistive bridge circuit will be presented in the course of the work. In comparison with known analogs, the used chip struc- ture has both strengths and weaknesses [9-13], but the critical- ity of its parametric values of output characteristics is heavily dependent on the application area of the sensing element. g 𝑅𝑖(∆𝑃) = (1 + 1 2 ∙𝜋44 ∙𝜎(∆𝑃)) ∙𝑅𝑖0, (1) (1) ∆𝑉𝑜𝑢𝑡(∆𝑃) = (𝑅1(∆𝑃)) ∙(𝑅3(∆𝑃)) −(𝑅2(∆𝑃)) ∙(𝑅4(∆𝑃)) (𝑅1(∆𝑃) + 𝑅2(∆𝑃)) ∙(𝑅3(∆𝑃) + 𝑅4(∆𝑃)) 𝑉𝑖𝑛, (2) 𝑆= ∆𝑉𝑜𝑢𝑡(∆𝑃) 𝑉𝑖𝑛· ∆𝑃, (3) 2𝐾𝑁𝐿𝑗(∆𝑃) = ∆𝑉𝑜𝑢𝑡(∆𝑃) −∆𝑉𝑜𝑢𝑡(0,5 𝑘𝑃𝑎) −𝑉0 0,5 𝑘𝑃𝑎 · ∆𝑃 ∆𝑉𝑜𝑢𝑡(0,5 𝑘𝑃𝑎) ∙100%. (4) (3) where ∆P –applied pressure rating, Ri (∆P) and Ri0 – PR rating (i = 1, 2, 3, 4) when ∆P is applied/ not applied, respectively; π44 – main piezoresistive coefficient for PR; σ(∆P) – MS rat- 2 IEEE SENSORS JOURNAL 8. Photolithography of contact windows; 8. Photolithography of contact windows; 8. Photolithography of contact windows; ing at ∆P; ∆Vout(∆P) – output signal of a bridge circuit at ∆P; Vin – bridge circuit supply voltage (5 V); S – sensitivity; 2KNL –output signal nonlinearity error; V0 – output signal at ∆P = 0.0 кPa; ∆Vout(0.5 kPa) – output signal at ∆P = 0.5 кPa. 11. Wafer dicing. The analysis of possible deformation/deflection of the membrane resulted in determination of the required distances for free movement of the sensing element mechanical struc- ture up to the stops (elements of a silicon assembly), needed to raise, in the operating pressure range, the threshold of burst pressure from both sides of the chip (required: Pburst > 300 кPa) [23, 24]. The deflection of the membrane at the rated voltage is ∆h0,5 = 2.7 µm. The bottom stop is made as an in- termediate element between the chip and the base; it is bonded across the area of the chip frame and has via holes to apply pressure from the base. The top stop has a shape of a regular parallelepiped and is bonded from both sides of the chip, where there are no metallic pads; it also has unbonded regions to apply pressure from the topside of the chip (Figure 4). The gap htouch model = 5...23 µm between the chip membrane and the bottom and top stops is ensured by the thickness of a low- temperature bonding glass. The gap may vary to let the mem- brane move freely in the range of the rated pressure and have a margin up to the point close to destruction [25, 26]. That means that touching should occur at Ptouch model = 1.0…4.5 кPa. If the applied pressure exceeds the rated values, the chip membrane (before the moment it breaks) touches the stop and the area with a surface-to-surface contact is reallocated, which stops MS growth. III. TECHNOLOGY The main process steps of fabrication of pressure sensor ul- trahigh sensitivity chips (Figure 5 and Table II) include: 1. Wafer thermal oxidation; 1. Wafer thermal oxidation; 1. Wafer thermal oxidation; 2. Sequence of micro cycles consisting of photolithography and follow-up doping of impurity of; a. Boron for high-alloy conducting areas of resistors (p+- type); I. INTRODUCTION Table I provides geometrical parameters of the structure defined in the process of their variation. Figure 2 shows maps of mechan- ical stresses and the membrane deflection at ∆P = 0.5 кPa. Figure 3 shows the dependence of sensitivity and nonlinearity on the applied pressure for three thicknesses of the membrane (membrane geometry parameter with the highest depend- ence). Theoretical values of the output characteristics for the optimum thickness of the membrane (W = 8 µm) are Smodel = 34.0 mV/кPa/V and 2KNLmodel= 0.68 %FS. 9. Sputtering of Al-Si (1.5 %) WAl-Si = 0,8 µm; 10. Photolithography on metal surface; IV. OUTPUT CHARACTERISTICS Prior to testing, all samples of pressure sensors are subject- ed to thermo- and barocycling in order to remove residual MS from chip bonding. The studies were carried out at the circuit supply voltage Vin of 5.0 V for differential pressure in a range of -0.5 to + 0.5 кPa, with the element topside upwards. The primary parameters for ultrahigh sensitivity chips are sensitivi- ty and nonlinearity that are to be measured at voltage supply from either side because of a certain asymmetry of the charac- teristics. The samples were divided in 3 groups with regard to the membrane thickness W: No.1 – 5…8 µm, No.2 – 8…9 µm, No.3 – 9…11 µm. Besides differences in the membrane thickness, there are spreads in the width of a gap between RIs b. Boron for low-alloy areas of resistors (p--type); c. Phosphorus for a substrate contact (n+-type). 3. Si3N4 (PECVD) deposition as protection at membrane wet etching; 4. Membrane photolithography on the backside of a wafer (RIE); 5. Anisotropic wet etching of a membrane in 30% solution of potassium hydroxide at a temperature of 85 °C; 6. Anisotropic wet etching in HF:HNO3:CH3COOH solution (2:9:4); 7. Si3N4 removal; 7. Si3N4 removal; 3 M. BASOV, D. PRIGODSKIY: INVESTIGATION OF HIGH SENSITIVITY PIEZORESISTIVE PRESSURE SENSORS FOR -0.5…+0.5 KPA M. BASOV, D. PRIGODSKIY: INVESTIGATION OF HIGH SENSITIVITY PIEZORESISTIVE PRESSURE SENSORS FOR -0.5…+0.5 KPA topside or the backside. Average values of sensitivity and nonlinearity are S = 34.5 mV/кPa/V and 2KNL = 0.81 %FS; ∆D = ±3 µm and the length of a RI edge ∆Z = ±60 µm in each group of the samples. topside or the backside. Average values of sensitivity and nonlinearity are S = 34.5 mV/кPa/V and 2KNL = 0.81 %FS; g p p The study of group No.3 is not relevant because of a low sensitivity S < 30 mV/кPa/V. Group No.1 with the required values of sensitivity showed that the application of these sam- ples also lost its relevance due to a high nonlinearity error 2KNL > 1.5 %FS. Group No.1 was divided into two subgroups based on the sensitivity rated values (No.1.1 – S ≈ 40…60 mV/кPa/V and No.1.2 – S ≈ 60…90 mV/кPa/V) in order to determine in further studies how greatly, relative to group No.2, an increase of sensitivity affects: 1) nonlinearity for this range, 2) zero signal ∆V0(σr) due to residual MS from a SiO2 layer. IV. OUTPUT CHARACTERISTICS Figure 7 shows the output signal dependence on the applied pressure ∆Vout(∆P) for 10 average statistical samples in three types of groups. Table III contains the main output parameters; ∆V0(σr) is calculated as follows: • Obtained errors TCZ < 0.1 %FS and TCS < 0.3 %FS, compensated by an external signal processing circuit (ASIC), are sufficiently acceptable; • THZ and THS increase at positive temperatures as com- pared with negative temperatures. General values of tem- perature hysteresis are relatively low for this ultralow pres- sure range and do not exceed 0.6 %FS [46-48]; • Short-term time stability and signal variation due to the effect of mechanical overload are up to 0.3 %FS; • A zero signal at turning the sensing element changes rather considerably, therefore the sensor can be used as accel- erometer. On the other hand, this effect has a negative im- pact on its direct application as the crystal location and in- fluence of the environmental vibration should be taken into consideration. ∆𝑉0(𝜎𝑟) = (∆𝑉𝑜𝑢𝑡(−0,1 𝑘𝑃𝑎) −∆𝑉𝑜𝑢𝑡(+0,1 𝑘𝑃𝑎) 2 ) −𝑉0, (5) The selected structure of an assembly was analyzed with regard to free travel of the membrane of a chip of group No.2 up to the stops. As seen from Figure 8, the membrane travel remains free until the moment of touching Ptouch samples = 2…5 кPa, when pressure is applied from either side of the chip. These chip samples of group No.2 were tested for struc- ture destruction in the absence of stops in the assembly. The membrane is irretrievably deformed at P burst without stops ≈ 9 кPa independently of the direction in which pressure is applied. When stops are used, the area of surface-to-surface contact between the membrane and stops is gradually reallocated at additional overload pressure and sensor is destroyed at a pres- sure above Pburst = 450 кPa. where ∆Vout(-0.1 kPa) – output signal at ∆P = 0.1 кPa applied from the topside, ∆Vout(+0.1 kPa) – output signal at ∆P = 0.1 кPa applied from the backside. Based on the analysis of ∆V0(σr), it may be concluded that residual MSs from SiO2 bend the thinned part of the membrane towards the topside of the chip, or upwards (Figure 4), which is more evident on sub- group No.1.2. IV. OUTPUT CHARACTERISTICS In order to exclude the effect of MSs, arising from bonding the chip with the top stop by low-temperature glass, similar samples of subgroup No.1.2 were combined in an assembly without a top stop and demonstrated the identical bias effect of an output signal ∆V0(σr). The observed effect that also manifests itself at a higher level of sensitivity, when pressure is applied from the topside, is kind of a “flap” of a thin stressed wafer. For elimination of the deficiency the pro- cess should be further modified to achieve the final trade-off relationship between MSs of different signs from SiO2 and Si3N4 films [42-45]. Additional application of wet stop-etching and an increase in the length of a RI edge would make it pos- sible not only to considerably reduce a spread in sensitivity, but to minimize the existing nonlinearity errors as well. where ∆Vout(-0.1 kPa) – output signal at ∆P = 0.1 кPa applied from the topside, ∆Vout(+0.1 kPa) – output signal at ∆P = 0.1 кPa applied from the backside. Based on the analysis of ∆V0(σr), it may be concluded that residual MSs from SiO2 bend the thinned part of the membrane towards the topside of the chip, or upwards (Figure 4), which is more evident on sub- group No.1.2. In order to exclude the effect of MSs, arising from bonding the chip with the top stop by low-temperature glass, similar samples of subgroup No.1.2 were combined in an assembly without a top stop and demonstrated the identical bias effect of an output signal ∆V0(σr). The observed effect that also manifests itself at a higher level of sensitivity, when pressure is applied from the topside, is kind of a “flap” of a thin stressed wafer. For elimination of the deficiency the pro- cess should be further modified to achieve the final trade-off relationship between MSs of different signs from SiO2 and Si3N4 films [42-45]. Additional application of wet stop-etching and an increase in the length of a RI edge would make it pos- sible not only to considerably reduce a spread in sensitivity, but to minimize the existing nonlinearity errors as well. V. CONCLUSION Based on the developed mathematical model, the optimum geometry of a membrane structure has been defined and an ultrahigh sensitivity chip of a pressure sensor, operating in a ultralow differential pressure range of -0.5 to +0.5 кPa with S = 34.5 ± 4.1 mV/кPa/V and 2KNL = 0.81 ± 0.50 %FS, has been realized. The difference in output characteristics measured when pressure is applied from the topside and the backside is relatively small (the difference in sensitivity is 2%) for the selected design of a chip. The samples show a certain spread in the rated values of sensitivity and sensor errors that can be minimized during further technological upgrading. The errors of temperature characteristics, time stability and overload abil- ity of chips are also sufficiently small for the ultralow pressure range (Table 4). The RI inertial mass allows considering this chip as an ultrahigh sensitivity accelerometer. Additionally, an advantage has been achieved as the threshold of burst pressure Pburst was raised above 450 кPa owing to the use of stop ele- ments in the assembly. Table IV contains characteristics of group No.2 samples (62 samples) with an optimum membrane thickness W of 8 to 9 µm. The calculation of parameters as relative values associat- ed with temperature characteristics, time stability and impact of overload pressure is carried out when pressure is applied from the membrane. Temperature parameters are measured in two separate temperature subranges: negative subrange from - 30 °С to +20 °С, positive subrange from +20 °С to +60° С. Testing of mechanical strength is performed at an overload pressure Pproof of 30 кPa (pressure is applied from both sides of the chip). Time stability of an output signal was monitored within the first 9 hours after supplying power to the chip elec- tric circuits. The effect of RI inertial mass at turning the chip by 180° was considered, which indirectly describes sensitivity of sensing elements, e.g., an accelerometer. With the analysis of output characteristics performed, it may be noted: The presented structure of a pressure sensor may be valid for researches, involved in investigations in this field, from the viewpoint of further upgrade and fabrication of pressure sen- sors chips with ultrahigh sensitivity and low errors. REFERENCES Kuhl, A square-diaphragm piezoresistive pressure sensor with a rectangular central boss for low-pressure ranges, IEEE Transactions on Electron Devices 40 (1993) 1754-1759. [10] S. Marco, J. Samitier, O. Ruiz, J.R. Morante, J. Esteved, High- Performance Piezoresistive Pressure Sensors for Biomedical Applica- tions Using Very Thin Structured Membranes, IOP Measurement Sci- ence and Technology 7 (1996) 1195-1203. [33] A.A. Barlian, W.T. Park, J.R. Mallon, A.J. Rastegar, B.L. Pruitt, Re- view: Semiconductor piezoresistance for microsystems, Proceedings of the IEEE, 97 (2009) 513–552. [11] Yu, Z., Zhao, Y., Sun, L., Tian, B., Jiang, Z., 2013. 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A Bossed Diaphragm Piezoresistive Pres- sure Sensor with a Peninsula-Island Structure for the Ultra-Low- Pressure Range with High Sensitivity. IOP Measurement Science and Technology. 27, 124012. REFERENCES Map of MS distribution (а) and membrane deflection (b) for the select- ed membrane geometry at pressure 0.5 кPa (b) (a) films PECVD deposition: a review, Journal of Optoelectronics and Ad- vanced Materials 4 (2011) 387-394. [45] Nie, M., Bao, H., 2016. A theoretical model and analusis of composite membrane of a piezoresistive pressure sensor. AIP Advances. 6, 105302. [46] Liu, Y., Wang, H., Zhao, W., Qin, H., Fang, X., 2016. Thermal- performance instability in piezoresistive sensors: Inducement and im- provement. Sensors. 16, 1984. [47] J.A. Chiou, S. Chen, Thermal hysteresis analysis of MEMS pressure sensors, Journal of Microelectromechanical Systems 14 (2005) 782-787. [48] H.N. Chiang, T.L. Chou, C.T. Lin, K.N. Chiang, Investigation of the hysteresis phenomenon of a silicon-based piezoresistive pressure sensor, IEEE Conference International Microsystems, Packaging, Assembly and Circuits Technology (2007), 165-168. (b) (a) Fig. 2. Map of MS distribution (а) and membrane deflection (b) for the select- ed membrane geometry at pressure 0.5 кPa sensing elements. Mikhail Basov was born in Domodedovo, Russian Federation, in 1989. He received the B.S. degrees in Nano- and Microelec- tronics engineering from the National Nuclear Research University “Moscow Physics Engineering Institute”, in 2012. Mikhail Basov was born in Domodedovo, Russian Federation, in 1989. He received the B.S. degrees in Nano- and Microelec- tronics engineering from the National Nuclear Research University “Moscow Physics Engineering Institute”, in 2012. Mikhail Basov was born in Domodedovo, Russian Federation, in 1989. He received the B.S. degrees in Nano- and Microelec- tronics engineering from the National Nuclear Research University “Moscow Physics Engineering Institute”, in 2012. Fig. 3. Sensitivity (solid line) and nonlinearity (dot line) dependence on the pressure at variation of the membrane thickness W Starting from 2010 he works as R&D engineer on advanced MEMS pressure His Ph.D. thesis is titled “High-Sensitivity Pressure Sensing Chip Utilizing Bipolar- Junction Transistors”. sensing elements. Denis M. Prigodskiy graduated from the National Research Technological Univer- sity “MISIS”, Moscow, Russia in 2008, in specialty microelectronics, engineer. He was a researcher at RPE “Kvant”, Mos- cow from 2008 to 2012 and was engaged in heterostructural photoelectric solar cells for space applications. Since 2012, he has been working as a leading research engineer at the “All-Russian Research Insti- tute of Automatics named after N.L. Dukhov”, Moscow, and has been working on semiconductor devices and silicon-based pressure sensors. REFERENCES Sensors. 17, 1965. [38] K. Petersen, P. Barth, J. Poydock, J. Brown, J. J. Mallon, J. Bryzek, Silicon fusion bonding for pressure sensors, Tech. Digest 1988 IEEE Solid-State Sensor and Actuator Workshop (1988) 144–147. [17] N. Arjunan, S. Thangavelu, Modeling and Analysis of a Multi Bossed Beam Membrane Sensor for Environmental Applications, Transactions on Electrical and Electronic Materials 18 (2017) 25–29. [39] S. Guo, H. Eriksen, K. Childress, A. Fink, M. Hoffman, High tempera- ture smart-cut SOI pressure sensor, Sensors and Actuators A: Physical 154 (2009) 255–260. [18] R.H. Johnson, S. Karbassi, U. Sridhar, B. Speldrich, A High-Sensitivity Ribbed and Bossed Pressure Transducer, Sensors and Actuators A: Physical 35 (1992) 93-99. [40] D.M. Prigodskiy, M.V. 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Guan, F. Yang, W. Wang, X. Huang, B. Jiang, D. Zhang, The Design and Analysis of Piezoresistive Shuriken-Structured Diaphragm Micro- Pressure Sensors, Journal of Microelectromechanical Systems PP(99) (2016) pp. 1-9. [22] A.A. Barlian, W.T. Park, J.R. Mallon, A.J. Rastegar, B.L. Pruitt, Re- view: Semiconductor piezoresistance for microsystems, Proceedings of the IEEE, 97 (2009) 513–552. [44] C. Iliescu, M. Avram, B. Chen, A. Popescu, V. Dumitrescu, D.P. Poe- nar, A. Sterian, D. Vrtacnik, S. Amon, P. Sterian, Residual stress in thin 5 M. BASOV, D. PRIGODSKIY: INVESTIGATION OF HIGH SENSITIVITY PIEZORESISTIVE PRESSURE SENSORS FOR -0.5…+0.5 KPA (a) (b) Fig. 2. REFERENCES He is author and co-author of 10 publications and 4 patents in the field of semiconductor devices and MEMS technologies. Fig. 3. Sensitivity (solid line) and nonlinearity (dot line) dependence on the pressure at variation of the membrane thickness W Fig. 4. Schematic representation of an assembly comprising a chip, stops and a base (a) (b) Fig. 1. Schematic representation of an ultrahigh sensitivity pressure sensor chip: a) topside, b) backside (a) (b) Fig. 4. Schematic representation of an assembly comprising a chip, stops and a base (b) (a) Fig. 1. Schematic representation of an ultrahigh sensitivity pressure sensor chip: a) topside, b) backside 6 IEEE SENSORS JOURNAL Fig. 5. Main process steps for an ultrahigh sensitivity pressure sensor chip (section А-А from Fig. 1) (b) (c) Fig. 7. Variation of an output signal versus the applied pressure ∆Vout(∆P) for groups: а) No.2, b) No.1.1, c) No.1.2 (b) (c) (b) (b) (b) (c) Fig. 5. Main process steps for an ultrahigh sensitivity pressure sensor chip (section А-А from Fig. 1) (c) (a) (b) Fig. 6. Examples of images of the obtained samples: a) rigid center geometry relative to PR regions, b) assembly in the package (a) (b) Fig. 7. Variation of an output signal versus the applied pressure ∆Vout(∆P) for groups: а) No.2, b) No.1.1, c) No.1.2 Fig. 8. Variation of an output signal versus the pressure for several typical samples of ultrahigh sensitivity pressure sensors (b) (a) Fig. 6. Examples of images of the obtained samples: a) rigid center geometry relative to PR regions, b) assembly in the package (a) (a) Fig. 8. Variation of an output signal versus the pressure for several typical samples of ultrahigh sensitivity pressure sensors TABLE I TABLE I MAIN GEOMETRICAL PARAMETERS OF THE SENSING ELEMENT Geometrical Parameter Size, μm L 6150 Q1/Q2 1122/3366 W 8 H 420 A 4200 D 18 Z 400 Y 690 G 285 (a) 7 M. BASOV, D. PRIGODSKIY: INVESTIGATION OF HIGH SENSITIVITY PIEZORESISTIVE PRESSURE SENSORS FOR -0.5…+0.5 KPA 7 TABLE II DIFFUSION PARAMETERS OF ULTRA-HIGH-SENSITIVITY PRESSURE SENSOR Process step Parameters Oxidation 1100 ºC, 15 min.-20 min.-15 min (dry-wet-dry) Forming high-doped P+ areas Diffusion from unlimited source: 1050 ºC, 55 min. Drive-in and oxidation: 1150 ºC, 5 min – 15 min. – 5 min (dry-wet-dry) Forming P- resistors Ion implantation: D = 8.0·1014 cm-2, E = 50 keV Impurity activation and oxidation: 1100 ºC for 45 min in inert atmosphere followed by oxidation 1000 ºC, 5 min – 35 min. – 5 min. (dry- wet-dry) Forming N+ areas Diffusion from unlimited source: 1000 ºC, 5+30+10 min. (Ar/O2+Ar/O2/POCl3+Ar/O2) Drive-in and oxidation: 1000 ºC, 5 min – 30 min. – 10 min (dry-wet-dry) TABLE III INFLUENCE OF TECHNOLOGICAL ERRORS Parameters Type of chip №2 №1.1 №1.2 Sensitivity S, mV/V/kPa Backside 34,2 ± 3,9 49,2 ± 8,6 73,2 ± 12,9 Topside 34,8 ± 4,2 50,5 ± 8,9 75,2 ± 14,0 Nonlinearity 2KNL, %FS Backside 0,77 ± 0,48 3,5 ± 2,1 9,5 ± 2,9 Topside 0,85 ± 0,52 4,5 ± 2,3 10,0 ± 3,2 ∆V0(σr), mV 0,26 ± 0,26 1,61 ± 0,59 8,72 ± 6,22 TABLE IV PERFORMANCE OF ULTRA-HIGH-SENSITIVITY PRESSURE SENSOR Parameters Value Sensitivity S, mV/V/kPa Backside 34,2 ± 3,9 Topside 34,8 ± 4,2 Nonlinearity 2KNL, %FS Backside 0,75 ± 0,48 Topside 0,87 ± 0,52 Zero pressure output signal (Offset) V0, mV/V < 5 TCZ (–30…+20 °С), %FS/°С 0,055 ± 0,051 TCZ (+20…+60 °С), %FS/°С 0,048 ± 0,044 TCS (–30…+20 °С), %FS/°С 0,280 ± 0,021 TCS (+20…+60 °С), %FS/°С 0,234 ± 0,026 Zero thermal hysteresis (THZ) (–30…+20°С), %FS 0,21 ± 0,13 (+20…+60°С), %FS 0,34 ± 0,25 Span thermal hysteresis (THS) (–30…+20°С), %FS 0,12 ± 0,11 (+20…+60°С), %FS 0,21 ± 0,18 Long-term instability of zero offset, %FS 0,18 ± 0,11 of pressure sensitivity, %FS 0,10 ± 0,09 Burst pressure Pburst, kPa > 450 Changing after proof pressure Pproof zero, %FS 0,19 ± 0,11 span, %FS 0,11 ± 0,08 Zero offset after turning on 180°, µV/V 52 ± 10 Number of samples in statistics 62 V, D. TABLE I PRIGODSKIY: INVESTIGATION OF HIGH SENSITIVITY PIEZORESISTIVE PRESSURE SENSORS FOR -0.5…+0.5 KPA IGODSKIY: INVESTIGATION OF HIGH SENSITIVITY PIEZORESISTIVE PRESSURE SENSORS FOR -0.5…+0.5 KPA TABLE IV ANCE OF ULTRA-HIGH-SENSITIVITY PRESSURE SENSOR TABLE IV ERFORMANCE OF ULTRA-HIGH-SENSITIVITY PRESSURE SENSOR TABLE III INFLUENCE OF TECHNOLOGICAL ERRORS Parameters Type of chip №2 №1.1 №1.2 Sensitivity S, mV/V/kPa Backside 34,2 ± 3,9 49,2 ± 8,6 73,2 ± 12,9 Topside 34,8 ± 4,2 50,5 ± 8,9 75,2 ± 14,0 Nonlinearity 2KNL, %FS Backside 0,77 ± 0,48 3,5 ± 2,1 9,5 ± 2,9 Topside 0,85 ± 0,52 4,5 ± 2,3 10,0 ± 3,2 ∆V0(σr), mV 0,26 ± 0,26 1,61 ± 0,59 8,72 ± 6,22
https://openalex.org/W2810411552	https://journal.lppmunindra.ac.id/index.php/SAP/article/download/1723/1337	Indonesian	null	Pengembangan Aplikasi Multimedia Pembelajaran Interaktif untuk Mata Pelajaran Bahasa Indonesia	SAP (Susunan Artikel Pendidikan)	2,017	cc-by	4,889	p-ISSN: 2527-967X e-ISSN: 2549-2845 p-ISSN: 2527-967X e-ISSN: 2549-2845 p-ISSN: 2527-967X e-ISSN: 2549-2845 Jurnal SAP Vol. 2 No. 1 Agustus 2017 ABSTRAK Perkembangan teknologi yang sangat pesat khususnya komputer, membawa dampak dalam dunia pendidikan dalam memanfaatkan komputer. Komputer dapat digunakan sebagai salah satu media pembelajaran yang interaktif. Untuk memudahkan dalam penyampaian materi pembelajaran kepada siswa maka materi ini dikemas secara menarik dalam bentuk multimedia yang bersifat mendidik dan menghibur bagi mereka. Tujuan penelitian ini adalah membuat aplikasi pembelajaran Bahasa Indonesia Interaktif berbasis multimedia sebagai alternatif media pembelajaran yang mudah dipelajari dan dipahami oleh siswa dengan konsep edutainmentpada Bimbel D-LLA COURSE. Aplikasi ini dibuat dengan menggunakan software Macromedia Flash.Pembuatan aplikasi ini menggunakan metode observasi dan studi pustaka.Berdasarkan hasil penelitian yang sudah dilakukan diperoleh kesimpulan bahwa aplikasi pembelajaran bahasa Indonesia Interaktif ini sudah berhasil dibuat yang terdiri dari enam menu yaitu : 4 jenis latihan (mengenal huruf di keyboard, belajar pemenggalan kata, tebak gambar dan menyusun huruf) dan 2 jenis permainan yaitu ( menangkap kata dalam telur, mencocokkan gambar dan kata) serta dilakukan pengujian kelayakan aplikasi mengunakan SQL. Kata kunci:, Media pembelajaran , Bahasa Indonesia , Multimedia , Edutainment , SQL ABSTRACT The increasingly rapid development of technology, especially computer, creates an impact on the use of computer in education. Computers can be used as one of interactive learning media. To facilitate the delivery of learning materials to students, there must be an interesting packaging of materials in the form of multimedia that is educative and entertaining for them. The objective of this research is to make an application of interactive multimedia-based Indonesian language learning as an alternative learning medium that is easy to learn and understand by students with an edutainment concept at Bimbel D-LLA COURSE. The application is made by using software Macromedia Flash. It is made using observation and literature review methods. From the result of the research, it can be concluded that the application of interactive Indonesian language learning has been successfully made with six menus, namely: 4 types of exercises (knowing letters on keyboard, learning syllabification, guessing images and arranging letters) and 2 types of games (catching words on eggs, matching pictures and words). Besides, the fitness of this application has also been tested using SQL. ords:Learning Media , Indonesian Language , Multimedia , Edutainment , SQL Keywords:Learning Media , Indonesian Language , Multimedia , Edutainment , SQL PENGEMBANGAN APLIKASI MULTIMEDIA PEMBELAJARAN INTERAKTIF UNTUK MATA PELAJARAN BAHASA INDONESIA Syamsiah Program Studi Informatika, Universitas Indraprasta PGRI Email: ncham.unindra08@gmail.com Pendahuluan Perkembangan teknologi yang pesat mempengaruhi peran sebuah komputer itu sendiri. Pada awalnya penggunaan komputer hanya difokuskan pada proses pengolahan data. Akan dijadikan sebagai media informasi dalam berbagai bidang salah satunya adalah bidang pendidikan.Pemanfaatan komputer sebagai media pembelajaran dalam dunia pendidikan sudah mulai digunakan di lembaga bimbingan belajar (bimbel). Sebagaimana seorang pengajar di suatu bimbel, komputer dapat digunakan sebagai media dalam proses belajar mengajar yang mempunyai fungsi sebagai media tutorial sehingga akan sangat membantu dalam proses penyampaian dan pemahaman terhadap materi yang disampaikan. 53 p-ISSN: 2527-967X e-ISSN: 2549-2845 Jurnal SAP Vol. 2 No. 1 Agustus 2017 p-ISSN: 2527-967X e-ISSN: 2549-2845 Jurnal SAP Vol. 2 No. 1 Agustus 2017 Pemberian materi bahasa Indonesia pada bimbel sejauh ini hanyalah dengan sarana papan tulis dengan kapur atau pun spidol untuk memberikan contoh atau gambaran kepada siswa. Dengan penyampaian materi pelajaran seperti disebutkan diatas, kualitas ilmu yang tersampaikan kepada siswacenderung monoton dan kurang maksimal.Siswa cenderung sulit memahami ataupun menerima pelajaran terasa membosankan,kaku dan kurang interaktif. Komputer yang memiliki kamampuan multimedia mampu menyajikan informasi secara audio visual yang menarik dan interaktif.Belajar dengan media komputer dapat diperkenalkan sejak dini kepada anak, yakni dengan programprogram aplikasi yang bersifat memadukan unsur pendidikan (education) dengan unsur hiburan (entertainment) atau sering dikenal dengan sebutan edutainment. Perpaduan tersebut akan melahirkan suasana belajar yang mengasyikkan bagi anak dengan tidak mengurangi nilai pembelajaran materi Bahasa Indonesia. Media pembelajaran bahasa Indonesia yang interaktif di sajikan dalam beberapa pemecahan masalah dari soal serta latihan-latihan dari materi yang ada. Dengan menerapkan metode pembelajaran interaktif melalui aplikasi yang berfokus pada proses pembelajaran bahasa Indonesia yang dilakukan secara praktek langsung, maka diharapkan akan meningkatkan kualitas proses pembelajaran. Dalam dunia pendidikanpun manfaat multimedia sudah dirasakan oleh semua pihak khususnya siswa. Siswa dapat menggunakan fasilitas ini untuk melakukan pembelajaran secaran interaktif sehingga membuat pelajaran bahasa Indonesia menjadi lebih menarik dan mudah dipahami. Tujuan penelitian ini adalah membuat suatu aplikasi pembelajaran bahasa Indonesia yang interaktif berbasis multimedia untuk lembaga bimbingan belajar (Bimbel) sebagai alternatif sistem pembelajaran yang menarik dan mudah dipahami oleh siswadengan konsep edutainment. Penelitian ini diharapkan dapat memberikan manfaat baik secara teoretis maupun secara praktis. Penelitian ini diharapkan dapat memberikan manfaat baik secara teoretis maupun secara praktis. a. Seara teoretis, penelitian ini dapat digunakan sebagai referensi alternatif dalam mengembangkan media pembelajaran interaktif yangdapat digunakan sebagai sarana belajar bahasa Indonesia. b. Secara praktis: b. Secara praktis: 1. Pendahuluan Manfaat bagi pengajar dan bimbel, hasil penelitian ini berupa produk CD pembelajaran interaktif yang diharapkan dapat memberikan sumbangan pemikiran bagi para pengajar dan bimbel, sehingga produk ini dapat dijadikan bahan ajar bagi siswauntukbelajar bahasa indonesia dengan cara yang menyenangkan dan intraktif. j g y g y g 2. Manfaat bagi siswa, hasil penelitian ini diharapkan dapat meningkatkan pemahaman, rasa cinta dan bangga terhadap bahasa Indonesia. 3. Bagi peneliti, penelitian ini diharapkan dapat memperluas pengetahuan tentang pengembangan media pembelajaran interaktif yang dapat dijadikan sebagai sarana belajar siswa yang interaktif dan menyenangkan Tinjauan Pusataka Pembelajaran Bahasa Indonesia 3. Teknik Pembelajaran 3. Teknik Pembelajaran Teknik adalah jalan, alat, atau media yang digunakan oleh guru untuk mengarahkan kegiatan siswa kearah tujuan yang ingin dicapai. Dalam kamus Besar Bahasa Indonesia, teknik diartikan sebagai metode atau sistem mengerjakan sesuatu, cara membuat atau melakukan sesuatu yang berhubungan dengan seni (2005: 1158).Istilah teknik dalam pembelajaran bahasa mengacu pada pengertian implementasi perencanaan pengajaran di depan kelas, yaitu penyajian pelajaran dalam kelas tertentu dalam jam dan materi tertentu pula. Teknik mengajar berupa berbagai macam cara, kegiatan, dan kiat (trik) untuk menyajikan pelajaran dalam rangka mencapai tujuanpembelajaran. Teknik pembelajaran bersifat implementasi, individual dan situasional. 1. Pendekatan Pembelajaran 1. Pendekatan Pembelajaran Istilah pendekatan dalam pembelajaran bahasa Indonesia mengacu pada teori-teori tentang hakekat bahasa dan pembelajaran bahasa Indonesia yang berfungsi sebagai sumber landasan/prinsip pengajaran bahasa Indonesia. Teori tentang hakikat bahasa Indonesia mengemukakan asumsi-asumsi tentang hakikat bahasa, karakteristik bahasa, unsur-unsur bahasa, serta fungsi dan pemakaiannya sebagai media komunikasi dalam suatu masyarakat bahasa. Menurut Anthony (Slamet, 2007: 50) pendekatan mengacu kepada seperangkat asumsi yang saling berkaitan dan berhubungan dengan pengajaran.Pendekatan merupakan dasar teoritis untuk suatu metode. Pembelajaran Bahasa Indonesia Untuk mendapatkan pemahaman yang komprehensif tentang strategi pembelajaran Bahasa Indonesia dan efektivitasnya terhadap pencapaian tujuan belajar, kajian Pembelajaran Bahasa Indonesia ini akan difokuskan pada (1) pembelajaran bahasa, dan (2) strategi pembelajaran Bahasa Indonesia, meliputi metode dan teknik pembelajaran Bahasa Indonesia, a. Pembelajaran Bahasa Pembelajaran merupakan upaya membelajarkan siswa atau kegiatan pengupayaan ini akan mengakibatkan siswa dapat mempelajari sesuatu dengan cara efektif dan efisien. Upaya yang dilakukan dapat berupa analisis tujuan dan karakteristik studi dan siswa, j Pembelajaran merupakan upaya membelajarkan siswa atau kegiatan pengupayaan ini akan mengakibatkan siswa dapat mempelajari sesuatu dengan cara efektif dan efisien. j p p y j g p g p y akan mengakibatkan siswa dapat mempelajari sesuatu dengan cara efektif dan efisien. Upaya yang dilakukan dapat berupa analisis tujuan dan karakteristik studi dan siswa, g p p j g Upaya yang dilakukan dapat berupa analisis tujuan dan karakteristik studi dan sisw 54 p-ISSN: 2527-967X e-ISSN: 2549-2845 p-ISSN: 2527-967X e-ISSN: 2549-2845 2. Metode Pembelajaran 2. Metode Pembelajaran Istilah metode berarti perencanaan secara menyeluruh untuk menyajikan materi pelajaran bahasa secara teratur. Istilah ini bersifat prosedural, dalam arti penerapan suatu metode dalam pembelajaran bahasa dikerjakan dengan melalui langkah yang teratur dan secara bertahap, dimulai dari penyusunan perencanaan pengajaran, penyajian pengajaran, proses belajar mengajar dan penilaian hasil belajar. Metode pembelajaran berarti carayang dipakai oleh guru agar tujuan pembelajaran dapat dicapai secara efektif dan efesien. Menurut (Sutikno, 2009: 88).Metode pembelajaran adalah cara menyajikan materi pelajaran yang dilakukan oleh pendidik agar terjadi proses pembelajaran pada diri siswa dalam upaya untuk mencapai tujuan. Jurnal SAP Vol. 2 No. 1 Agustus 2017 Jurnal SAP Vol. 2 No. 1 Agustus 2017 analisis sumber belajar, menetapkan strategi pengorganisasian, isi pembelajaran, menetapkan strategi penyampaian pembelajaran, menetapkan strategi pengelolaan pembelajaran, dan menetapkan prosedur pengukuran hasil pembelajaran. analisis sumber belajar, menetapkan strategi pengorganisasian, isi pembelajaran, menetapkan strategi penyampaian pembelajaran, menetapkan strategi pengelolaan pembelajaran, dan menetapkan prosedur pengukuran hasil pembelajaran. b. Strategi Pembelajaran Bahasa Indonesia Pembicaraan mengenai strategi pembelajaran bahasa tidak terlepas dari pembicaraan mengenai pendekatan, metode, dan teknik mengajar.Secara umum strategi adalah suatu garis besar haluan dalam bertindak untuk mencapai tujuan yang sudah ditentukan (Pupuh &Sobry, 2007: 3). Menurut Kemp (Senjaya, 2008) mengemukakan bahwa strategi pembelajaran adalah suatu kegiatan pembelajaran yang harus dikerjakan guru dan siswa supaya tujuan pembelajaran dapat dicapai secara efektif dan efisien, menurut (Sumarno, 2011) Strategi pembelajaran dapat diartikan sebagai kegiatan yang dipilih oleh pembelajar atau instruktur dalam proses pembelajaran yang dapat memberikan kemudahan fasilitas kepada pembelajar menuju kepada tercapainya tujuan pembelajaran tertentu yang sudah ditetapkan. p-ISSN: 2527-967X e-ISSN: 2549-2845 Jurnal SAP Vol. 2 No. 1 Agustus 2017 Jurnal SAP Vol. 2 No. 1 Agustus 2017 p-ISSN: 2527-967X e-ISSN: 2549-2845 tengah. Dalam bahasa Indonesia kata medium diartikan sebagai “antara’ atau “sedang” untuk menjelaskan media pembelajaran adalah segala sesuatu yang dapat digunakan untuk menyalurkan pesan dari pengirim ke penerima pesan. Berdasarkan pernyataan tersebut dapat disimpulkan bahwa media pembelajaran adalah alat bantu yang digunakan oleh guru sebagai alat bantu mengajar. Berdasarkan beberapa pendapat di atas, dapat disimpulkan pengertian media pembelajaran sebagai alat bantu mengajar untuk menyampaikan materi agar pesan lebih mudah diterima dan menjadikan siswa lebih termotivasi dan aktif. tengah. Dalam bahasa Indonesia kata medium diartikan sebagai “antara’ atau “sedang” untuk menjelaskan media pembelajaran adalah segala sesuatu yang dapat digunakan untuk menyalurkan pesan dari pengirim ke penerima pesan. Berdasarkan pernyataan tersebut dapat disimpulkan bahwa media pembelajaran adalah alat bantu yang digunakan oleh guru sebagai alat bantu mengajar. Berdasarkan beberapa pendapat di atas, dapat disimpulkan pengertian media pembelajaran sebagai alat bantu mengajar untuk menyampaikan materi agar pesan lebih mudah diterima dan menjadikan siswa lebih termotivasi dan aktif. Pengertian Multimedia Multimedia berasal dari teater, yaitu pertunjukan yang memanfaatkan lebih dari satu medium di panggung yang mencakup monitor video, synthesized band, dan karya seni manusia sebagaibagian dari pertunjukan. Pengertian kedua mensyaratkan adanya sinkronisasi berbagai media tadi dengan bantuan komputer, Membedakkan dengan pengertian multimedia yang pertama yang memanfaatkan berbagai media yang terpisah dan berdiri sendiri (Hamalik, 2005:34). Unsur-Unsur Multimedia Multimedia memiliki unsur sebagai berikut (Haryadi, 2006:10) Teks Bentuk data multimedia yang paling mudah disimpan dan dikendalikan adalah teks. Kebutuhan teks bergantung pada kegunaan aplikasi multimedia. a. Gambar Gambar dapat meringkas dan menyajikan data kompleks dengan cara yang baru dan lebih berguna. Gambar sering kali muncul sebagai backdrop (latar belakang) yang mempermanis teks. b. Audio Multimedia tanpa bunyi hanya disebut unimedia, bukan multimedia. Masing-masing kemampuan membutuhkan teknologi,perangkatkeras, dan perangkat lunak untuk menjalankannya. Ada tigabelas jenis objek bunyi yang bisa digunakan dalam produksi multimedia, yakni format waveform audio, aiff dat, ibf, mod, rni, sbi, snd, voc, au, MIDI soundtrack, compact disk audio, dan MP3 file. b. Audio c. Video Video menyediakan sumber daya yang kaya dan hidup bagi aplikasi multimedia. Ada empat macam video yang dapat digunakan sebagai objek link dalam aplikasi multimedia :live video feeds, videotape, videodisc, dan digital video. d. Animasi Dalam multimedia, animasi merupakan penggunaan komputer untuk menciptakan gerak pada layar. Ada Sembilan macam yaitu animasi sel, animasi frame, animasi sprite,animasilintasan, animasi splin, animasi vector, animasi karakter, animasi computational, dan morphing. Pengertian Media Pembelajaran g j Media adalah bentukjamak dari medium yang berasal dari bahasa latin medius yang berarti 55 p-ISSN: 2527-967X e-ISSN: 2549-2845 Metode Pengumpulan Data a. Diskusi Interaktif Diskusi interaktif merupakan metode pengumpulan data dengan cara mengajukan pertanyaan secara lisan kepada pihakyang bersangkutan.Dalam hal ini pihak Bimbel D- lla Cause sebagai objek penelitian. b. Studi Kepustakaan Studi pustaka adalah penelitian untukmencari teori/konsep/generalisasi yang dapat digunakan sebagai landasan teori dalam penelitian.Untukmencari metodologi yang sesuai dilakukan dengan teknik membandingkan antara teori yang ada dengan fakta dilapangan. Literatur yang didapat oleh penulis adalah modul referensi yang digunakan guru, jurnal ilmiah dan data yang diperoleh dari internet dengan sumber terpercaya. c. Kuisioner Kuisioner merupakan teknik pengumpulan data yang dilakukan dengan cara memberi seperangkat pertanyaan atau pernyataan tertulis kepada responden untuk dijawabnya. Responden yang dimaksud disini yaitu pengajar dan pemilik bimbel. c. Kuisioner Kuisioner merupakan teknik pengumpulan data yang dilakukan dengan cara memberi seperangkat pertanyaan atau pernyataan tertulis kepada responden untuk dijawabnya. Responden yang dimaksud disini yaitu pengajar dan pemilik bimbel. Software Quality Assurance (SQA) Untuk memastikan bahwa perangkat lunak yang dibuat memiliki standar minimal kualitas, maka salah satu metode untuk pengukuran kualitas perangkat lunak secara kuantitatif adalah metode SQA (Software Quality Assurance). Tabel.1 Metric of Software Quality Assurance (SQA) No Metrik Deskripsi Bobot 1 Auditability Memenuhi standard atau tidak 0.1 2 Accuracy Keakuratan komputasi 0.15 3 Completeness Kelengkapan 0.1 4 Error Tolerance Toleransi terhadap kesalahan 0.1 5 Execution Efficiency Kinerja Eksekusi 0.1 6 Operability Kemudahan untuk dioperasikan 0.15 7 Simplicity Kemudahan untuk difahami 0.15 8 Training Kemudahan pembelajaran fasilitas Help 0.15 Ada 8 buah kriteria yang dapat digunakan untuk mengukur kualitas sebuah perangkat lunak secara kuantitatif.Seperti terlihat pada table.1 Tabel.1 Metric of Software Quality Assurance (SQA) Metodologi Penelitian Metode Pengumpulan Data Jurnal SAP Vol. 2 No. 1 Agustus 2017 Jurnal SAP Vol. 2 No. 1 Agustus 2017 p-ISSN: 2527-967X e-ISSN: 2549-2845 Jurnal SAP Vol. 2 No. 1 Agustus 2017 p-ISSN: 2527-967X e-ISSN: 2549-2845 Macromedia Flash sering digunakan untuk membuat animasi dan untuk keperluan lain seperti membuat game dan tutorial. Karena Macromedia dapat menampilkan teks, gambar, animasi, dan audio secara bersama maka sangat mungkin apabila Macromedia Flash digunakan sebagai sarana pengembangan media pembelajaran. Dapat disimpulkan bahwa Macromedia Flash merupakan software yang dapat dikembangkan menjadi media pembelajaran yang interaktif. Software Quality Assurance (SQA) Macromedia Flash Macromedia Flash merupakan aplikasi yang digunakan untuk melakukan desain dan membangun perangkat presentasi, publikasi, atau aplikasi lainnya yang membutuhkan ketersediaan sarana interaksi dengan penggunanya. Proyek yang dibangun dengan Flash bias terdiri atas teks,gambar,animasi sederhana, video, atau efek-efek khusus lainnya. Dikse (2010:1) berpendapat bahwa Macromedia Flash dibuat oleh perusahaan software macromedia untuk keperluan membuat suatu aplikasi webyang interaktif dan menarik. 56 Jurnal SAP Vol. 2 No. 1 Agustus 2017 menggunakan. Angket ini digunakan untuk mendapatkan data tentang kelayakan penggunaan aplikasi pembelajaran bahasa Indonesia yang interaktif berbasis multimedia sebagai media dalam proses belajar mengajar. a. Latihan,terdiri dari 4 jenis latihan yaitu : a. Latihan,terdiri dari 4 jenis latihan yaitu : 1. Mengenal Huruf di Keyboard 2. Belajar Pemenggalan Kata 3. Tebak Gambar 4. Menyusun Huruf b. Permainan, terdiri dari 2 jenis permainan yaitu : 1. Kata dalam Telur 1. Kata dalam Telur 2. Mencocokkan Gambar dan Kata Aplikasi " aplikasi media pembelajaran bahasaIndonesia yang interaktif berbasis multimedia" memiliki antarmuka sebagai berikut : Hasil dan Pembahasan Produk penelitian yang dihasilkan adalah Perancangan Media Pembelajaran Interakif untuk mata pelajaran bahasa Indonesia. Pelajaran yang diberikan melalui 2 cara yaitu : Tampilan Pembuka p Tampilan pembuka muncul pada saat pengguna membuka program pertama kali. Gambar 1. Tamplan Menu Utama Gambar 1. Tamplan Menu Utama Sumber Data a. Sumber literature Pengumpulan materi mata pelajaran diambil dari modul yang sudah dipakai dalam yanglain. Pengambilan script serta pedoman pembuatan modul interaktif diambil dari beberapa buku-buku panduan belajar macromedia flash. b. Sumber data primer Cara pengambilan sumber data primer di sini adalah mengambil data dengan b. Sumber data primer Cara pengambilan sumber data primer di sini adalah mengambil data dengan 57 p-ISSN: 2527-967X e-ISSN: 2549-2845 p-ISSN: 2527-967X e-ISSN: 2549-2845 a. Tombol Latihan Apabila tombol latihan ditekan, maka halaman akan berganti ke menu latihan. Menu latihan memiliki 4 (empat) jenis latihan yang dapat dimainkan. Sebaiknya pengguna memulai latihan secara urut dimulai dari latihan 1.Pada halaman menu latihan dan halaman masing- masing latihan terdapat tombol kembali. e. Tombol Keluar e. Tombol Keluar Apabila tombol keluar ditekan, pengguna akan keluar dari aplikasi. e. Tombol Keluar Apabila tombol keluar ditekan, pengguna akan keluar dari aplikasi. c. Tombol Daftar Nilai c. Tombol Daftar Nilai Apabila tombol daftar nilai ditekan, maka halaman akan berganti ke halaman daftar nilai. Halaman daftar nilai adalah daftar nilai 10 (sepuluh) terbaik yang diperoleh oleh pengguna dalam setiap menu. Pada halaman ini terdapat tombol kembali. d. Tombol Bantuan Apabila tombol bantuan ditekan, maka halaman akan berganti ke menu bantuan. Menu bantuan berisikan informasi singkat pemakaian aplikasi online "educational game belajar membaca dan menulis untuk pemula". Menu Utama Menu Utama Menu utama merupakan bagian utama dari aplikasi dimana terdapat 5(lima) tombol navigasi dari submenu lainnya. Tombol navigasi tersebut adalah: Menu Utama Menu utama merupakan bagian utama dari aplikasi dimana terdapat 5(lima) tombol navigasi dari submenu lainnya. Tombol navigasi tersebut adalah: a. Tombol Latihan Apabila tombol latihan ditekan, maka halaman akan berganti ke menu latihan. Menu latihan memiliki 4 (empat) jenis latihan yang dapat dimainkan. Sebaiknya pengguna memulai latihan secara urut dimulai dari latihan 1.Pada halaman menu latihan dan halaman masing- masing latihan terdapat tombol kembali. a. Tombol Latihan Apabila tombol latihan ditekan, maka halaman akan berganti ke menu latihan. Menu latihan memiliki 4 (empat) jenis latihan yang dapat dimainkan. Sebaiknya pengguna memulai latihan secara urut dimulai dari latihan 1.Pada halaman menu latihan dan halaman masing- masing latihan terdapat tombol kembali. 58 Jurnal SAP Vol. 2 No. 1 Agustus 2017 Jurnal SAP Vol. 2 No. 1 Agustus 2017 p-ISSN: 2527-967X e-ISSN: 2549-2845 a. Mengenal Huruf di Keyboard Latihan ini bermaksud untuk mengajarkan pengguna dalam mengenal huruf dalam abjad, dan penggabungan huruf.Gambar keyboard sebagai petunjuk huruf yang muncul, suara akan dibunyikan sesuai dengan huruf yang muncul. Pemberian soal dibuat secara terurut kemudian soal diacak. Pada latihan ini, terdapat total waktu dan nilai yang sudah ditempuh selama latihan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik. Setiap memulai latihan dan pindah soal berikutnya, pengguna diminta menekan spasi. Jurnal SAP Vol. 2 No. 1 Agustus 2017 b. Tombol Permainan Apabila tombol permainan ditekan, maka halaman akan berganti ke menu permainan. Menu permainan memiliki 2 (dua) jenis permainan yang dapat dimainkan.Pada halaman menu permainan dan halaman masing- masing permainan terdapat tombol kembali. b. Tombol Permainan Apabila tombol permainan ditekan, maka halaman akan berganti ke menu permainan. Menu permainan memiliki 2 (dua) jenis permainan yang dapat dimainkan.Pada halaman menu permainan dan halaman masing- masing permainan terdapat tombol kembali. b. Belajar Pemenggalan Kata j gg Latihan ini bermaksud mengajarkan pengguna cara memenggal kata untuk mempermudah proses membaca secara utuh. Latihan ini dibagi menjadi 2 (dua) level yaitu : Latihan ini bermaksud mengajarkan pengguna cara memenggal kata untuk mempermudah proses membaca secara utuh. Latihan ini dibagi menjadi 2 (dua) level yaitu : 1. Level 1 Dalam penyampaiannya digunakan gambar, teks dan suara. Gambar yang muncul disertai dengan tulisan dari nama gambar tersebut. Pengguna diminta menirukan tulisan di tempat/ kotak yang sudah disediakan yang secara otomatis sudah dipenggal.Pemberian soal dibuat secara terurut dengan peningkatan kosakata berdasarkan kesulitan pembacaan. 2. Level 2 Dalam penyampaiannya digunakan gambar dan suara. Gambar yang muncul tidak disertai dengan tulisan dari nama gambar tersebut. Pengguna diminta menuliskan nama dari gambar tersebut pada tempat/ kotak yang sudah disediakan yang secara otomatis sudah dipenggal. Pada level ini, merupakan pengulangan materi level 1 dan penambahan kosakata baru untuk memperbanyak pembendaharaan kata.Pemberian soal dibuat secara acak. Pada latihan ini, setiap 1 (satu) huruf yang dituliskan benar, suara akan dibunyikan. Tetapi bila tulisan salah, suara tidak dibunyikan sampai pengguna menuliskan huruf dengan benar. Terdapat total waktu dan nilai yang sudah ditempuh selama latihan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik.Setiap memulai latihan dan pindah soal berikutnya, pengguna diminta menekan spasi. c. Tebak Gambar Latihan ini bermaksud menghapalkan bentuk serta memahami gambar dan kata yang sudah diberikan pada latihan 1 (satu) dan 2 (dua).Dalam penyampaiannya digunakan gambar dan teks dimana pengguna diberikan soal pilihan ganda yang dapat dipilih salah satu jawaban yang dianggap benar dan pemberian soal dibuat secara acak. Pada latihan ini, terdapat total waktu dan nilai yang sudah ditempuh selama latihan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik.Setiap memulai latihan dan pindah soal berikutnya, pengguna diminta menekan spasi. d. Menyusun Huruf Latihan ini bermaksud mengasah kemampuan untuk menyusun huruf- huruf yang sudah diacak sesuai dengan suara yang dibunyikan.Terdapat 2 (dua) level pada latihan ini yaitu : 1. Level 1 Dalam penyampaiannya digunakan gambar, teks dan suara.Terdapat gambar, huruf- huruf yang sudah diacak dan suara yang dibunyikan.Pengguna diminta menyusun huruf- huruf tersebut sesuai dengan gambar dan suara yang dibunyikan.Pada level ini, ditambahkan materi kosakata baru untuk memperbanyak pembendaharaan kata yang berguna dalam proses belajar membaca. 2. Level 2 Dalam penyampaiannya digunakan teks dan suara.Huruf- huruf yang sudah diacak muncul beserta suara yang sudah dibunyikan.Pengguna diminta menyusun huruf- huruf tersebut sesuai dengan suara yang dibunyikan. Jurnal SAP Vol. 2 No. 1 Agustus 2017 p-ISSN: 2527-967X e-ISSN: 2549-2845 p-ISSN: 2527-967X e-ISSN: 2549-2845 Menu Latihan Menu Latihan Pada menu latihan terdapat 4 (empat) tombol navigasi, bila tombol ditekan maka halaman akan berganti ke latihan yang sudah dipilih. Pilihan latihan tersebut adalah: Gambar 2. Tamplan Menu Latihan Gambar 2. Tamplan Menu Latihan a. Mengenal Huruf di Keyboard a. Mengenal Huruf di Keyboard Latihan ini bermaksud untuk mengajarkan pengguna dalam mengenal huruf dalam abjad, dan penggabungan huruf.Gambar keyboard sebagai petunjuk huruf yang muncul, suara akan dibunyikan sesuai dengan huruf yang muncul. Pemberian soal dibuat secara terurut kemudian soal diacak. Pada latihan ini, terdapat total waktu dan nilai yang sudah ditempuh selama latihan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik. Setiap memulai latihan dan pindah soal berikutnya, pengguna diminta menekan spasi. a. Mengenal Huruf di Keyboard Latihan ini bermaksud untuk mengajarkan pengguna dalam mengenal huruf dalam abjad, dan penggabungan huruf.Gambar keyboard sebagai petunjuk huruf yang muncul, suara akan dibunyikan sesuai dengan huruf yang muncul. Pemberian soal dibuat secara terurut kemudian soal diacak. Pada latihan ini, terdapat total waktu dan nilai yang sudah ditempuh selama latihan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik. Setiap memulai latihan dan pindah soal berikutnya, pengguna diminta menekan spasi. 59 59 Jurnal SAP Vol. 2 No. 1 Agustus 2017 Jurnal SAP Vol. 2 No. 1 Agustus 2017 berikutnya, pengguna diminta menekan spasi. berikutnya, pengguna diminta menekan spasi. b. Belajar Pemenggalan Kata Pada level ini materi yang diberikan merupakan pengulangan materi level 1.Pada latihan ini, terdapat total waktu dan nilai yang sudah ditempuh selama latihan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik.Setiap memulai latihan dan pindah soal 60 p-ISSN: 2527-967X e-ISSN: 2549-2845 p-ISSN: 2527-967X e-ISSN: 2549-2845 Jurnal SAP Vol. 2 No. 1 Agustus 2017 Menu Permainan Menu Permainan Pada menu permainan terdapat 2 (dua) tombol navigasi, bila tombol ditekan maka halaman akan berganti ke permainan yang sudah dipilih. Pilihan permainan tersebut adalah : Gambar 3. Tamplan Menu Permainan Gambar 3. Tamplan Menu Permainan a. Menangkap Kata Dalam Telur a. Menangkap Kata Dalam Telur Permainan ini bermaksud untuk memperdalam kosakata yang sudah diberikan selama latihan lewat permainan.Pada permainan ini, soal yang muncul berupa tulisan dan suara dimana pengguna diminta untuk menuliskan kata yang ada pada telur.Kecepatan dan jarak antara masing- masing soal yang muncul semakin cepat dan dekat.Pengguna tidak diharuskan menjawab soal dari yang pertama kali muncul.Materi yang diberikan merupakan pengulangan yang terdapat pada materi latihan. Pada permainan ini, terdapat total waktu dan nilai yang sudah ditempuh selama permainan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik.Setiap memulai permainan dan pindah soal berikutnya, pengguna diminta menekan spasi. a. Menangkap Kata Dalam Telur Permainan ini bermaksud untuk memperdalam kosakata yang sudah diberikan selama latihan lewat permainan.Pada permainan ini, soal yang muncul berupa tulisan dan suara dimana pengguna diminta untuk menuliskan kata yang ada pada telur.Kecepatan dan jarak antara masing- masing soal yang muncul semakin cepat dan dekat.Pengguna tidak diharuskan menjawab soal dari yang pertama kali muncul.Materi yang diberikan merupakan pengulangan yang terdapat pada materi latihan. Pada permainan ini, terdapat total waktu dan nilai yang sudah ditempuh selama permainan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik.Setiap memulai permainan dan pindah soal berikutnya, pengguna diminta menekan spasi. Analisis Aplikasi (SQA) Untuk mengetahui kualitas aplikasi pembelajaran bahasa Indonesia yang interaktif berbasismultimediamaka digunakan Software Quality Assurance (SQA). Komponen dari SQA yang digunakan pada pengujian ini dapat dilihat pada tabel.1.Ada 5 komponen dalamsoftware quality assurance (SQA) yang digunakan. Dari 5 komponen tersebut Auditability (0.25),Completeness (0.15),Operability (0.25),Simplicity (0.15) dan Training (0.25) akan dibuat 5 pertanyaan untuk angket yang akan disebarkan kepada 5 orang pengamat yang merupakan user yang diambil secara acak. Tabel 2. Hasil Evaluasi Software Quality Assurance (SQA) User Skor Metrik Skor 1 2 3 4 5 1 100 80 80 100 100 97 2 100 80 80 60 100 91 3 100 80 100 80 80 94 4 80 60 80 100 80 84 5 100 60 80 100 100 94 Rata-Rata 92 Tabel 2. Hasil Evaluasi Software Quality Assurance (SQA) Skor1 = (1000.25)+ (800.15)+ (800.25)+ (1000.15)+ (1000.25)=97.00 Skor2 = (1000.25)+ (800.15)+ (800.25)+ (600.15)+ (1000.25)=91.00 Skor3 = (1000.25)+ (800.15)+ (1000.25)+ (800.15)+ (800.25)=94.00 Skor4 = (800.25)+ (600.15)+ (800.25)+ (1000.15)+ (800.25)=84.00 Skor5 = (1000.25)+ (600.15)+ (800.25)+ (1000.15)+ (1000.25)=94.00 Rata-rata =((97.00)+ (91.00)+ (94.00)+ 84.00)+ (92.00))/5=92.00 Skor1 = (1000.25)+ (800.15)+ (800.25)+ (1000.15)+ (1000.25)=97.00 Skor rata-rata yang dihasilkan adalah 92.00, sedangkan nilai optimal untuk sebuah perangkat lunak yang memenuhi standar kualitas berdasarkan uji SQA adalah 80.00 Skor rata-rata yang dihasilkan adalah 92.00, sedangkan nilai optimal untuk sebuah perangkat lunak yang memenuhi standar kualitas berdasarkan uji SQA adalah 80.00 Tombol Kembali Tombol kembali terdapat pada setiap masing- masing halaman yang berfungsi untuk kembali ke halaman yang sudah dibuka sebelumnya. Jurnal SAP Vol. 2 No. 1 Agustus 2017 p-ISSN: 2527-967X e-ISSN: 2549-2845 Jurnal SAP Vol. 2 No. 1 Agustus 2017 Menu Level Menu level terdapat pada latihan pemenggalan kata dan latihan menyusun huruf. Pada menu ini terdiri dari 2 (dua) tombol navigasi yaitu tombol level 1 dan tombol level 2. Menu Level Menu level terdapat pada latihan pemenggalan kata dan latihan menyusun huruf. Pada menu ini terdiri dari 2 (dua) tombol navigasi yaitu tombol level 1 dan tombol level 2. b. Mencocokkan Gambar dan Kata b. Mencocokkan Gambar dan Kata Permainan ini bermaksud untuk menghapal bentuk dan memahami gambar dengan memasangkan setiap gambar dengan kata yang sesuai. Pada permainan ini, soal yang diberikan merupakan pengulangan dari kosakata pada latihan dengan menambahkan kosakata baru untuk menambah pembendaharaan kata. Setiap ditemukan gambar dan kata yang sesuai, suara akan dibunyikan.Pada permainan ini, terdapat total waktu dan nilai yang sudah ditempuh selama permainan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik.Setiap memulai permainan dan pindah soal berikutnya, pengguna diminta menekan spasi. b. Mencocokkan Gambar dan Kata Permainan ini bermaksud untuk menghapal bentuk dan memahami gambar dengan memasangkan setiap gambar dengan kata yang sesuai. Pada permainan ini, soal yang diberikan merupakan pengulangan dari kosakata pada latihan dengan menambahkan kosakata baru untuk menambah pembendaharaan kata. Setiap ditemukan gambar dan kata yang sesuai, suara akan dibunyikan.Pada permainan ini, terdapat total waktu dan nilai yang sudah ditempuh selama permainan yang akan tercatat dalam daftar nilai bila memperoleh total terbaik.Setiap memulai permainan dan pindah soal berikutnya, pengguna diminta menekan spasi. 61 p-ISSN: 2527-967X e-ISSN: 2549-2845 Jurnal SAP Vol. 2 No. 1 Agustus 2017 Simpulan Dan Saran Simpulan Simpulan Kesimpulan yang dapat diambil dari penelitian tentang Aplikasi Media Pembelajaran Bahasa Indonesia Interaktif adalah sebagai berikut : p Kesimpulan yang dapat diambil dari penelitian tentang Aplikasi Media Pembelajaran Baha Indonesia Interaktif adalah sebagai berikut : 1. Sudah dihasilkan sebuah produk penelitian berupa “Aplikasi Multimedia Pembelajaran”dan sudah digunakan oleh pengajarmata pelajaranbahasa indonnesia di bimbel D-LLA. 2. Aplikasi berbasis multimedia ini dianggap layak berdasarkan nilai skor 92.00, sehingga dapat digunakan untuk membantu Guru dalam menyampaikan materi dan juga 62 Jurnal SAP Vol. 2 No. 1 Agustus 2017 p-ISSN: 2527-967X e-ISSN: 2549-2845 p-ISSN: 2527-967X e-ISSN: 2549-2845 miminimalkan rasa bosan siswa karena pelajaran dikelas yang monoton dengan konsep edutainment. Saran Dalam pembuatan aplikasi pembelajaran bahasa Indonesia yang interaktif berbasis multimedia ini belumlah sempurna. Penulis menyadari masih banyak kekurangan yang terdapat pada aplikasi pembelajaran Bahasa Indonesia ini. Oleh sebab itu penulis berharap penelitian inidapat dilanjutkan untuk mendapatkan hasil yang lebih baik dari sebelumnya. Berikut adalah saran yang dapat diberikan untuk pengembangan aplikasi pembelajaran bahasa Indonesia yang interaktif berbasis, yakni: a. Memberikan tampilan animasi yang lebih menarik dan lebih interaktif. b. Menambah fitur-fitur pembelajaran pada aplikasi misalnya sinonin, Antonim dan lainnya. b. Menambah fitur-fitur pembelajaran pada aplikasi misalnya sinonin, Antonim da lainnya. c. Menambah jumlah level/ tingkat padatiap tipe soal. c. Menambah jumlah level/ tingkat padatiap tipe soal. Daftar Pustaka Departemen Pendidikan Nasional RI. . (2005). Pedoman Perpustakaan Perguruan Tinggi Jakarta: Departemen Pendidikan Nasional RI Direktorat Jendral Pendidikan Tinggi. Departemen Pendidikan Nasional RI. . (2005). Pedoman Perpustakaan Perguruan Tinggi Jakarta: Departemen Pendidikan Nasional RI Direktorat Jendral Pendidikan Tinggi. Dikse, I Wayan. (2010).Animasi dengan Flash 8.Yogyakarta: Graha Ilmu Dikse, I Wayan. (2010).Animasi dengan Flash 8.Yogyakarta: Graha Ilmu Hamalik, Umar. (2005).Psikologi Belajar dan Mengajar. Bandung: Sinar Baru. Haryadi. (2006).Pengembangan Media Pembelajaran KimiaBerbantuan Komputer Haryadi. (2006).Pengembangan Media Pembelajaran KimiaBerbantuan Komputer Tentang Kimia Unsur Untuk Siswa Kelas XII. Yogyakarta: UNY. Haryadi. (2006).Pengembangan Media Pembelajaran KimiaBerbantuan K Tentang Kimia Unsur Untuk Siswa Kelas XII. Yogyakarta: UNY. y ( ) g g j Tentang Kimia Unsur Untuk Siswa Kelas XII. Yogyakarta: UNY. Machfudz, Imam. (2002). Metode Pengajaran Bahasa IndonesiaKomunikatif. Jurnal Bahasa dan Sastra UM dan Sastra UM Puput, F., &Sutikno, M., S., (2007). Strategi Belajar Mengajar Melalui Penanaman Konsep Umum & Kosep Islami. Bandung: PT RefikaAditama Sanjaya, Winna. (2008) Strategi Pembelajaran Berorientasi Standar Proses Pendidikan. Jakarta : Kencana Prenada Media Grouf Sumarno, Alim. (2011). Pemanfaatan ICT Dalam Proses Merancang Dan.Mengimplementasikan Model Pembelajaran Inovatif Designed Student. Centred2011Strategi PembelajaranInovatif Kontemporer. Jakarta: PT.Bumi Aksara. g j f p Slamet St. Y., (2007).Dasar-dasar Pembelajaran Bahasa dan Sastra Indonesia di Sekolah. Dasar, Surakarta: LPP UNS, hal. 50. Sutikno, M. Sobry, (2009). Belajar dan Pembelajaran, Prospect. Bandung: PT RefikaAditama 63
https://openalex.org/W2893480441	https://eprints.whiterose.ac.uk/135903/14/08476165.pdf	English	null	Bounds on GreenTouch GreenMeter Network Energy Efficiency	Journal of lightwave technology	2,018	cc-by	9,602	Bounds on GreenTouch GreenMeter Network Energy Efﬁciency Musa , Taisir E. H. El-Gorashi, and Jaafar M. H. Elmirghani, Senior Member, IEEE Mohamed O. I. Musa , Taisir E. H. El-Gorashi, and Jaafar M. H. Elmirghani, Senior Me Abstract—In this paper, we validate the energy efﬁciency im- provements in core networks obtained through mixed integer linear programming (MILP) optimization models as part of the GreenMeter study carried out by the GreenTouch consortium by developing closed form expressions and bounds for the power con- sumption of core networks. We consider nonbypass, bypass, mixed line rates, and physical topology optimization energy efﬁciency schemes. In addition to validating the optimization model results by setting bounds on the power consumption, these bounds can predict network performance at operating conditions highly com- plex for the MILP models. The derivation of a single bound that includes all the measures proved intractable and therefore each measure is evaluated separately. into sleep mode and placing protection resources into a low power state mode when not utilized. In [5] and [6] the mixed line rate is investigated where links with different speeds are assigned, adapting to the variability in network and demands. Optimizing the physical network topology has proven also to yield considerable energy savings as shown in [7]. In [8]–[10], the content distribution problem in the core network considering data centres as well as peer to peer trafﬁc was thoroughly in- vestigated and the connection of virtually allocated data centre resources over virtually connected network has been studied in [11]. The same optimization techniques have been used in [12] for greening big data networks, and in [13] for energy efﬁcient data centres with dis-aggregated servers. Index Terms—Energy Efﬁciency, IP/WDM, MILP model, Greentouch, GreenMeter. In [3] we produced a collective mixed integer linear program- ming optimization model incorporating the mentioned energy efﬁciency schemes, and evaluated the collective mixed integer linear programming (MILP) model over the AT&T topology. In this paper we extend our work by developing bounds on the power consumption of core networks for the non-bypass, bypass, mixed line rate and topology optimization scenarios to verify the energy savings of these schemes individually obtained through MILP optimization models and to provide simple an- alytic bounds and closed form expressions that can be used to obtain results for individual measures or a set of measures. I. INTRODUCTION T HERE has been a growing demand for advanced energy efﬁcient schemes in the wake of the exponential growth of Internet trafﬁc resulting from the popularity of data intensive applications and the widespread use of Internet connected de- vices. To tackle this, the GreenTouch consortium was formed with the ambitious endeavor to cut energy consumption levels in mobile access, ﬁxed access and core networks in the year 2020 by a factor of 1000 compared to 2010 levels. T y p In the white papers in [1], [2], the Greentouch consortium published the results of a thorough study on implementing a set of technologies and solution for an end-to-end energy re- duction in mobile access, ﬁxed access and core networks and is referred to as the GreenMeter. The core networks category con- sidered multiple layer solutions and the results are summarized in [3] combining the individual solutions. The solutions include the bypass approach, the mixed line rates, physical network topology optimization, the optimization of distributed clouds for content distribution, and virtualization of network equipment. In [4] the bypass approach, where the trafﬁc bypasses electri- cal conversion at the intermediate nodes to which the trafﬁc is not destined, is considered, as well putting idle components The remainder of this paper is organized as follows: In Section II, we present the analytic bounds for the IP over WDM network under non-bypass and sleep mode, followed by the by- pass case under sleep mode in Section III. In Section IV we develop the bounds for the mixed line rate and energy efﬁcient protection and in Section V we develop the bounds for the topology optimization scenario. The paper is ﬁnally concluded in Section VI. 5395 5395 JOURNAL OF LIGHTWAVE TECHNOLOGY, VOL. 36, NO. 23, DECEMBER 1, 2018 Bounds on GreenTouch GreenMeter Network Energy Efﬁciency These formulas are valuable as they give insights and predictions where the MILP struggles at different scenarios and network conditions, such as big network sizes. The derivation of a sin- gle bound that includes all the measures proved intractable. We have developed similar bounds for the network coding enabled energy efﬁcient survivable core networks in [14] and [15]. Manuscript received March 29, 2018; revised July 29, 2018; accepted September 18, 2018. Date of publication September 28, 2018; date of cur- rent version November 2, 2018. This work was supported by the Engineer- ing and Physical Sciences Research Council (EPSRC), through INTERNET (EP/H040536/1) and STAR (EP/K016873/1) projects. (Corresponding author: Mohamed O. I. Musa.) Manuscript received March 29, 2018; revised July 29, 2018; accepted September 18, 2018. Date of publication September 28, 2018; date of cur- rent version November 2, 2018. This work was supported by the Engineer- ing and Physical Sciences Research Council (EPSRC), through INTERNET (EP/H040536/1) and STAR (EP/K016873/1) projects. (Corresponding author: Mohamed O. I. Musa.) The authors are with the School of Electronic and Electrical Engineer- ing, University of Leeds, Leeds LS2 9JT, U.K. (e-mail:,m.musa@leeds.ac.uk; t.e.h.elgorashi@leeds.ac.uk; j.m.h.elmirghani@leeds.ac.uk). Color versions of one or more of the ﬁgures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identiﬁer 10.1109/JLT.2018.2871602 der a Creative Commons Attribution 3.0 License. For more information, see http://creativecommons.org/licenses/by/3.0/ This work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativecomm The authors are with the School of Electronic and Electrical Engineer- ing, University of Leeds, Leeds LS2 9JT, U.K. (e-mail:,m.musa@leeds.ac.uk; t.e.h.elgorashi@leeds.ac.uk; j.m.h.elmirghani@leeds.ac.uk). Color versions of one or more of the ﬁgures in this paper are available online at http://ieeexplore.ieee.org. II. BOUNDS ON POWER CONSUMPTION OF CORE NETWORKS UNDER NON-BYPASS AND SLEEP MODE MEASURES Here we develop upper and lower bounds for the IP over WDM power consumption under the non-bypass approach con- sidering sleep, energy efﬁcient and inefﬁcient protection and random and equal trafﬁc demands. In the energy efﬁcient JOURNAL OF LIGHTWAVE TECHNOLOGY, VOL. 36, NO. 23, DECEMBER 1, 2018 5396 This EFDFA power consumption is bounded by the fol- lowing bounds: protection scheme, protection resources are switched off when idle as opposed to the energy inefﬁcient protection where they are left active even when idle. m∈N n∈Nm pf Am,nFm,n ≥pf A(min) W m∈N n∈Nm λm,n (6) m∈N n∈Nm pf Am,nFm,n Let N be the set of IP/WDM nodes and Nm be the set of neighboring nodes of node m. There are W wavelengths per ﬁber and each wavelength supports a data rate B. Between each node pair (m, n) exists a number of EDFAs denoted as Am,n, a number of regenerators denoted as Gm,n and a number of optical ﬁbres referred to as Fm,n. The average number of EDFAs and regenerators in the network is A and G respectively. N N pf Am,nFm,n ≥pf A(min) W m∈N n∈Nm λm,n (6) m∈N n∈Nm pf Am,nFm,n ≥pf A(min) W m∈N n∈Nm λm,n (6) A F (6) N N pf Am,nFm,n Let pr, pt, pf , px, pg and ps be the power consump- tion of a router port, a transponder, an EDFA, a multi- plexer/demultiplexer, an optical regenerator and an optical switch respectively. ≤pf A(max) W m∈N n∈Nm λm,n (7) (7) For a given trafﬁc demand Ls,d between a node pair (s, d), the network assigns a path with a total hop count of hs,d. ¯L denotes the average trafﬁc demand in the network and h is the average hop count of paths in the network. The result of routing all demands gives a total number of wavelengths of λm,n on a physical link (m, n). where A(max) = max m,n∈N Am,n (8) A(min) = min m,n∈N Am,n (9) (8) (9) The total power consumption of the network under the non- bypass approach with sleep and energy efﬁcient protection scheme is given as the sum of the following components: are number of EDFAs associated with the links of maxi- mum and the minimum distances, respectively. II. BOUNDS ON POWER CONSUMPTION OF CORE NETWORKS UNDER NON-BYPASS AND SLEEP MODE MEASURES 5) The power consumption of optical switches 3) The power consumption of EDFAs 3) The power consumption of EDFAs 5) The power consumption of optical switches 5) The power consumption of optical switches m∈N n∈Nm pf Am,nFm,n (5) ηn s∈N ps = ηnNps (15) m∈N n∈Nm pf Am,nFm,n (5) ηn s∈N ps = ηnNps (15) (5) ηn s∈N ps = ηnNps (15) (5) (15) MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY 5397 where the total number of demands in the network is N(N −1) and h is the average hop count of the paths in the network, given The total power consumption, after grouping terms and rear- ranging, is upper bounded by: as: P ≤ηn pr B s∈N d∈N Ls,d + 1 B s∈N d∈N Ls,dhs,d ηnpr + ηnpt + pf A(max) W + pgG(max) + ηnNps (16) as: h = N Under equal trafﬁc de hop count. Under unequ two large cities may ha compared to two small P ≤ηn pr B s∈N d∈N Ls,d + 1 B s∈N d∈N Ls,dhs,d ηnpr + ηnpt + pf A(max) W + pgG(max) + ηnNps (16) h = 1 N(N −1) s∈N d∈N s̸=d hs,d (26) (26) Under equal trafﬁc demands it sufﬁces to consider the average hop count. Under unequal trafﬁc demand two nodes representing two large cities may have larger trafﬁc demand between them compared to two small cities. In this case it is important to consider the number of hops over which each trafﬁc demand is routed. Therefore we introduce upper and lower bounds on the power consumption where all trafﬁc is assumed to travel the maximum hop count h(max) or the minimum hop count h(min), respectively between each node pair. These bound are given as (16) and lower bounded by: and lower bounded by: P ≥ηn pr B s∈N d∈N Ls,d + 1 B s∈N d∈N Ls,dhs,d ηnpr + ηnpt + pf A(min) W + pgG(min) + ηnNps (17) p is routed. Therefore we introduce upper and lower bounds on the power consumption where all trafﬁc is assumed to travel the maximum hop count h(max) or the minimum hop count h(min), respectively between each node pair. II. BOUNDS ON POWER CONSUMPTION OF CORE NETWORKS UNDER NON-BYPASS AND SLEEP MODE MEASURES The total power consumption bounds can be given by: P ≥ηn pr B s∈N d∈N Ls,d Considering energy inefﬁcient protection resources, i.e., pro- visioned resources are doubled, the total power consumption becomes bounded by: + Ec(min) s∈N d∈N Ls,dhs,d + ηnNps (20) (20) P ≥2N(N −1)¯L ηn pr B + Ec(min)h(min) + ηnNps (32) P ≤2N(N −1)¯L ηn pr B + Ec(max)h(max) + ηnNps (33) (32) P ≤ηn pr B s∈N d∈N Ls,d + Ec(max) s∈N d∈N Ls,dhs,d + ηnNps (21) (21) N d∈N Ls,dhs,d + ηnNps (21) B (33) (33) (33) or equal trafﬁc demands, i.e.,: Ls,d = ¯L (22) ∀s, d ∈N, s ̸= d (23) total power consumption bounds are given as: P ≥N(N −1)¯L ηn pr B + Ec(min)h + ηnNps (24) P ≤N(N −1)¯L ηn pr B + Ec(max)h + ηnNps (25) It can be seen from Figure 1 that the MILP results are found to be much closer to the lower bound than the upper bound as most of the links in the AT&T network are comparable in length to the shortest link length and hence the number of EDFAs and regenerators are close to their minimum values. This makes the values generated by the lower bound represent a very good and quick approximation to the consumed power. The bounds in Figure 2 for unequal trafﬁc additionally include the hop count component as shown in equation 30 and 31 where the maximum hop count and the minimum possible hop count are used for the For equal trafﬁc demands, i.e.,: or equal trafﬁc demands, i.e.,: Ls,d = ¯L (22) ∀s, d ∈N, s ̸= d (23) total power consumption bounds are given as: It can be seen from Figure 1 that the MILP results are found to be much closer to the lower bound than the upper bound as most of the links in the AT&T network are comparable in length to the shortest link length and hence the number of EDFAs and regenerators are close to their minimum values. II. BOUNDS ON POWER CONSUMPTION OF CORE NETWORKS UNDER NON-BYPASS AND SLEEP MODE MEASURES These bound are given as P ≥ηn pr B s∈N d∈N Ls,d + ηnNps + Ec( i )h( i ) Ls,d (27) P ≥ηn pr B s∈N d∈N Ls,d + 1 B s∈N d∈N Ls,dhs,d ηnpr + ηnpt + pf A(min) W + pgG(min) + ηnNps (17) P ≥ηn pr B s∈N d∈N Ls,d + ηnNps (17) (17) + Ec(min)h(min) s∈N,d∈N Ls,d (27) (27) Let: Ec(min) = 1 B ηnpr + ηnpt + pf A(min) W + pgG(min) (18) s∈ ,d∈ P ≤ηn pr B s∈N d∈N Ls,d + ηnNps + Ec(max)h(max) s∈N,d∈N Ls,d (28) Ec(min) = 1 B ηnpr + ηnpt + pf A(min) W + pgG(min) (18) Ec(min) = 1 B ηnpr + ηnpt Ec(min) = 1 B ηnpr + ηnpt pf A( ) P ≤ηn pr B s∈N d∈N Ls,d + ηnNps G(min) (18) ti fﬁ + Ec(max)h(max) s∈N,d∈N Ls,d (28) (18) (28) represents the non-bypass lower energy consumption coefﬁ- cient, and represents the non-bypass lower energy consumption coefﬁ- cient, and s∈N,d∈N Given that: 1 Given that: Given that: ¯L = 1 N(N −1) s∈N d∈N Ls,d (29) Ec(max) = 1 B ηnpr + ηnpt + pf A(max) W + pgG(max) (19) (29) Ec(max) = 1 B ηnpr + ηnpt The bounds can be simpliﬁed to: The bounds can be simpliﬁed to: + pf A(max) W + pgG(max) (19) (19) P ≥N(N −1)¯L ηn pr B + Ec(min)h(min) + ηnNps (30) P ≤N(N −1)¯L ηn pr B + Ec(max)h(max) + ηnNps (31) represents the non-bypass higher energy consumption coefﬁcient. II. BOUNDS ON POWER CONSUMPTION OF CORE NETWORKS UNDER NON-BYPASS AND SLEEP MODE MEASURES mum and the minimum distances, respecti 1) The power consumption of router ports p 4) The power consumption of regenerators = ηn ⎛ ⎝ s∈N prQs + m∈N,n∈Nm prλm,n ⎞ ⎠ (1) m∈N n∈Nm pgGm,nλm,n (10) (10) (1) The regenerators power consumption is bounded by the following bounds: The regenerators power consumption is bounded by the following bounds: Where ηn is the IP/WDM power usage effectiveness and Qs is the number of aggregation ports at node s. This can be rewritten as: Where ηn is the IP/WDM power usage effectiveness and Qs is the number of aggregation ports at node s. This can be rewritten as: m∈N n∈Nm pgGm,nλm,n m∈N n∈Nm pgGm,nλm,n ηn ⎛ ⎝pr B s∈N d∈N Ls,d + pr B s∈N,d∈N Ls,dhs,d ⎞ ⎠ (2) ≥pgG(min) m∈N n∈Nm λm,n (11) (2) (11) m∈N n∈Nm pgGm,nλm,n where hs,d is the hop count of the path traversed by de- mand (s, d) ≤pgG(max) m∈N n∈Nm λm,n (12) (12) 2) The power consumption of transponders = ηn m∈N n∈Nm ptλm,n (3) = ηn m∈N n∈Nm ptλm,n (3) where (3) (3) where G(max) = max m,n∈N Gm,n (13) G(min) = min m,n∈N Gm,n (14) (13) This can be rewritten as This can be rewritten as (14) ηn pt B s∈N d∈N Ls,dhs,d (4) ar (4) are the number of regenerators associated with the links of maximum and the minimum distances, respectively. are the number of regenerators associated with the links of maximum and the minimum distances, respectively. II. BOUNDS ON POWER CONSUMPTION OF CORE NETWORKS UNDER NON-BYPASS AND SLEEP MODE MEASURES The power consumption of router ports: The power consumption of router ports: ηn s∈N prQs + s∈N d∈N pr B Ls,d = 2ηn pr B s∈N d∈N Ls,d (34) (34) which can be rewritten as: which can be rewritten as: 2ηn pr B N(N −1)¯L (35) (35) Using the same derivation approach as the one used in the non- bypass case we have: Fig. 1. Power Consumption with Bounds of the Original AT&T Topology, 2020 BAU+GT Components, 40 Gbps, Non-bypass, Sleep and Energy efﬁcient Protection Considering Equal Trafﬁc Demands. P ≤2ηn pr B N(N −1)¯L + ηnNps Fig. 2. Power Consumption and its bounds of the Original AT&T Topol- ogy, 2020 BAU+GT Components, 40 Gbps, bypass, Sleep and energy efﬁcient Protection Considering Unequal Trafﬁc Demands. + Eb(max) s∈N,d∈N Ls,dhs,d (36) (36) P ≥2ηn pr B N(N −1)¯L + ηnNps + Eb(min) s∈N,d∈N Ls,dhs,d (37) (37) where Eb(max) = 1 B ηnpt + pf A(max) W + pgG(max) (38) Eb(min) = 1 B ηnpt + pf A(min) W + pgG(min) (39) (38) (39) are the bypass upper and lower energy consumption coefﬁcient, respectively. For equal trafﬁc demands, the total power con- sumption is bounded by: Fig. 2. Power Consumption and its bounds of the Original AT&T Topol- ogy, 2020 BAU+GT Components, 40 Gbps, bypass, Sleep and energy efﬁcient Protection Considering Unequal Trafﬁc Demands. P ≥N(N −1)¯L 2ηn pr B + Eb(min)h + ηnNps (40) P ≤N(N −1)¯L 2ηn pr B + Eb(max)h + ηnNps (41) (40) (41) upper and lower bounds respectively. Therefore, the trafﬁc plays a role in estimating where the power consumed lies between the two bounds. However, given that the average hop count of the AT&T topology is 2.65 and the maximum and minimum hop count are 5 and 1 respectively, and with the tendency of all paths to select the shortest possible paths, this makes the MILP results even closer to the lower bound. Similar to the non-bypass case, an upper bound and lower bound on the total power consumption of unequal trafﬁc de- mands is attained when all trafﬁc demands are routed through the minimum hop count h(min) and the maximum hop count h(max), respectively: P ≥2ηn pr B N(N −1)¯L + Eb(min)h(min) s∈N,d∈N Ls,d II. BOUNDS ON POWER CONSUMPTION OF CORE NETWORKS UNDER NON-BYPASS AND SLEEP MODE MEASURES This makes the values generated by the lower bound represent a very good and Ls,d = ¯L ∀s, d ∈N, s ̸= d Ls,d = ¯L (22) ∀s, d ∈N, s ̸= d (23) ∀s, d ∈N, s ̸= d The total power consumption bounds are given as: The total power consumption bounds are given as: P ≥N(N −1)¯L ηn pr B + Ec(min)h + ηnNps (24) P ≤N(N −1)¯L ηn pr B + Ec(max)h + ηnNps (25) g y p y g quick approximation to the consumed power. The bounds in Figure 2 for unequal trafﬁc additionally include the hop count component as shown in equation 30 and 31 where the maximum hop count and the minimum possible hop count are used for the P ≥N(N −1)¯L ηn pr B + Ec(min)h + ηnNps P ≤N(N −1)¯L ηn pr B + Ec(max)h + ηnNps P ≥N(N −1)¯L ηn pr B + Ec(min)h + ηnNps (24) P ≤N(N −1)¯L ηn pr B + Ec(max)h + ηnNps (25) quick approximation to the consumed power. The bounds in Figure 2 for unequal trafﬁc additionally include the hop count component as shown in equation 30 and 31 where the maximum hop count and the minimum possible hop count are used for the P ≥N(N −1)¯L ηn pr B + Ec(min)h + ηnNps (24) P ≤N(N −1)¯L ηn pr B + Ec(max)h + ηnNps (25) quick approximation to the consumed power. The bounds in Figure 2 for unequal trafﬁc additionally include the hop count component as shown in equation 30 and 31 where the maximum hop count and the minimum possible hop count are used for the 5398 JOURNAL OF LIGHTWAVE TECHNOLOGY, VOL. 36, NO. 23, DECEMBER 1, 2018 Fig. 1. Power Consumption with Bounds of the Original AT&T Topology, 2020 BAU+GT Components, 40 Gbps, Non-bypass, Sleep and Energy efﬁcient Protection Considering Equal Trafﬁc Demands. The bounds on the power consumption of the bypass IP over WDM network can be obtained in a similar derivation to the non-bypass case. Router ports are only used at the source and destination nodes of each demand. MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY 5399 Fig. 3. Power Consumption and its bounds of the Original AT&T Topology, 2020 BAU+GT Components, 40 Gbps, bypass, Sleep and Energy Efﬁcient Protection considering Equal Trafﬁc Demands. Figure 3 and Figure 4 show the upper and lower bound of the bypass IP over WDM network power consumption and compare them to the MILP model results using equal trafﬁc and the general trafﬁc case, respectively. As with the non-bypass case, the MILP results are closer to the lower bound than the upper bound. IV. BOUNDS ON POWER CONSUMPTION UNDER MIXED LINE RATES, SLEEP AND ENERGY EFFICIENT PROTECTION Here we develop upper and lower bounds, as well as an ap- proximating expression of the IP over WDM network power consumption under the optimum MLR solution considering the bypass and non-bypass approach, sleep, energy efﬁcient protec- tion and equal average trafﬁc demands. The total power consumption of a non-bypass IP over WDM network using mixed line rates can be approximated by: Fig. 3. Power Consumption and its bounds of the Original AT&T Topology, 2020 BAU+GT Components, 40 Gbps, bypass, Sleep and Energy Efﬁcient Protection considering Equal Trafﬁc Demands. P = ηn pr B s∈N d∈N Ls,d + m∈N n∈Nm fp( ˜wmn) + ηn m∈N n∈Nm ft( ˜wmn) + pf A B m∈N n∈Nm λm,n + Npsηn + G m∈N n∈Nm fg( ˜wmn) (48 Fig. 4. Power Consumption and its bounds of the Original AT&T Topol- ogy, 2020 BAU+GT Components, 40 Gbps, bypass, Sleep and energy efﬁcient Protection Considering Unequal Trafﬁc Demands. + pf A B m∈N n∈Nm λm,n + Npsηn + G m∈N n∈Nm fg( ˜wmn) (48) (48) Where fp( ˜wmn) is the function relating the power consumption at a given selected line rate ˜wmn for router ports. Similarly, ft( ˜wmn) and fg( ˜wmn) are the corresponding functions for the transponder and the regenerator. This function is concave due to the increase in energy efﬁciency per bit as the selected line rate increases. Using Jensen’s inequality, given a concave function f(x): Fig. 4. Power Consumption and its bounds of the Original AT&T Topol- ogy, 2020 BAU+GT Components, 40 Gbps, bypass, Sleep and energy efﬁcient Protection Considering Unequal Trafﬁc Demands. ft m∈N n∈Nm ˜wmn ≥ m∈N n∈Nm ft( ˜wmn) (49) III. BOUNDS ON POWER CONSUMPTION OF CORE NETWORKS UNDER BYPASS AND SLEEP MODE MEASURES + ηnNps (42) P ≤2ηn pr B N(N −1)¯L + Eb(max)h(max) s∈N,d∈N Ls,d (42) Here, similar to the non-bypass approach in Section II, we develop upper and lower bounds for the IP over WDM power consumption under the bypass approach considering sleep, en- ergy efﬁcient and inefﬁcient protection and random and equal average trafﬁc demands. P ≤2ηn pr B N(N −1)¯L + Eb(max)h(max) s∈N,d∈N Ls,d + ηnNps (43) (43) + ηnNps This can be simpliﬁed to: TABLE I POWER CONSUMPTION VALUES FOR THE DIFFERENT CORE NETWORK COMPONENTS AS ESTIMATED IN 2020 [3] TABLE I POWER CONSUMPTION VALUES FOR THE DIFFERENT CORE NETWORK COMPONENTS AS ESTIMATED IN 2020 [3] After rearranging the terms: After rearranging the terms: g g P ≤N(N −1)¯L ηn pr B + pf A WB h + ηn B fp N(N −1)¯Lh + ηn B ft N(N −1)¯Lh + Npsηn + G B fg N(N −1)¯Lh (53) Let : u = N(N −1)¯Lh (54) hen P ≤ ηn pr Bh + pf A WB u + ηn B fp(u) + ηn B ft(u) + Npsηn + G B fg(u) (55) P ≤N(N −1)¯L ηn pr B + pf A WB h + ηn B fp N(N −1)¯Lh + ηn B ft N(N −1)¯Lh + Npsηn + G B fg N(N −1)¯Lh (5 Let : Let : then P ≤ ηn pr Bh + pf A WB u + ηn B fp(u) + ηn B ft(u) + Npsηn + G B fg(u) (55) port), and the worst power savings when using the least efﬁcient port. Note that fp(u), ft(u) and fg(u) are obtained from Table I where a linear interpolation is assumed in between the given data rates (40 Gb/s, 100 Gb/s, 400 Gb/s and 1 Tb/s). The value p⇓ r and p⇑ r are the power consumption of the lowest considered port (i.e., 40 Gbps) and the highest considered port (i.e., 1 Tbps). The same applies to the transponders (p⇓ t and p⇑ t ) and the regenerators (p⇓ g and p⇑ g). Therefore the power consump- tion at a given line rate is bounded for the three equipment as follows: (55) Figure 5 shows the power consumption obtained from the MILP model and the analytical upper bound for the 2020 network using non-bypass and MLR assuming equal trafﬁc demands. Equation (56) presents a bound that tries to approach the optimum power consumption value. To get upper and lower bounds, the concave function representing the power consump- tion vs the data rate is approximated as a linear function with a slope corresponding to the lowest port power consumption and the highest port power consumption, respectively. This can be simpliﬁed to: This can be simpliﬁed to: (49) P ≥N(N −1)¯L 2ηn pr B + Eb(min)h(min) + ηnNps (44) P ≤N(N −1)¯L 2ηn pr B + Eb(max)h(max) + ηnNps Therefore, the power consumption is upper bounded by: P ≤ηn pr B s∈N d∈N Ls,d + ηnfp m∈N n∈Nm ˜wmn + ηnft m∈N n∈Nm ˜wmn + pf A B m∈N n∈Nm λm,n + Npsηn (45) (45) Considering energy inefﬁcient protection resources, the total power consumption becomes bounded by: Considering energy inefﬁcient protection resources, the total power consumption becomes bounded by: P ≥2N(N −1)¯L 2ηn pr B + Eb(min)h(min) + ηnNps (46 + pf A B m∈N n∈Nm λm,n + Npsηn (46) + Gfg m∈N n∈Nm ˜wmn (50) P ≤2N(N −1)¯L 2ηn pr B + Eb(max)h(max) + ηnNps (50) (47) JOURNAL OF LIGHTWAVE TECHNOLOGY, VOL. 36, NO. 23, DECEMBER 1, 2018 5400 which can be shown to be equal to: which can be shown to be equal to: which can be shown to be equal to: P ≤ηn pr B s∈N d∈N Ls,d + ηn B fp s∈N d∈N Ls,dhs,d + ηn B ft s∈N d∈N Ls,dhs,d + pf A WB s∈N d∈N Ls,dhs,d + Npsηn + G B fg s∈N d∈N Ls,dhs,d (51) P ≤ηn pr B s∈N d∈N Ls,d + ηn B fp s∈N d∈N Ls,dhs,d + ηn B ft s∈N d∈N Ls,dhs,d + pf A WB s∈N d∈N Ls,dhs,d + Npsηn + G B fg s∈N d∈N Ls,dhs,d (51 (51) Assuming equal trafﬁc demands: P ≤ηn pr B N(N −1)¯L + ηn B fp N(N −1)¯Lh + ηn B ft N(N −1)¯Lh + pf A WB N(N −1)¯Lh + Npsηn + G B fg N(N −1)¯Lh Fig. 5. Power Consumption and its bounds of the Original AT&T Topology, 2020 BAU+GT Components, with MLR, sleep and Energy Efﬁcient Protection using Equal Trafﬁc. V. BOUNDS AND CLOSED FORMS OF POWER CONSUMPTION UNDER TOPOLOGY OPTIMIZATION, BYPASS, SLEEP AND ENERGY EFFICIENT PROTECTION + G m∈N n∈Nm p⇓ g ˜wmn (59) (59) Here we develop upper and lower bounds on the power con- sumption of the optimum energy efﬁcient topology for IP over WDM network under the bypass approach considering sleep, energy efﬁcient protection and equal trafﬁc demands. We also develop closed form expressions for the power consumption of various regular topologies and present a uniﬁed polynomial for- mula with the corresponding coefﬁcients for each topology. A topology optimized to minimize the network power consump- tion is decided based on the trafﬁc demands Ls,d,t and distance between nodes. The trafﬁc demands determine the number of router ports and transponders at each hop while the distance de- termines the number of EDFAs and regenerators. This can be simpliﬁed to: BOUNDS AND CLOSED FORMS OF POWER CONSUMPTION UNDER TOPOLOGY OPTIMIZATION BYPASS SLEEP AND P ≥ηn pr B s∈N d∈N Ls,d + m∈N n∈N p⇓ r ˜wmn P ≥ 2ηn p⇓ r Bh + pf A WB + ηn B p⇓ t + G B p⇓ g u + Npsηn (65) P ≥ηn pr B s∈N d∈N Ls,d + m∈N n∈Nm p⇓ r ˜wmn P ≥ 2ηn p⇓ r Bh + pf A WB + ηn B p⇓ t + And similarly the upper bound is giv ∈N n∈N p⇓ r ˜wmn P ≥ 2ηn p⇓ r Bh + pf A WB + ηn B p⇓ t + G B p⇓ g u + Npsηn (65) (65) And similarly, the upper bound is given as: And similarly, the upper bound is given as: P ≤ 2ηn p⇑ r Bh + pf A WB + ηn B p⇑ t + G B p⇑ g u + Npsηn (66) (66) + pf A B m∈N n∈Nm λm,n + Npsηn V. BOUNDS AND CLOSED FORMS OF POWER CONSUMPTION UNDER TOPOLOGY OPTIMIZATION, BYPASS, SLEEP AND ENERGY EFFICIENT PROTECTION Starting from equation (21), the power consumption of the optimal topology can be given as: Which gives: P ≥ηn pr B s∈N d∈N Ls,d + ηn B p⇓ r s∈N d∈N Ls,dhs,d + p⇓ t ηn B s∈N d∈N Ls,dhs,d + pf A WB s∈N d∈N Ls,dhs,d + Npsηn + G B p⇓ g s∈N d∈N Ls,dhs,d (6 (60) Assuming equal trafﬁc demands: P = min(ηn pr B N(N −1)¯L + s∈N d∈N EcLs,dhs,d) = ηn pr B N(N −1)¯L + s∈N d∈N EcLs,d min(hs,d) (67) P ≥ηn pr B N(N −1)¯L + ηn B p⇓ r N(N −1)¯Lh + ηn B p⇓ t N(N −1)¯Lh + pf A WB N(N −1)¯Lh + Npsηn + G B p⇓ g N(N −1)¯Lh (67) where where Ec = ηnpr + ηnpt + pf A W + pgG (68) (68) (61) (61) Assuming equal demands, the total power is given as: Which gives: Which gives: P = ηn pr B N(N −1)¯L + ¯L s∈N d∈N min(Echs,d) (69) (69) P ≥ ηn pr Bh + pf A WB + ηn B p⇓ r + ηn B p⇓ t + +G B p⇓ g u The optimal topology power consumption lies between the fol- lowing bounds, where we assumed all links to deploy the max- imum and minimum number of regenerators and EDFAs: (62) + Npsηn And similarly, the upper bound is given as: And similarly, the upper bound is given as: P ≥N(N −1)¯L ηn pr B + Ec(min)hop (70) P ≤N(N −1)¯L ηn pr B + Ec(max)hop (71) (70) P ≤ ηn pr Bh + pf A WB + ηn B p⇑ r + ηn B p⇑ t + G B p⇑ g u (71) (63) + Npsηn where hop is the average hop count of the optimal topology. This can be simpliﬁed to: This means that the MLR has the best power savings if all ports can use the most efﬁcient ports (i.e., proportionally using the highest p⇓ r ˜wmn ≥fp( ˜wmn) ≥p⇑ r ˜wmn (56) p⇓ t ˜wmn ≥ft( ˜wmn) ≥p⇑ t ˜wmn (57) p⇓ g ˜wmn ≥fg( ˜wmn) ≥p⇑ g ˜wmn (58) p⇓ r ˜wmn ≥fp( ˜wmn) ≥p⇑ r ˜wmn (56) p⇓ t ˜wmn ≥ft( ˜wmn) ≥p⇑ t ˜wmn (57) p⇓ g ˜wmn ≥fg( ˜wmn) ≥p⇑ g ˜wmn (58) (56) (58) For the router ports, transponders and regenerators respectively. Therefore, starting from equation (49), the lower bound is MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY 5401 given as: which is simpliﬁed to: which is simpliﬁed to: given as: given as: iven as: P ≥ηn pr B s∈N d∈N Ls,d + m∈N n∈Nm p⇓ r ˜wmn which is simpliﬁed to: P ≥ 2ηn p⇓ r Bh + pf A WB + ηn B p⇓ t + G B p⇓ g u + Npsηn (65) And similarly, the upper bound is given as: P ≥ηn pr B s∈N d∈N Ls,d + m∈N n∈Nm p⇓ r ˜wmn + ηn m∈N n∈Nm p⇓ t ˜wmn p P ≥ 2ηn p⇓ r Bh + pf A WB + ηn B p⇓ t + G B p⇓ g u + Npsηn (65) And similarly, the upper bound is given as: P ≤ 2ηn p⇑ r Bh + pf A WB + ηn B p⇑ t + G B p⇑ g u + Npsηn (66) P ≥ηn pr B s∈N d∈N Ls,d + m∈N n∈Nm p⇓ r ˜wmn + ηn m∈N n∈Nm p⇓ t ˜wmn + pf A B m∈N n∈Nm λm,n + Npsηn ⇓ p P ≥ 2ηn p⇓ r Bh + pf A WB + ηn B p⇓ t + G B p⇓ g u + Npsηn (65) And similarly, the upper bound is given as: P ≤ 2ηn p⇑ r Bh + pf A WB + ηn B p⇑ t + G B p⇑ g u + Npsηn (66) V. V. BOUNDS AND CLOSED FORMS OF POWER CONSUMPTION UNDER TOPOLOGY OPTIMIZATION, BYPASS, SLEEP AND ENERGY EFFICIENT PROTECTION (76) This means that the power consumption of the star topology is a polynomial in the network size, N, of the order 2. This means that the power consumption of the star topology is a polynomial in the network size, N, of the order 2. B. Ring Topology (N odd) B. Ring Topology (N odd) s∈N d∈N s̸=d hs,d = 2N 1 + 2 + 3 + · · · + N −1 2 = 2N 1 2 N −1 2 N −1 2 + 1 = N(N 2 −1) 4 (77) (77) Fig. 7. Power Consumption of the regular topologies with 2020 BAU+GT Components, 40 Gbps and Sleep, considering equal trafﬁc demands. Fig. 8. The hop counts of a 4-node line topology. where each node takes an incremental number of hops to reach nodes on one side, and similarly for the other side, and this is repeated for all nodes. Note here the total number of nodes on each side to reach be a node is N −1 2 . Fig. 8. The hop counts of a 4-node line topology. h = N(N 2 −1) 4N(N −1) = N + 1 4 (78) P = N(N −1)¯L ηn pr B + Ec N + 1 4 = ηn 4 ¯LN 3 + pr B ¯LN 2 − ηn pr B + Ec 4 N ¯L (79) (78) hop. Therefore a full mesh with links of the maximum distance will have a higher power consumption that the optimal topology: P ≤N(N −1)¯L ηn pr B + Ec(max) (73) (73) (79) Figure 6 shows that the power consumption of the optimal topology given by the MILP model is bounded by the lower bound for the optimal topology given by equation (72) and upper bounded by the equation (73). C. Ring Topology (N Even) C. Ring Topology (N Even) C. Ring Topology (N Even) C. Ring Topology (N Even) C. V. BOUNDS AND CLOSED FORMS OF POWER CONSUMPTION UNDER TOPOLOGY OPTIMIZATION, BYPASS, SLEEP AND ENERGY EFFICIENT PROTECTION Similarly, The power consumption of the MLR approach under the bypass approach can be shown to have the following bounds: The lower bound: The power consumption of the optimal topology is lower bounded by the power consumption of a full mesh, i.e., h = 1 where all links are of the minimum distance between nodes given as: The lower bound: P ≥2N(N −1)¯Lηn B p⇓ r + ηn B p⇓ t (N −1)¯Lh + pf A WB N(N −1)¯Lh + Npsηn + G B p⇓ gN(N −1)¯Lh P ≥N(N −1)¯L ηn pr B + Ec(min) (72) (72) The optimal topology connects node pairs with a direct link unless the power consumption of regenerators and EDFAs in the direct link is greater than the power consumption of an additional The optimal topology connects node pairs with a direct link unless the power consumption of regenerators and EDFAs in the direct link is greater than the power consumption of an additional (64) 5402 JOURNAL OF LIGHTWAVE TECHNOLOGY, VOL. 36, NO. 23, DECEMBER 1, 2018 Fig. 6. Power Consumption of the optimal topology and its bounds, 2020 BAU+GT Components, 40 Gbps, bypass, sleep and energy efﬁcient Protection, considering equal trafﬁc demands. and same trafﬁc as the other topologies. For link distances, the average link distance was used. and same trafﬁc as the other topologies. For link distances, the average link distance was used. A. Star Topology s∈N d∈N s̸=d hs,d =2(N −1) + 2(N −1)(N −1)=2(N −1)2 (74) hs,d =2(N −1) + 2(N −1)(N −1)=2(N −1)2 s∈N d∈N s̸=d (74) (74) where the ﬁrst term 2(N −1) is the total hops from each node to the center of the star considering bidirectional demands, and the second term 2(N −1)(N −2) is for the demands from nodes to each other excluding the center of the star. h = 2(N −1)2 N(N −1) = 2(N −1) N (75) P = N(N −1)¯L ηp pr B + pc 2(N −1) N = 2pc ¯LN 2 + ηp pr B −2pc ¯LN + 2pc −ηp pr B ¯L (76) Fig. 6. Power Consumption of the optimal topology and its bounds, 2020 BAU+GT Components, 40 Gbps, bypass, sleep and energy efﬁcient Protection, considering equal trafﬁc demands. (75) Fig. 7. Power Consumption of the regular topologies with 2020 BAU+GT Components, 40 Gbps and Sleep, considering equal trafﬁc demands. MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY 5403 TABLE II REGULAR TOPOLOGIES POLYNOMIAL COEFFICIENTS TABLE III SUMMARY OF THE BOUNDS FOR THE DIFFERENT SCENARIOS e node. The number of nodes on each side after removing TABLE II REGULAR TOPOLOGIES POLYNOMIAL COEFFICIENTS TABLE III SUMMARY OF THE BOUNDS FOR THE DIFFERENT SCENARIOS a single node. The number of nodes on each side after removing a node to make it symmetric is N 2 −1. And the hop count to the removed node is counted for by the term N 2 . h = N 3 4N(N −1) = N 2 4(N −1) (81) P = N(N −1)¯L ηn pr B + Ec N 2 4(N −1) = Ec 4 ¯LN 3 + ηn pr B ¯LN 2 −ηn pr B N ¯L (82) D. Line Topology The total number of hops used by all demands is given by equation (83), which can be explained with the aid of Figure 8. To the right is a 4-node line topology and the array to the left Fig. 9. Power Consumption of the different topologies at different n 2020 BAU+GT Components, 40Gbps, bypass, Sleep and energy efﬁc tection, considering equal trafﬁc demands. a single node. The number of nodes on each side after removing a node to make it symmetric is N 2 −1. And the hop count to the removed node is counted for by the term N 2 . h = N 3 4N(N −1) = N 2 4(N −1) (81) P = N(N −1)¯L ηn pr B + Ec N 2 4(N −1) = Ec 4 ¯LN 3 + ηn pr B ¯LN 2 −ηn pr B N ¯L (82) (81) (82) V. BOUNDS AND CLOSED FORMS OF POWER CONSUMPTION UNDER TOPOLOGY OPTIMIZATION, BYPASS, SLEEP AND ENERGY EFFICIENT PROTECTION Ring Topology (N Even) s∈N d∈N s̸=d hs,d = N 2 1 + 2 + 3 + · · · + N 2 −1 + N 2 s∈N d∈N s̸=d hs,d = N 2 1 + 2 + 3 + · · · + N 2 −1 + N 2 = N N 2 −1 N 2 + N 2 = N 3 4 (80) For different regular topologies (line, ring, and star topolo- gies) a closed form expression of the power consumption can be obtained using the closed form expressions of the hop count given below and substituting them in equation (69). The power consumption of the considered regular topologies at different times of the day considering equal trafﬁc demands is shown in Figure 7 where the regular topologies used the same set of equip- ment, same power consumption values, same number of nodes, = N N 2 −1 N 2 + N 2 = N 3 4 (80) (80) Because of the even number of nodes, the number of nodes on one side is higher than the number of nodes on the other side by Because of the even number of nodes, the number of nodes on one side is higher than the number of nodes on the other side by REFERENCES (86) [1] J. M. H. Elmirghani, T. Klein, K. Hinton, T. E. H. El-Gorashi, A. Q. Lawey1, and X. Dong, “GreenTouch GreenMeter core network power consumption models and results,” in Proc. IEEE Online Conf. Green Commun., 2014, pp. 1–8. The total power for the aforementioned topologies can be given as a 3rd order polynomial: [2] J. M. H. Elmirghani et al., “Energy efﬁciency measures for future core networks,” in Proc. Opt. Fiber Commun. Conf. Exhib., Mar. 19–23, 2015, Paper Th1I-4. P = aN 3 + bN 2 + cN + d (87) (87) [3] J. M. H. Elmirghani et al., “GreenTouch GreenMeter core network energy efﬁciency improvement measures and optimization,” IEEE/OSA J. Opt. Commun. Netw., vol. 10, no. 2, pp. A250–A269, Feb. 2018. In Table II we summarize the values for the coefﬁcients a, b, c, and d for the different regular topologies. The table, alongside Figure 9 show that the star and the full mesh topologies have lower power consumption than other topologies as the most sig- niﬁcant polynomial coefﬁcient is zero (a = 0). It shows also that the full mesh topology has a close but lower power consumption to the star topology, which gets better as the number of nodes increases because the b and c coefﬁcients are higher in the star topology. It shows that the line topology consumes more power than the two variants of the ring topology, and the highest power consumption over other topologies due to the higher signiﬁcant polynomial coefﬁcients. [4] G. Shen and R. S. Tucker, “Energy-minimized design for IP over WDM networks,” IEEE/OSA J. Optical Commun. Netw., vol. 1. no. 1, pp. 176–186, Jun. 2009. pp [5] K. Axel, U. Gebhard, and F. Ilchmann, “Energy and cost efﬁciency of adaptive and mixed-line-rate IP over DWDM networks,” J. Lightw. Tech- nol., vol. 30. no. 2, pp. 215–221, Jan. 2012. [6] Taisir E. H. El-Gorashi, X. Dong, and J. M. H. Elmirghani, “Green optical orthogonal frequency-division multiplexing networks,” IET Optoelectron- ics, vol. 8. no. 3, pp. 137–148, Jun. 2014. [7] X. Dong, T. E. H. El-gorashi, and J. M. H. Elmirghani, “On the energy efﬁciency of physical topology design for IP over WDM networks,” J. Lightw. Technol., vol. 30, no. 12, pp. 1931–1942, Jun. 2012. [8] X. Dong, T. El-Gorashi, and J. M. H. Elmirghani, “Green IP over WDM networks with data centers,” J. Lightw. Technol., vol. 29, no. 12, pp. REFERENCES 1861–1880, Jun. 2011. In Table III we summarize the lower and upper bounds for the different approaches considered in this paper. This table will be useful for quick and easy reference to the bounds and the reader can refer to previous sections for more details about the derivation steps and assumption. The table is divided into two parts where the ﬁrst part considers the non-bypass case and the second is the bypass case. In each part, incremental scenarios that build on the basic approach are shown in sequence. [9] A. Lawey, T. El-Gorashi, and J. Elmirghani, “Distributed energy efﬁ- cient clouds over core networks,” J. Lightw. Technol., vol. 32, no. 7, pp. 1261–1281, Apr. 2014. [10] A. Lawey, T. El-Gorashi, and J. Elmirghani, “Bittorrent content dis- tribution in optical networks,” J. Lightw. Technol., vol. 32, no. 21, pp. 4209–4225, Nov. 2014. [11] L. Nonde, T. El-Gorashi, and J. Elmirghani, “Energy efﬁcient virtual network embedding for cloud networks,” J. Lightw. Technol., vol. 33, no. 9, pp. 1828–1849, May 2015. [12] A. M. Al-Salim, A. Q. Lawey, T. E. H. El-Gorashi, and J. M. H. Elmirghani, “Energy efﬁcient big data networks: impact of volume and variety,” IEEE Trans. Netw. Service Manage., vol. 15, no. 1, pp. 458–474, Mar. 2018. JOURNAL OF LIGHTWAVE TECHNOLOGY, VOL. 36, NO. 23, DECEMBER 1, 2018 JOURNAL OF LIGHTWAVE TECHNOLOGY, VOL. 36, NO. 23, DECEMBER 1, 2018 5404 core network energy efﬁciency models. The bounds and formu- las give insights into the behavior of the systems and facilitate predictions at situations where the MILP model is too complex. They also give approximations to the optimum results found by the MILP models. The power consumption of the optimum scenario generally lies close to the lower bound while the upper bound is far from the optimum case because it takes into con- sideration extreme cases that are not usually found in practice. We have also shown that the full-mesh topology is a very good estimate of the optimal network topology and in general, regular topologies can be represented as a polynomial of the third or- der, with different coefﬁcients for different topologies. We also provide closed form expressions for the network paths average hop counts for regular topologies. shows the hop count of a routed trafﬁc between each source and destination node pair. The following equation calculates the total hop count by realizing the asymmetry in this matrix, and realizing that the total hop count follows this pattern: hop count 1 exists 6 times, hop count 2 exists 4 times and hop count 3 exists twice. The following equation generalizes it to a N number of nodes. s∈N d∈N s̸=d hs,d = 2 N −1 i=1 i(N −i) = 2N N −1 i=1 i −2 N −1 i=1 i2 = N(N −1)(N + 1) 3 (83) h = N(N −1)(N + 1) 3N(N −1) = N + 1 3 (84) = N(N −1)(N + 1) 3 (83) h = N(N −1)(N + 1) 3N(N −1) = N + 1 3 (84) (83) (83) where we used the fact that: D. Line Topology The total number of hops used by all demands is given by equation (83), which can be explained with the aid of Figure 8. To the right is a 4-node line topology and the array to the left Fig. 9. Power Consumption of the different topologies at different node sizes, 2020 BAU+GT Components, 40Gbps, bypass, Sleep and energy efﬁcient Pro- tection, considering equal trafﬁc demands. ACKNOWLEDGMENT n 1 i2 = n(n + 1)(2n + 1) 6 (85) n 1 i2 = n(n + 1)(2n + 1) 6 (85) erefore P = N(N −1)¯L ηn pr B + Ec N + 1 4 = Ec 4 ¯LN 3 + ηn pr B ¯LN 2 − ηn pr B + Ec 4 N ¯L (86) The authors would also like to acknowledge broad discussions with members of the GreenTouch Wired, Core and Access Net- works working group. All data are provided in full in the results section of this paper. (85) therefore P = N(N −1)¯L ηn pr B + Ec N + 1 4 = Ec 4 ¯LN 3 + ηn pr B ¯LN 2 − ηn pr B + Ec 4 N ¯L (86) MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY Conf., Los Angeles, CA, USA, 2012, Paper OM2G-1. p [18] F. Amarilli, S. B. Chang, S. Korotky, and K. Satzke, “GreenTouch appli- cation taxonomy,” version V2, GreenTouch Conﬁdential, Jun. 1, 2012. [19] Cisco, “Cisco CRS 16-Slot Chassis Power Systems,” Jan. 2009. [On- line]. 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Available: https://www.cisco. com/c/en/us/products/collateral/routers/carrier-routing-system/data ‘sheet’ c78-478689.html [24] Cisco, “Cisco CRS 100 gigabit Ethernet interface modules,” Jan. 2014. [Online]. Available: https://www.cisco.com/en/US/prod/collateral/ routers/ps5763/CRS-1x100GE’ DS.pdf [25] Cisco, “Cisco CRS-X 4-port 100GE LAN/OTN interface module,” Oct. 2013. [Online]. Available: https://www.cisco.com/en/US/prod/colla teral/routers/ps5763/data ‘sheet’ c78-728888.pdf Mohamed O. I. Musa received the B.Sc. (ﬁrst-class Hons.) degree in electrical and electronic engineering from the University of Khartoum, Khartoum, Sudan, in 2009, the M.Sc. degree (with distinction) in broadband wireless and optical communication from the University of Leeds, Leeds, U.K., in 2011, and the Ph.D. in energy efﬁcient network coding in optical networks from the University of Leeds, Leeds, U.K., in 2016. His current research in- terests include ICT energy optimization, network coding and energy efﬁcient routing protocols in optical networks. He currently chairs working group for the IEEE P1928.1 standard, and is the secretary for the IEEE Green ICT Standards Committee. Taisir E. H. El-Gorashi received the B.S. (ﬁrst-class Hons.) degree in electrical and electronic engineering from the University of Khartoum, Khartoum, Sudan, in 2004, the M.Sc. (with distinction) degree in photonic and communication systems from the University of Wales, Swansea, U.K., in 2005, and the Ph.D. degree in optical networking from the University of Leeds, Leeds, U.K., in 2010. She is currently a Lecturer of optical networks with the School of Electrical and Electronic Engineering, University of Leeds. She was a Postdoctoral Research Fellow at the University of Leeds from 2010 to 2014, where she focused on the energy efﬁciency of optical networks investigating the use of renewable energy in core networks, green IP over WDM networks with data centers, en- ergy efﬁcient physical topology design, energy efﬁciency of content distribution networks, distributed cloud computing, network virtualization, and big data. MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY He is and has been on the technical program committee of the 34 IEEE ICC/GLOBECOM conferences between 1995 and 2016 including 15 times as Symposium Chair. He has given over 55 invited and keynote talks over the past 8 years. He received the IEEE Commu- nications Society Hal Sobol Award, the IEEE Comsoc Chapter Achievement Award for excellence in chapter activities (both in international competition in 2005), the University of Wales Swansea Outstanding Research Achievement Award, 2006; and received in international competition: the IEEE Communi- cations Society Signal Processing and Communication Electronics outstanding service award, 2009, a best paper award at IEEE ICC2013. Related to Green Communications he received the IEEE Comsoc Transmission Access and Opti- cal Systems outstanding Service Award 2015 in recognition of Leadership and Contributions to the Area of Green Communications, the GreenTouch 1000x award in 2015 for pioneering research contributions to the ﬁeld of energy ef- ﬁciency in telecommunications, the IET 2016 Premium Award for best paper in IET Optoelectronics, and shared the 2016 Edison Award in the collective disruption category with a team of 6 from GreenTouch for their joint work on the GreenMeter. He is currently the Editor of IET Optoelectronics and Jour- nal of Optical Communications, and was the Editor of IEEE COMMUNICATIONS SURVEYS AND TUTORIALS and IEEE JOURNAL ON SELECTED AREAS IN COMMU- NICATIONS series on Green Communications and Networking. He was Co-Chair of the GreenTouch Wired, Core and Access Networks Working Group, an ad- viser to the Commonwealth Scholarship Commission, member of the Royal Society International Joint Projects Panel and member of the Engineering and Physical Sciences Research Council (EPSRC) College. He has been awarded in excess of 22 million in grants to date from EPSRC, the EU and industry and has held prestigious fellowships funded by the Royal Society and by BT. He was an IEEE Comsoc Distinguished Lecturer 2013–2016. His work led to 5 IEEE standards with a focus on energy efﬁciency, where he currently heads the work group responsible for IEEE P1925.1, IEEE P1926.1, IEEE P1927.1, IEEE P1928.1, and IEEE P1929.1, resulting in signiﬁcant impact through industrial and academic uptake. [16] Cisco, “Cisco Visual Networking Index: Forecast and Methodology, 2014–2019,” Cisco White Paper [Online]. Available: http://www. cisco.com/c/en/us/solutions/collateral/service-provider/ipngn-ip-next- generation-network/white ‘paper’ c11-481360.html [17] S. K. Korotky, Trafﬁc trends: Drivers and measures of cost-effective and energy-efﬁcient technologies and architectures for backbone optical net- works,” in Proc. Opt. Fiber Commun. MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY [15] M. Musa, T. Elgorashi, and J. Elmirghani, “Energy efﬁcient survivable IP- over-WDM networks with network coding,” IEEE/OSA J. Opt. Commun. Netw., vol. 9, no. 3, pp. 207–217, Mar. 2017. Jaafar M. H. Elmirghani received the B.Sc. (ﬁrst-class Hons.) degree in elec- trical engineering from the University of Khartoum, Khartoum, Sudan, in 1989 and was awarded all 4 prizes in the Department for Academic Distinction, the Ph.D. degree in the synchronization of optical systems and optical receiver de- sign from the University of Huddersﬁeld, Huddersﬁeld, U.K., in 1994, and the D.Sc. degree in communication systems and networks from the University of Leeds, Leeds, U.K., in 2014. He is the Director of the Institute of Integrated Information Systems within the School of Electronic and Electrical Engineer- ing, University of Leeds, Leeds, U.K. He joined Leeds in 2007 and prior to that (2000–2007) as the Chair in Optical Communications with the Univer- sity of Wales Swansea, he founded, developed, and directed the Institute of Advanced Telecommunications and the Technium Digital (TD), a technology incubator/spin-off hub. He has provided outstanding leadership in a number of large research projects at the IAT and TD. He has coauthored Photonic Switch- ing Technology: Systems and Networks (Wiley) and has authored more than 450 papers. His research interests include optical systems and networks. He is a Fellow of the IET, Chartered Engineer, and a Fellow of the Institute of Physics. He was the Chairman of the IEEE Comsoc Transmission Access and Optical Systems technical committee and was the Chairman of the IEEE Comsoc Sig- nal Processing and Communications Electronics technical committee, and an editor of the IEEE COMMUNICATIONS MAGAZINE. He was a Founding Chair of the Advanced Signal Processing for Communication Symposium which started at IEEE GLOBECOM99 and has continued since at every ICC and GLOBE- COM. He was also Founding Chair of the ﬁrst IEEE ICC/GLOBECOM optical symposium at GLOBECOM00, the Future Photonic Network Technologies, Architectures, and Protocols Symposium. He chaired this symposium, which continues to date under different names. He was the founding chair of the ﬁrst Green Track at ICC/GLOBECOM at GLOBECOM 2011, and is Chair of the IEEE Green ICT initiative within the IEEE Technical Activities Board Future Directions Committee, a pan IEEE Societies initiative responsible for Green ICT activities across IEEE, 2012-present. VI. CONCLUSIONS [13] H. M. Mohammad Ali, T. E. H. El-Gorashi, A. Q. Lawey, and J. M. H. Elmirghani, “Future energy efﬁcient data centers with disaggregated servers,” J. Lightw. Technol., vol. 35, no. 24, pp. 5361–5380, Dec. 2017. In this paper we developed bounds on the power consumption of core networks for the non-bypass, bypass, MLR and topol- ogy optimization scenarios to verify the individual elements of the optimization models produced in our previous GreenTouch [14] M. Musa, T. Elgorashi, and J. Elmirghani, “Bounds for energy efﬁcient survivable IP over WDM networks with network coding,” IEEE/OSA J. Opt. Commun. Netw., vol. 10, no. 5, pp. 471–481, May 2018. 5405 MUSA et al.: BOUNDS ON GREENTOUCH GREENMETER NETWORK ENERGY EFFICIENCY In 2012, she was a BT Research Fellow, where she developed an energy efﬁcient hybrid wireless optical broadband access network and explored the dynamics of TV viewing behavior and program popularity. The energy efﬁciency techniques developed during her postdoctoral research contributed three out of the eight carefully chosen core network energy efﬁciency improvement measures rec- ommended by the GreenTouch consortium for every operator network world- wide. Her work led to several invited talks at GreenTouch, Bell Labs, the Optical Network Design and Modeling Conference, the Optical Fiber Communications Conference, the International Conference on Computer Communications, and the EU Future Internet Assembly in 2013 and collaboration with Alcatel Lucent and Huawei.
https://openalex.org/W2002207189	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0107585&type=printable	English	null	Parkin (PARK 2) Mutations Are Rare in Czech Patients with Early-Onset Parkinson's Disease	PloS one	2,014	cc-by	5,126	Abstract uthors confirm that all data underlying the findings are fully available without restriction. All relevant data are included within the paper Funding: This work was supported by the Czech Ministry of Health, IGA NT/11331 (OF, MM, and ER); Czech Ministry of Health, RVO-VFN64165/2012 (DZ and PM); Charles University in Prague, PRVOUK P26/LF1/4 (JR and ER); and Charles University in Prague, UNCE 204011/2012 (DZ, JK, and PM). 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. * Email: fiala@inep.cz intronic regions. The parkin gene encodes a 465-amino acid protein, which contains a ubiquitin-like domain at the N terminus and a RING (Really Interesting New Gene) domain composed of three RING finger motifs (RING0, 1, and 2). RING1 and 2 are separated by a sequence without any recognizable domain structure named IBR (in between-RING) [8]. Parkin exhibits E3 ubiquitin ligase activity and mediates the ubiquitination of a number of proteins, thus targeting them for proteasomal degradation. Parkin has been reported to influence mitochondrial fusion, mitophagy and mitochondrial transport through ubiquity- lation of mitofusin, voltage dependent anion channel 1 (VDAC1) and Miro/Milton complex, respectively. Parkin has also been shown to be important in the regulation of mitochondrial biogenesis [9]. Ondrej Fiala1,2, Daniela Zahorakova3, Lenka Pospisilova3, Jana Kucerova3, Milada Matejckova4, Pavel Martasek3, Jan Roth1, Evzen Ruzicka1 1 Department of Neurology and Centre of Clinical Neuroscience, 1st Faculty of Medicine and General University Hospital, Charles University, Prague, Czech Republic, 2 Institute of Neuropsychiatric Care (INEP), Prague, Czech Republic, 3 Department of Pediatrics and Adolescent Medicine, 1st Faculty of Medicine, and General University Hospital, Charles University, Prague, Czech Republic, 4 Department of Pathology and Molecular Medicine, Thomayer’s University Hospital, Prague, Czech Republic Parkin (PARK 2) Mutations Are Rare in Czech Patients with Early-Onset Parkinson’s Disease Ondrej Fiala1,2, Daniela Zahorakova3, Lenka Pospisilova3, Jana Kucerova3, Milada Matejckova4, Pavel Martasek3, Jan Roth1, Evzen Ruzicka1 September 2014 \| Volume 9 \| Issue 9 \| e107585 Abstract Objective: The aim of the study is to determine the frequency of parkin allelic variants in Czech early-onset Parkinson’s disease patients and healthy controls. Methods: A total of 70 early-onset Parkinson’s disease patients (age at onset #40 years) and 75 controls were screened for the sequence variants and exon rearrangements in the parkin gene. Results: Parkin mutations were identified in five patients (7.1%): the p.R334C point mutation was present in one patient, four patients had exon deletions. The detected mutations were observed in the heterozygous state except one homozygous deletion of the exon 4. No mutations were obtained in control subjects. A novel sequence variant p.V380I (c.1138G.A) was identified in one control. Non-pathogenic polymorphisms p.S167N and p.D394N were seen in similar percentage in patients and controls, polymorphism p.V380L was almost twice as frequent in controls as in patients. Conclusions: Our study contributes to the growing body of evidence on the low frequency of the parkin mutations in the early-onset Parkinson’s disease suggesting the potential role of other genes in the pathogenesis of the disease. akova D, Pospisilova L, Kucerova J, Matejckova M, et al. (2014) Parkin (PARK 2) Mutations Are Rare in Czech Patients with Early-Onset ONE 9(9): e107585. doi:10.1371/journal.pone.0107585 Editor: Oscar Arias-Carrion, Hospital General Dr. Manuel Gea Gonza´lez, Mexico Copyright: 2014 Fiala 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: 2014 Fiala et al. This is an open-access article distributed under the terms of the Creative Commons Attrib 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 Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are included within the paper. Funding: This work was supported by the Czech Ministry of Health, IGA NT/11331 (OF, MM, and ER); Czech Ministry of Health, RVO-VFN64165/2012 (DZ and PM); Charles University in Prague, PRVOUK P26/LF1/4 (JR and ER); and Charles University in Prague, UNCE 204011/2012 (DZ, JK, and PM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Genotypic characteristics and frequency of allelic variants of the parkin gene For genetic analysis, a venous blood sample was collected and genomic DNA was isolated from peripheral blood leukocytes using a standard salting-out procedure. All 12 exons of the parkin gene were amplified from the patient’s genomic DNA by the polymerase chain reaction (PCR). For amplification, previously described primers were used [16]. All fragments were analyzed in both directions on a 3500xL Genetic Analyzer (Applied Biosys- tems). Parkin allelic variants were numbered relative to Genbank mRNA sequence (accession number NM_004562). Genotypic characteristics of the patients and controls are shown in Table 2, the frequency of allelic variants of the parkin gene is reported in Table 3. Previously described polymorphisms p.S167N and p.D394N were seen in similar percentage in patients (7.1%, 10.0%) and controls (9.3%, 8.0%). Polymorphism p.V380L was almost twice as frequent in controls (25.3%) as in patients (14.3%). A novel sequence variant p.V380I (c.1138G.A) was identified in one control (1.3%). One patient (1.4%) and three controls (4.0%) were carriers of two different polymorphisms (p.S167N + p.D394N, p.S167N + p.V380L, p.V380I + p.D394N). Disease-causing mutations (HGMD classification) p.A82E, p.R334C, and p.R402C occurred in three patients (4.3%) and one control (1.3%); from those subjects, a shared mutation with another polymorphism (p.A82E + p.D394N, p.V380L + p.R402C) was detected in one patient (1.4%) and one control (1.3%). Exon deletions were identified in four patients (5.7%). All allelic variants were observed in the heterozygous state, except two homozygous polymorphisms (p.V380L in patient, p.S167N in control) and one homozygous deletion (Ex4del). The patient with this mutation was also heterozygous for deletion of exons 2–3 and heterozygous for the p.V380L polymorphism; his family history was positive (Table 2, Patient 3). No statistically significant difference was found in the frequency of parkin allelic variants between patients and controls (Table 3). Controls were screened for the sequence variants identified in patients using high resolution melt analysis (HRM). The primers used for HRM were the same as those used to generate the PCR products for sequencing in patients. Reference samples of known genotypes were included into each sample group. The samples were then analyzed in Light Scanner instrument (Biofire Defence) using a melt range of 65uC to 98uC. Acquired data were analyzed by the supplied Call-IT 1.5 software using the auto group and high sensitivity settings. Introduction Parkinson’s disease (PD) is a chronic progressive neurodegen- erative disorder, clinically characterized by resting tremor, rigidity, and bradykinesia, as well as non-motor impairment such as cognitive deficit or autonomic dysfunction [1]. The prevalence of PD is about 0.3% in the entire population and more than 1% in the population over the age of 60 years [2]. The mean age at onset (AAO) of PD is usually between 60–70 years [3], however 3–5% of all patients with PD have onset before the age 40 years [4]. This rare form of the disease is referred as early-onset PD (EOPD); the incidence of EOPD is estimated 0.5 per 100 000 per year [5]. The clinical phenotype of EOPD differs from classic PD in several features such as slower disease progression, more frequent occurrence of dystonia, marked sleep benefit, excellent treatment response, and early development of levodopa-induced dyskinesia and motor fluctuations [4,6]. Among several genes whose mutations are associated with autosomal recessive EOPD, parkin mutations were shown as the most common [7]. More than 180 pathologic allelic variants of the parkin gene have been described with half of them being located in the region spanning exons 2 to 4. The exon rearrangements account for more than 50% of all parkin mutations [10,11]. Lu¨cking and colleagues reported the parkin mutations in 50% of familial as well as 18% of sporadic EOPD cases [7]. In contrast, other studies have shown a pathogenic mutation frequency as low as 1.6–8.6% [12–14]. The Parkin is one of the largest human genes spanning approx- imately 1.38 Mb. It consists of 12 coding exons separated by large PLOS ONE \| www.plosone.org 1 September 2014 \| Volume 9 \| Issue 9 \| e107585 Parkin Mutations in Czech Patients prevalence of the parkin mutations vary widely across studies, possibly due to a population-specific variability in allele frequen- cies. The aim of this study is to determine the frequency of parkin allelic variants in Czech EOPD patients and healthy controls. electronic tools MutPred and SIFT. The genotype frequencies in the general population were calculated from data of the Exome Variant Server (European American population). Genotypic characteristics and frequency of allelic variants of the parkin gene To identify exon rearrangements in the parkin gene, we analyzed gene dosage in patients and controls using multiplex ligation-dependent probe amplification (MLPA) using SALSA MLPA kit P051-C3-0313 and P052-C2-0313 (MRC-Holland). The MLPA assay was performed according to the manufacturer’s protocol. The fragments were analyzed on a 3130 Genetic Analyzer (Applied Biosystems) with the fragment analysis software Gene Mapper 4.0. For each sample, the relative peak area was calculated and compared with controls using the Coffalyser v.131211 software. Clinical characteristics of patients Clinical characteristics of EOPD patients are summarized in Table 1. The mean AAO was 35.064.9 years, the average disease duration amounted to 12.368.0. The mean H-N stage was 2.160.9. Dystonia occurred in more than half of the patients (55.7%), the half of the individuals (50.0%) referred marked sleep benefit. A relatively high percentage of patients reported hallucinations (18.6%). The most common sign of dysautonomia was hyperhidrosis (57.1%), followed by hypersalivation (42.9%), urinary dysfunction (28.6%), and constipation (27.1%). An excellent response to dopaminergic therapy was reported by two thirds of the cases (68.6%), levodopa-induced dyskinesia and motor fluctuations were registered in more than half of the patients (51.4% and 70.0%, respectively). Dyskinesia had developed after a mean interval of 5.4 years 64.1 after commencing dopaminergic treatment, motor fluctuations appeared even earlier (4.7 years 64.1). A positive family history was present in 10 (14.3%) individuals. Materials and Methods The Fisher Exact Test was used to assess differences in frequency of parkin allelic variants between patients and controls. Odds ratios (OR) are given with their 95% confidence intervals (CI). Statistical analysis was performed using the GraphPad Prism 6 software. Patients and controls A total of 70 unrelated Czech patients (47 males, 23 females; mean age 47.7 years) with EOPD (AAO #40 years) were recruited from the Movement Disorders Center, Prague, Czech Republic. The diagnosis of PD was based on the UK Brain Bank diagnostic criteria, not including the presence of family history as an exclusion criterion [15]. Controls were 75 healthy unrelated individuals (52 males, 23 females; mean age 45.4 years) recruited from the Department of Blood Transfusion, General University Hospital, Prague, Czech Republic. Written informed consent was obtained from all individuals. Phenotypic data were assessed by personal interview and neurological examination, both in patients and in controls. All controls had normal neurological examination and negative family history for parkinsonism. Family history was defined as positive if the proband had at least one affected first- or second-degree relative. Personal interview and clinical evaluations of EOPD patients included the severity of PD (Hoehn and Yahr stage; H-Y), the presence of dystonia, hallucinations, dysautono- mia (hyperhidrosis, hypersalivation, urinary dysfunction, consti- pation), and sleep benefit, as well as the response to dopaminergic therapy and the occurrence of levodopa-induced dyskinesia and motor fluctuations. The study was approved by the ethics committee of the General University Hospital, Prague, Czech Republic. September 2014 \| Volume 9 \| Issue 9 \| e107585 Discussion The basic clinical characteristics of Czech EOPD patients do not differ from previous descriptions of EOPD phenotype [4,6,17]. Our results confirmed a high occurrence of dystonia (55.7%), The identified allelic variants were classified according to terminology of the Human Gene Mutation Database (HGMD). The prediction of the pathogenicity was performed using September 2014 \| Volume 9 \| Issue 9 \| e107585 PLOS ONE \| www.plosone.org 2 Parkin Mutations in Czech Patients Table 1. Clinical characteristics of patients (n = 70). Characteristics Patients Male/Female (% male) 47/23 (67.1%) Age at onset (mean 6 SD) years 35.064.9 Age at examination (mean 6 SD) years 47.468.4 Disease duration (mean 6 SD) years 12.368.0 H-Y stage (mean 6 SD) 2.160.9 Positive family history 10 (14.3%) Dystonia 39 (55.7%) Hallucinations 13 (18.6%) Hyperhidrosis 40 (57.1%) Hypersalivation 30 (42.9%) Urinary dysfunction 20 (28.6%) Constipation 19 (27.1%) Sleep benefit 35 (50.0%) Excellent response to dopaminergic therapy 48 (68.6%) Levodopa-induced dyskinesia 36 (51.4%) Latency of dyskinesia (mean 6 SD) years 5.464.1 Motor fluctuations 49 (70.0%) Latency of motor fluctuations (mean 6 SD) years 4.764.1 doi:10.1371/journal.pone.0107585.t001 Table 1. Clinical characteristics of patients (n = 70). Characteristics Patients Male/Female (% male) 47/23 (67.1%) Age at onset (mean 6 SD) years 35.064.9 Age at examination (mean 6 SD) years 47.468.4 Disease duration (mean 6 SD) years 12.368.0 H-Y stage (mean 6 SD) 2.160.9 Positive family history 10 (14.3%) Dystonia 39 (55.7%) Hallucinations 13 (18.6%) Hyperhidrosis 40 (57.1%) Hypersalivation 30 (42.9%) Urinary dysfunction 20 (28.6%) Constipation 19 (27.1%) Sleep benefit 35 (50.0%) Excellent response to dopaminergic therapy 48 (68.6%) Levodopa-induced dyskinesia 36 (51.4%) Latency of dyskinesia (mean 6 SD) years 5.464.1 Motor fluctuations 49 (70.0%) Latency of motor fluctuations (mean 6 SD) years 4.764.1 doi:10.1371/journal.pone.0107585.t001 Table 1. Clinical characteristics of patients (n = 70). motor symptoms also prevail in late-onset PD [18] having negative impact on health-related quality of life [19]. marked sleep benefit (50.0%), an excellent response to dopami- nergic therapy (68.6%) as well as high rate of the levodopa- induced dyskinesia (51.4%) and motor fluctuations (70.0%) with early development after commencing the dopaminergic treatment (dyskinesia = 5.4 years; motor fluctuations = 4.7 years). The signs of dysautonomia (hyperhidrosis, hypersalivation, urinary dysfunc- tion, constipation) were also common, hallucinations occurred in a rather high percentage of patients (18.6%). The following non- Polymorphisms p.S167N and p.D394N were as common in patients as in controls. Discussion Although both polymorphisms are classified according to HGMD as disease-associated, meta-analytic studies did not show the association between these polymorphisms and PD risk [20,21]. Polymorphism p.V380L was seen almost twice more frequently in controls than in patients. It corresponds with the recent meta-analysis demonstrating association of the p.V380L Table 2. Genotypic characteristics of patients and controls. Individual Sequence variant/mutation Positive family history Patient 1 p.A82E het + p.D394N het no Control 1, Patient 2 p.S167N het + p.D394N het no Patient 3 p.V380L het + ex2-3del het + ex4del hom yes Control 2 p.S167N hom + p.V380L het no Control 3 p.V380I het + p.D394N het no Control 4 p.V380L het + p.R402C het no Patient 4–7, Control 5–10 p.S167N het patient 4 Patient 8 p.R334C het no Patient 9–16, Control 11–26 p.V380L het patient 9,10 Patient 17 p.V380L hom no Patient 18–22, Control 27–30 p.D394N het no Patient 23 p.R402C het no Patient 24 ex2del het no Patient 25 ex1-2del het no Patient 26 ex2-5del het no het - heterozygous, hom- homozygous. doi:10.1371/journal.pone.0107585.t002 PLOS ONE \| www.plosone.org 3 September 2014 \| Volume 9 \| Issue 9 \| e107585 Table 2. Genotypic characteristics of patients and controls. September 2014 \| Volume 9 \| Issue 9 \| e107585 September 2014 \| Volume 9 \| Issue 9 \| e107585 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Parkin Mutations in Czech Patients Table 3. Frequency of allelic variants of the parkin gene in patients (n = 70) and controls (n = 75). September 2014 \| Volume 9 \| Issue 9 \| e107585 Discussion Prediction analysis using MutPred and SIFT estimated a low probability of pathogenic effect (Table 3). One patient was a carrier of the p.A82E sequence variant (labeled as disease causing mutation in HGMD). This variant is very likely to be non-pathogenic as its presence has been previously observed in healthy controls [23]; it has a benign prediction analysis, and no effect of the p.A82E variant on the cellular distribution of the protein in vitro [24]. Even though the p.R402C allelic variant has been predicted as damaging and classified as disease causing mutation in HGMD (Table 3), its pathogenic status is equivocal since it has been also detected in healthy subject [25]. In our sample, heterozygous p.R402C variant was present in one patient and one control. The minor- allele frequency in European American population is estimated at 0.26% according to the Exome Variant Server (EVS). Therefore, we assume that p.R402C is a non-pathogenic silent substitution. In our previous pilot study [40], using less strict criteria for the AAO (#45 years), we reported parkin mutations and polymor- phisms among 45 Czech patients with EOPD, however their clinical significance was unclear due to the lack of control group. In the present case-control study, we are able to address this issue. Since we found previously a high percentage of the parkin polymorphisms in patients (34.9%), we suggested these polymor- phisms as possible risk factors for EOPD. In the light of the new results presented here, it appears however that the detected parkin polymorphisms (p.S167N, p.V380L, p.D394N) are not risk factors for EOPD. Mutations of the parkin gene were identified in five patients (7.1%): the p.R334C point mutation was present in one patient (1.4%), four patients (5.7%) had exon deletions. All deletions contained exon 2 encoding ubiquitin-like domain [26], which is crucial for a normal function of the protein and prevents its autoubiquitination [27]. No exon deletion was obtained in controls suggesting the possible pathogenic role of exon rearrangements in the etiology of EOPD. The detected mutations were observed only in the heterozygous state except one homozygous deletion of the exon 4. With regard to recessive inheritance of parkin type of EOPD, the presence of the homozygous or two compound heterozygous parkin mutations is required to cause the disease. Although the clinical significance of single heterozygous parkin mutations remains unclear, partial evidence indicates that heterozygous mutations also contribute to PD risk [28]. 14. Koziorowski D, Hoffman-Zacharska D, Slawek J, Szirkowiec W, Janik P, et al. (2010) Low frequency of the PARK2 gene mutations in Polish patients with the early-onset form of Parkinson disease. Parkinsonism Relat Disord 16: 136–138. Acknowledgments The authors are thankful to Mrs. Alena Puchmajerova and Mrs. Irena Starkova for their valuable technical support. The incidence of PD increases with age; it is five times higher at the age between 40–49 years than in the range of 30–39 years [5]. EOPD is considered a disease with AAO #40 years [8]. Nevertheless, many studies applied higher AAO, typically #45 [33–35] or #50 years [36–39]. The application of various criteria for AAO may cause misinterpretation when comparing data (e.g. the percentage of identified mutations) among studies. Thus, we adopted the most strict AAO criteria (#40 years) in order to Discussion Allelic variant Zygosity HGMD class SIFT/MutPred EVS genotype frequency Frequency in patients Frequency in controls Fisher P- value OR 95% CI SNPs/point mutations p.A82E het DM Tolerated/Low risk 0.51% 1 (1.4%) 0 (0.0%) 0.489 3.26 0.13–81.40 p.S167N het DP Tolerated/Very low risk 3.74% 5 (7.1%) 6 (8.0%) 1.0 0.89 0.26–3.04 p.S167N hom DP Tolerated/Very low risk 0.05% 0 (0.0%) 1 (1.3%) 1.0 0.35 0.01–8.80 p.R334C het DM Tolerated/Medium risk NA 1 (1.4%) 0 (0.0%) 0.483 3.26 0.13–81.40 p.V380L het DP Tolerated/Very low risk 28.51% 9 (12.9%) 19 (25.3%) 0.062 0.44 0.18–1.04 p.V380L hom DP Tolerated/Very low risk 2.84% 1 (1.4%) 0 (0.0%) 0.483 3.26 0.13–81.40 p.V380I het NA Tolerated/Low risk NA 0 (0.0%) 1 (1.3%) 1.0 0.35 0.01–8.80 p.D394N het DP Tolerated/Very low risk 8.53% 7 (10.0%) 6 (8.0)% 0.775 1.28 0.40–4.01 p.R402C het DM Damaging/High risk 0.51% 1 (1.4%) 1 (1.3%) 1.0 1.07 0.07–17.49 Exon rearrangements Ex1del het DM NA NA 1 (1.4%) 0 (0.0%) 0.483 3.26 0.13–81.40 Ex2del het DM NA NA 4 (5.7%) 0 (0.0%) 0.052 10.22 0.54–193.50 Ex3del het DM NA NA 2 (2.9%) 0 (0.0%) 0.231 5.51 0.26–116.90 Ex4del het DM NA NA 1 (1.4%) 0 (0.0%) 0.483 3.26 0.13–81.40 Ex4del hom DM NA NA 1 (1.4%) 0 (0.0%) 0.483 3.26 0.13–81.40 Ex5del het DM NA NA 1 (1.4%) 0 (0.0%) 0.483 3.26 0.13–81.40 CI - confidence interval, DM - disease causing mutation, DP - disease-associated polymorphism, EVS - Exome variant server, het - heterozygous, HGMD - Human gene mutation database, hom - homozygous, NA - not available, OR - odds ratio. 4 Parkin Mutations in Czech Patients polymorphism with decreased risk for PD [22], despite the fact that the p.V380L variant is listed as disease-associated polymor- phism in HGMD. preserve a more phenotypically homogeneous patient’s sample at the expense of its size. The relatively small size of our patient’s sample (n = 70) was a substantial limitation for the statistical analysis. Although there was no statistically significant difference in the frequency of parkin allelic variants between patients and controls, noticeable contrast can be seen in two cases: a twice higher frequency of heterozygous p.V380L polymorphism in controls (p = 0.062; OR = 0.44; 95% CI = 0.1821.04) and an exclusive occurrence of heterozygous deletion of exon 2 in four patients (p = 0.052; OR = 10.22; 95% CI = 0.542193.50). A novel allelic variant p.V380I (c.1138G.A) was identified in one control. 13. Kilarski LL, Pearson JP, Newsway V, Majounie E, Knipe MD, et al. (2012) Systematic review and UK-based study of PARK2 (parkin), PINK1, PARK7 (DJ-1) and LRRK2 in early-onset Parkinson’s disease. Mov Disord 27: 1522– 1529. Discussion Unaffect- ed carriers of heterozygous parkin mutations show presynaptic dopaminergic dysfunction in the striatum [29,30] and hyperecho- genicity of the substantia nigra [31,32]. These preclinical changes suggest that heterozygous parkin mutations might contribute to the pathogenesis of EOPD. Overall, our study contributes to the growing body of evidence on the low frequency of the parkin mutations in the EOPD [12,13,41,42], although it does not corroborate with the other studies indicating high prevalence of these mutations [7,35]. Furthermore, we have identified a low percentage of EOPD familiar cases positive for mutations in the parkin gene (only one case; 10%) in comparison to previous observations [7]. Possible explanations for this discrepancy could be a population-specific variability in allele frequencies, different AAO criteria, various ratios of sporadic and familiar cases, and different criteria for determining the allelic variants as mutations. 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Lancet Neurol 6: 652–662. 42. Mellick GD, Siebert GA, Funayama M, Buchanan DD, Li Y, et al. (2009) Screening PARK genes for mutations in early-onset Parkinson’s disease patients from Queensland, Australia. Parkinsonism Relat Disord 15: 105–109. 29. Hilker R, Klein C, Ghaemi M, Kis B, Strotmann T, et al. (2001) Positron emission tomographic analysis of the nigrostriatal dopaminergic system in PLOS ONE \| www.plosone.org September 2014 \| Volume 9 \| Issue 9 \| e107585 September 2014 \| Volume 9 \| Issue 9 \| e107585 6
https://openalex.org/W2095146796	https://thescholarship.ecu.edu/bitstream/10342/5522/1/pone.0073595.PMC3753306.pdf	English	null	Medical and Household Characteristics Associated with Methicillin Resistant Staphylococcus aureus Nasal Carriage among Patients Admitted to a Rural Tertiary Care Hospital	PloS one	2,013	cc-by	7,825	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Center for Research Resources (grant number UL1RR025747), the NC Occupational Safety and Health Education Research Center (5-52665), and the H. Michael and Barbara Arrighi Endowed Scholarship Fund. The content is the sole responsibility of the authors and does not represent the official views of the National Center for Research Resources, the National Institutes of Health, or the National Institutes for Occupational Safety and Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Keith Ramsey has provided consultancy for BD GeneOhm, is on the Speakers Bureaus of Cubist Pharmaceuticals and Sanofi-Pasteur, and has served as an expert witness in legal proceedings related to healthcare-associated infections, and received fees for his services. Pia D. M. MacDonald is employed by Social & Scientific Systems, Inc. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: schinasi@email.unc.edu complex and distinctions between CA and HA strains are increasingly difficult to disentangle. Leah Schinasi1, Steve Wing1, Pia D. M. MacDonald1,2, David B. Richardson1, Jill R. Stewart3, Kerri L.Augustino4,5, Delores L. Nobles4,5, Keith M. Ramsey4,5 1 Department of Epidemiology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2 Social & Scientific Systems, Inc., Durham, North Carolina, United States of America, 3 Department of Environmental Sciences and Engineering, The University of NC at Chapel Hill, Chapel Hill, North Carolina, United States of America, 4 Department of Infection Control, Vidant Medical Center, Greenville, North Carolina, United States of America, 5 Division of Infectious Diseases, The Brody School of Medicine at East Carolina University, Greenville, North Carolina, United States of America Abstract Background: Methicillin resistant Staphylococcus aureus (MRSA) poses a threat to patient safety and public health. Understanding how MRSA is acquired is important for prevention efforts. This study investigates risk factors for MRSA nasal carriage among patients at an eastern North Carolina hospital in 2011. Methods: Using a case-control design, hospitalized patients ages 18 – 65 years were enrolled between July 25, 2011 and December 15, 2011 at Vidant Medical Center, a tertiary care hospital that screens all admitted patients for nasal MRSA carriage. Cases, defined as MRSA nasal carriers, were age and gender matched to controls, non-MRSA carriers. In-hospital interviews were conducted, and medical records were reviewed to obtain information on medical and household exposures. Multivariable conditional logistic regression was used to derive odds ratio (OR) estimates of association between MRSA carriage and medical and household exposures. Results: In total, 117 cases and 119 controls were recruited to participate. Risk factors for MRSA carriage included having household members who took antibiotics or were hospitalized (OR: 3.27; 95% Confidence Interval (CI): 1.24–8.57) and prior hospitalization with a positive MRSA screen (OR: 3.21; 95% CI: 1.12–9.23). A lower proportion of cases than controls were previously hospitalized without a past positive MRSA screen (OR: 0.40; 95% CI: 0.19–0.87). Conclusion: These findings suggest that household exposures are important determinants of MRSA nasal carriage in hospitalized patients screened at admission. Citation: Schinasi L, Wing S, MacDonald PDM, Richardson DB, Stewart JR, et al. (2013) Medical and Household Characteristics Associated with Methicillin Resistant Staphylococcus aureus Nasal Carriage among Patients Admitted to a Rural Tertiary Care Hospital. PLoS ONE 8(8): e73595. doi:10.1371/journal.pone.0073595 Citation: Schinasi L, Wing S, MacDonald PDM, Richardson DB, Stewart JR, et al. (2013) Medical and Household Characteristics Associate Staphylococcus aureus Nasal Carriage among Patients Admitted to a Rural Tertiary Care Hospital. PLoS ONE 8(8): e73595. doi:10.137 Editor: Tara C. Smith, University of Iowa, United States of America Editor: Tara C. Smith, University of Iowa, United States of America Received December 7, 2012; Accepted July 26, 2013; Published August 26, 2013 Copyright: 2013 Schinasi 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 Schinasi et al. Medical and Household Characteristics Associated with Methicillin Resistant Staphylococcus aureus Nasal Carriage among Patients Admitted to a Rural Tertiary Care Hospital Leah Schinasi1, Steve Wing1, Pia D. M. MacDonald1,2, David B. Richardson1, Jill R. Stewart3, Kerri L.Augustino4,5, Delores L. Nobles4,5, Keith M. Ramsey4,5 Introduction When it was first identified in the 1960s, Methicillin resistant Staphylococcus aureus (MRSA) was a nosocomial pathogen [1]. In the 1990s, the epidemiology of this virulent pathogen changed; it was identified in people without recent medical contact [2]. Healthcare associated (HA) and community associated (CA) MRSA strains are genetically distinct [3]. However, CA strains have been detected in hospital outbreaks [4], and people without recent hospitalizations may carry HA strains [5]. Indeed, the epidemiology of MRSA is In addition to medical related exposures [6], household and environmental exposures are gaining recognition as potentially important determinants of transmission and acquisition [7,8]. For example, MRSA carriage or infection has been associated with living with household members with a history of infections [8,9] and with presence of MRSA in the household environment [8]. There is evidence that MRSA may be transmitted between humans and animals [9,10]. There have been reports of MRSA August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 1 August 2013 \| Volume 8 \| Issue 8 \| e73595 MRSA and Medical Household Exposures infections and outbreaks among athletics participants [11] and associations between smoking and MRSA infection [12]. Geno- typic, clinical and demographic differences in the epidemiology of MRSA have been observed among regions and sub-communities within the same area [13,14]. Therefore, region specific investi- gations of the sources of acquisition are important for informing infection prevention. Case identification Cases were defined as inpatients who screened positive for MRSA nasal carriage upon admission to the hospital. They were identified by reviewing daily reports from the electronic medical record, which captured MRSA screening results and demographic data on all admitted patients to the hospital. p Although there is a growing body of literature on risk factors for MRSA carriage in urban communities [8,15,16], data on predictors of carriage in rural areas remains limited. The eastern section of North Carolina is largely rural [17], and compared to the statewide population, a higher proportion of eastern North Carolina residents are Black and live in poverty–approximately 28% and 20% of eastern North Carolinians, respectively, versus 21% and 17% of North Carolina’s total population [18]. Vidant Medical Center (VMC) is an 861-bed teaching hospital of the Brody School of Medicine at East Carolina University and the tertiary care center for 29 eastern North Carolina counties. Since February of 2007, VMC has screened all patients for MRSA within 24 hours of admission using duplicate swabs of the anterior nares. Since the initiation of this program, all MRSA carriers have been placed on contact isolation, prescribed 5-day courses of mupirocin for application to their nares, and bathed with chlorhexidine soap [19]. Ethics statement The Institutional Review Boards at the University of North Carolina at Chapel Hill and East Carolina University approved this work. Participants provided written informed consent and signed Health Information Portability and Accountability Act authorization forms. Control identification Controls were defined as inpatients who screened negative for MRSA upon admission to the hospital. For every case, an eligible control was identified by reviewing the same daily reports of the electronic medical record that were used to identify cases. When more than one patient was eligible to serve as a control, a random number generator was used to select the control. If that potential control declined to participate or was discharged from the hospital before the interviewer approached them, another control was identified. Because of the above-stated objective of the parent study to investigate the relationship of MRSA carriage with occupational exposures, and because gender and age are strong potential determinants of occupational exposure and case status that could be controlled by design rather than analysis only, controls were matched to cases based on age (65 years) and gender. VMC’s screening program provided the opportunity to investigate risk factors for MRSA carriage among hospitalized patients in eastern North Carolina. The objective of this study was to investigate household exposures, hospitalization and/or MRSA screening history, demographic characteristics, and smoking history as predictors of MRSA nasal carriage in patients who were recently admitted and screened at VMC. To investigate differences in the epidemiology of the genetic subtypes of MRSA, a repetitive sequence-based polymerase chain reaction (rep-PCR) [20] was used to characterize isolates from study participants as CA or HA, and associations between exposures and CA or HA MRSA carriage were investigated. Interviews and medical record review Participant enrollment and structured interviews were conduct- ed with cases and controls from July – December 2011. The interviewer approached eligible cases and controls in their hospital rooms, introduced the study, and invited them to participate in a brief interview. The structured interview included questions about medical and antibiotic use history in the past 12 months for participants and their household member(s), indoor pets, sports participation or gym use in the past 2 weeks, smoking history in the past 12 months, and demographics. One author (L.S.) conducted all interviews. The following information was abstracted from medical records: prior MRSA screening results, surgery, hospitalizations, and antibiotic prescriptions within the past 12 months; and diagnoses listed in the discharge summary for the current hospitalization. If participants reported not taking antibiotics but their medical records showed antibiotic prescriptions, including mupirocin for previous MRSA carriage, the data were coded to reflect the record. If participants reported antibiotic use but their medical records showed no antibiotic prescriptions, participants’ reports were used. Because of the way the question about antibiotic use was asked during the interview, it was not possible to tell if participants self-reported mupirocin use as an antibiotic exposure; therefore, mupirocin prescriptions recorded in the medical chart were included with other antibiotic exposures. One author (L.S) abstracted all medical record data. Participant eligibility Eligible participants were admitted patients at VMC, screened for MRSA within 24 hours of their admission and English or Spanish speakers. The same eligibility criteria were applied to cases and controls. These data arise from a parent study, the objectives of which were to investigate relationships between occupational exposures to livestock and MRSA nasal carriage. To increase the probability of employment, participants were restricted to ages 18–65. To increase the probability of exposure to livestock for the parent study, participants were restricted to residents of swine-producing zip codes in which the permitted number of swine was equal to or greater than the median number listed with the North Carolina Division of Water Quality (North Carolina zip codes with 1,032,750 or more permitted swine). Of 1,083 zip codes in North Carolina [21], 176 were eligible. Statistical analysis Conditional logistic regression models, which adjusted for matching by age and gender, were used to estimate odds ratios (OR) and 95% confidence intervals (CIs). The following variables were examined in a multivariable model: education; self-reported race/ethnicity; visiting a gym or playing sports; tobacco smoking history; indoor cats or dogs; household member presence, hospitalization or antibiotic use; and participant hospitalization and MRSA screening history. These variables were selected a priori, based on the hypothesis that they might be associated with MRSA nasal carriage. Variables were coded to provide the best predication with the fewest degrees of freedom, as indicated by Akaike Information Criterion (AIC) statistics. The variable representing the presence, hospitalization and antibiotic use history of household members was coded as a 3-level categorical variable (0 = participant lived alone, 1 = participant lived with 1 or more household members, but none used antibiotics in the past 4 weeks or were hospitalized in the past 12 months, 2 = participant lived with 1 or more household members who used antibiotics in the past 4 weeks or were hospitalized in the past 12 months). Similarly, history of participant hospitalization and MRSA screening was coded as a 3-level categorical variable (0 = not hospitalized in the past 12 months, 1 = hospitalized but never screened positive for MRSA in the past 12 months, 2 = hospital- ized and screened positive for MRSA at least once in the past 12 months). These variables were coded to create mutually exclusive categories that could be included in the same multivariable model. All the other variables included in the multivariable model were coded dichotomously. Five cases had concomitant MRSA clinical infections— abscesses (n = 2), cellulitis/abscess, pneumonia, and bacteremia (Table S1). Thirteen (11.1%) cases and 3 (3.4%) controls had cellulitis or soft tissue infection diagnoses. Seven (6.0%) cases and 3 (2.5%) controls had diarrhea. Thirty-eight (31.9%) cases and 31 (26.5%) controls had diabetes. Odds ratios were derived from a conditional, multivariable model (Table 2). Cases had a higher odds of having less than a high school degree (OR: 2.04, 95% CI: 0.75–5.50), being a non- white race, having indoor cats or dogs (OR: 1.79; 95% CI: 0.88– 3.63), smoking cigarettes in the past 12 months (OR: 1.60; 95% CI: 0.80–3.19), and visiting a gym or playing sports in the past 2 weeks (OR: 2.64; 95% CI: 0.64–10.88). MRSA and Medical Household Exposures MRSA and Medical Household Exposures DNA was extracted using an UltraCleanTM Microbial DNA Isolation Kit (Mo Bio Laboratories, Solana Beach, CA). The NanoDropH ND-1000 Spectrophotometer (Isogen, Ijssel stein, The Netherlands) was used to estimate the genomic DNA concentrations. Extracts were diluted to give a final DNA concentration of 35 ng/ml. DNA was extracted using an UltraCleanTM Microbial DNA Isolation Kit (Mo Bio Laboratories, Solana Beach, CA). The NanoDropH ND-1000 Spectrophotometer (Isogen, Ijssel stein, The Netherlands) was used to estimate the genomic DNA concentrations. Extracts were diluted to give a final DNA concentration of 35 ng/ml. Because patients were not always available to be interviewed due to rapid turnover or medical treatments and surgery, and because not all eligible patients agreed to participate, at the end of data collection some participants were left without a matching case/control. To avoid double loss of information, in analysis, case and control matched sets were pooled. Pooled sets were created by identifying the appropriate gender/age match who was admitted within the shortest amount of time of the unmatched case/control. The use of the same control for more than one case has been described previously as a valid method that should not bias measures of association [24]. The DiversiLabH Staphylococcus kit for DNA fingerprinting (bioMerieux, Boxtel, Netherlands), a repetitive sequence-based PCR (rep-PCR), was used to amplify regions between repetitive, noncoding sequences in DNA samples. The DiversilabH system is a commercially available strain typing kit that is popular in clinical settings because it is less time consuming compared to pulsed-field gel electrophoresis and, unlike sequence based typing methods such as Staphylococcus Protein A (spa) or multi locus sequence typing, does not require access to DNA sequencing facilities [20]. The protocol was run according to manufacturer specifications. Software from the DiversiLabH system (version v.r.3.3.40) was used for genotyping [20]. DiversiLabH produces a dendogram, a graph of fluorescence intensity that corresponds to banding patterns, and a similarity matrix. The rep-PCR profiles were compared to the DiversiLabH MRSA library, which contains 70 samples of 14 representative USA pulsed field gel electrophoresis types [3]. Fingerprints with no banding differences were consid- ered matches and assigned the library type. Isolates without a match in the library were considered non-typeable, or no match. MRSA isolates were subsequently classified as CA, HA or non- matches. All statistical analyses were performed using Statistical Analysis Software version 9.3. Results Of 164 cases and 190 controls invited, 121 (73.8%) and 122 (64.2%) consented to participate, respectively. Subsequently, 4 cases and 3 controls were excluded based on having addresses outside the eligible zip codes, leaving 117 cases and 119 controls for analysis. One hundred (89.3%) matched sets had 1 case per control. Because cases and controls were pooled to avoid loss of information, 7 sets (6.3%) had 2 controls per case and 5 (4.5%) had 2 cases per control. All participants lived in 22 counties in eastern North Carolina or in the eastern-most portion of central NC. Sixty-eight (57.1%) controls and 67 (57.3%) cases were female (Table 1). Fifty-eight controls (48.7%) and 54 (46.2%) cases identified as non-Hispanic white. In the past 12 months, 94 cases (80.3%) and 93 controls (78.2%) used or were prescribed antibiotics. Cases and controls had similar numbers of household members. MRSA detection and typing VMC’s clinical microbiology laboratory used the BD Gen- OhmH MRSA polymerase chain reaction (PCR) to test nasal swabs for MRSA [22]. The BD GeneOhmH real-time PCR identifies MRSA by targeting the junction of the SCCmec right extremity (SRE) with a section of the orfX gene, which is specific to S. aureus [23]. Swabs were stored at 4uC for 24 – 48 hours, then transferred to the infection control laboratory and streaked onto a CHROMagarH MRSA plate (CHROM agar Microbiology, Paris, France). The CHROMagarH plate was incubated for 24 – 48 hours at 37uC. Mauve colonies were identified as MRSA. August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org 2 Statistical analysis Cases had a higher odds of past hospitalization with a positive MRSA screen (OR: 3.21; 95% CI: 1.12–9.23) but a lower odds of hospitalization without a positive screen in the past 12 months (OR: 0.40; 95% CI: 0.19– 0.87). Additionally, cases had 3.27 times the odds of living with someone who had used antibiotics in the past 4 weeks and/or was hospitalized in the past 12 months (95% CI: 1.24–8.57). Of 117 cases, 108 duplicate swabs were available for culturing and 49 (45.4%) grew MRSA colonies. Using the multivariable model, the 49 culture positive cases were compared to 52 matched controls (Table S2). Because of the small numbers of participants in these analyses, the resulting measures of association were imprecise. Being hospitalized but never screening positive for MRSA was negatively associated with MRSA carriage (OR: 0.29; 95% CI: 0.06–1.39). Living with someone who used antibiotics in the past 4 weeks and/or was hospitalized in the past year (OR: 4.76; 95% CI: 0.88–25.72), having less than a high school degree (OR: 2.65, 95% CI: 0.53–13.24); and having indoor pets (OR: 1.26; 95% CI: 0.40–3.99) was associated with a higher odds of MRSA carriage. Similar proportions of cultured cases and controls had prior hospitalization in addition to prior MRSA carriage (OR: In addition to the analysis of the full data set, cases whose isolates grew MRSA colonies were compared to their matched controls. Also, the relationship between HA or CA MRSA carriage and the explanatory variables was examined. In the analysis of CA and HA carriers, samples sizes were small; therefore, each explanatory variable was investigated in a separate model. PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e73595 August 2013 \| Volume 8 \| Issue 8 \| e73595 3 MRSA and Medical Household Exposures Table 1. Characteristics of methicillin resistant Staphylococcus aureus nasal carriers and their gender and age matched controls. No. (%) Controls Cases (n = 119) (n = 117) Female 68 57.1 67 57.3 Age, y 18–29 29 24.4 24 20.5 30–39 15 12.6 16 13.7 40–49 17 14.3 22 18.8 50–59 40 33.6 37 31.6 60–65 18 15.1 18 15.4 Race or ethnicitya Non-Hispanic White 58 48.7 54 46.2 Non-Hispanic Black 55 46.2 53 45.3 Hispanic or Latino 3 2.5 4 3.4 Other 3 2.5 6 5.1 No. Discussion From the inception of the universal MRSA screening program at VMC in February of 2007 until December of 2011, approximately 7.9% of nearly 225,000 screened patients, exclud- ing the neonatal population, were identified as MRSA nasal carriers by PCR. We compared participants who were hospitalized and screened positive for MRSA on a previous occasion within the past year to participants who were not hospitalized in the past year; MRSA carriage identified by PCR predicted nasal carriage at later hospital admission. VMC prescribes mupirocin to MRSA carriers to decolonize their nares; patients complete the treatment either while they are hospitalized or as outpatients. Of 36 participants who screened positive for MRSA carriage on a previous visit to VMC within the past year, all but 1 were prescribed mupirocin after their last positive MRSA screen. This suggests that MRSA carriers who retested positive were re- colonized in the community. The finding that past MRSA carriage predicted a positive MRSA screen upon readmission could be partially explained by carriage of mupirocin resistant MRSA strains. Based on point prevalence estimates, low- and high-level mupirocin resistance was detected in 3.5% and 6.0% of isolates collected from VMC patients in 2011, respectively (unpublished data). Therefore, it is unlikely that mupirocin resistance fully explains these results. 1.03; 95% CI: 0.25–4.24). Also, lower proportions of culture positive cases than controls were non-Hispanic white (OR: 0.89; 95% CI: 0.26–3.09), smoked cigarettes in the past year (OR: 0.61; 95% CI: 0.14–2.62), and lived with someone who did not use antibiotics and was not hospitalized (OR: 0.60; 95% CI: 0.15– 2.46). Because of sparse data, the effect of gym use or sports participation in the past 2 weeks was not estimable. 1.03; 95% CI: 0.25–4.24). Also, lower proportions of culture positive cases than controls were non-Hispanic white (OR: 0.89; 95% CI: 0.26–3.09), smoked cigarettes in the past year (OR: 0.61; 95% CI: 0.14–2.62), and lived with someone who did not use antibiotics and was not hospitalized (OR: 0.60; 95% CI: 0.15– 2.46). Because of sparse data, the effect of gym use or sports participation in the past 2 weeks was not estimable. Participants who were hospitalized in the past 12 months without a prior positive MRSA screen had lower odds of MRSA carriage than participants who were not hospitalized in the past year. Previous research suggests that past hospitalization is associated with MRSA carriage [15,26,27]. Statistical analysis household members Mean and standard error 3.2 5.5 3.1 1.9 Min and max 0 .10 0 .10 Antibiotic use, past 12 mo.b 93 78.2 94 80.3 Surgery, past 12 mo. 40 33.6 46 39.3 Hospitalized, past 12 mo. 70 58.8 71 60.7 Abbreviations: y, year; mo, month; min, minimum; max, maximum. aRace/ethnicity was self-reported by participants during the interview. The other category includes participants identifying their race or ethnicity as Asian, American Indian, or other. bAny participant who was previously prescribed mupirocin for MRSA nasal carriage within the past year was coded as having taken antimicrobials. doi:10.1371/journal.pone.0073595.t001 CI: 0.14–2.51); were a non-white race (OR: 1.49; 95% CI: 0.32– 6.91; OR: 0.60; 95% CI: 0.14–2.51); and had household members who did not use antibiotics in the past 4 weeks and were not hospitalized in the past year (OR: 1.19; 95% CI: 0.19–7.53 and OR: 0.89; 95% CI:0.18–4.48). Compared to controls, higher proportions of CA and HA carriers had less than a high school degree (OR: 2.56; 95% CI: 0.46–14.29; OR: 1.50; 95% CI: 0.25– 8.98) and smoked cigarettes in the past year (OR: 2.36; 95% CI: 0.44–12.56; OR: 1.67; 95% CI: 0.40–6.97). Higher proportions of CA carriers had household members who used antibiotics in the past 4 weeks and/or were hospitalized in the past 12 months (OR: 3.64; 95% CI: 0.34–39.22). Due to sparse data, this effect was not estimable for HA carriers, and the effect of gym use/sports participation was not estimable for CA or HA carriers. CI: 0.14–2.51); were a non-white race (OR: 1.49; 95% CI: 0.32– 6.91; OR: 0.60; 95% CI: 0.14–2.51); and had household members who did not use antibiotics in the past 4 weeks and were not hospitalized in the past year (OR: 1.19; 95% CI: 0.19–7.53 and OR: 0.89; 95% CI:0.18–4.48). Compared to controls, higher proportions of CA and HA carriers had less than a high school degree (OR: 2.56; 95% CI: 0.46–14.29; OR: 1.50; 95% CI: 0.25– 8.98) and smoked cigarettes in the past year (OR: 2.36; 95% CI: 0.44–12.56; OR: 1.67; 95% CI: 0.40–6.97). Higher proportions of CA carriers had household members who used antibiotics in the past 4 weeks and/or were hospitalized in the past 12 months (OR: 3.64; 95% CI: 0.34–39.22). Due to sparse data, this effect was not estimable for HA carriers, and the effect of gym use/sports participation was not estimable for CA or HA carriers. Discussion Patients previously hospitalized with a negative MRSA PCR on admission may be less susceptible or less exposed in the community. August 2013 \| Volume 8 \| Issue 8 \| e73595 Molecular typing Patterns of MRSA carriage are commonly classified as persistent, intermittent, and absent. Previous research has shown that a high percentage of patients who initially screen negative for MRSA return to the hospital with negative screens on future visits, whereas high proportions of those who screen positive on an initial visit show patterns of persistent or intermittent carriage [28]. Although results should be interpreted tentatively since most of the measures of association were imprecise due to sparse data, results from our study contribute to evidence that the household environment may affect MRSA acquisition [7,8,29,30] and could play a role in intermittent or persistent carriage. Compared to living alone, living with household members who either used antibiotics or were hospitalized was positively associated with MRSA carriage. It is possible that household members acquired MRSA through past medical related exposures and then exposed MRSA carriers in this study. Of 49 isolates typed via rep-PCR, 7 (14.3%) did not match types in the DiversiLabH library. Twenty (40.8%) isolates matched the CA strain, USA300. The other 22 isolates matched HA strains; 15 (30.6%) were USA100, 2 (4.1%) were USA500, 3 (6.1%) were USA800, 1 (2.0%) was USA200, and 1 (2.0%) was USA600. None of the non-typeable strains had a pattern that matched the livestock associated multi locus sequence type 398 strain in the DiversiLabH typing system [25]. CA and HA carriers were compared with controls using conditional logistic regression models (Table S3). Because there were only 20 CA isolates and 22 HA isolates in these analyses, the resulting OR estimates were imprecise. However, a higher proportion of CA carriers but a lower proportion of HA carriers were previously hospitalized with a past positive MRSA screen (OR: 7.67; 95% CI: 0.86–68.38; OR: 0.54; 95% CI: 0.10–2.90); had indoor pets (OR: 1.93; 95% CI: 0.49–7.58; OR: 0.60; 95% August 2013 \| Volume 8 \| Issue 8 \| e73595 August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org 4 MRSA and Medical Household Exposures Table 2. Estimates of association of methicillin resistant Staphylococcus aureus (MRSA) nasal carriage identified by polymerase chain reaction with medical and household exposures from a multivariable logistic model conditioned on age and gender. No. Molecular typing Estimates of association of methicillin resistant Staphylococcus aureus (MRSA) nasal carriage identified by polymerase chain reaction with medical and household exposures from a multivariable logistic model conditioned on age and gender. No household members No household members Household members used antibiotics in the past 4 weeks and/or hospitalized in the past 12 mo. 25 21 breviation: months, mo.; odds ratio, OR; confidence interval, CI. , ; , ; , Non-white or Hispanic includes non-Hispanic black, Hispanic/Latino, Asian, American Indian, or other race/ethnicities. The gym visitation/sports participation variable reflects the 2 weeks prior to the hospital admission, even though 2 cases and 1 control were screened for MRSA 9 or more days before their current hospital admission. E d i h d l 3 l l i l i bl , ; , ; , aNon-white or Hispanic includes non-Hispanic black, Hispanic/Latino, Asian, American Indian, or other race/ethnicities. bThe gym visitation/sports participation variable reflects the 2 weeks prior to the hospital admission, even though 2 cases and 1 control were screened for MRSA 9 or more days before their current hospital admission. aNon-white or Hispanic includes non-Hispanic black, Hispanic/Latino, Asian, American Indian, or other race/ethnicities. bThe gym visitation/sports participation variable reflects the 2 weeks prior to the hospital admission, even though 2 cases more days before their current hospital admission. Entered into the model as a 3-level categorical variable. oi:10.1371/journal.pone.0073595.t002 doi:10.1371/journal.pone.0073595.t002 methodology has been shown to be more sensitive than selective media, which might account for some of the higher detection [22]. However, these data could also raise concerns about false positives among the cases identified by PCR, which might have detected methicillin sensitive S. aureus containing SCCmec fragments that lack the mecA gene, non-viable bacteria, or DNA residue that remained following decolonization [23,32,33,34]. The percentage of PCR positive swabs that did not grow colonies was similar to that reported in some but not all studies. For example, in a neonatal intensive care unit, MRSA was detected by PCR in 21 of 435 (4.8%) nasal swabs, and 11 (52.4%) of the PCR positive swabs grew MRSA colonies when cultured [35]. In another study, 123 of 599 nasal swabs were positive by PCR, of which 77 (62.6%) were positive by culture without enrichment [36]. Molecular typing In a third study, 606 nose, throat, and groin/perineum swabs were collected from 202 patients; 120 MRSA specimens were detected by PCR, of which 85 (70.8%) were detected by direct culture [33]. In our study, the delay from the time when the duplicate swabs from PCR positives were transported and plated onto selective media varied from 24 – 48 hours, which could have reduced subsequent culture yields. Additionally, a higher proportion of cases might have grown colonies on selective media had overnight enrichment been used, which has been shown to improve the sensitivity of cultures [37]. Of 49 culture positive isolates, 22 (44.9%) matched HA types, 20 (40.8%) matched CA types and 7 (14.3%) did not have a match in the DiversiLabH MRSA library. To explore potential differ- ences in the epidemiology of HA versus CA MRSA, we examined associations between carriage of each MRSA type and the exposure variables. Because of small numbers of participants in these analyses, the measures of association were imprecise. However, a higher proportion of CA carriers than controls were hospitalized and screened positive for MRSA previously. Other studies have reported that CA strains may be carried by those with a history of hospitalization [5]. Based on small numbers, the proportion of culture positive cases carrying CA strains was high compared to that reported by others [5,15]. The high proportion of cases carrying CA MRSA in our study could be attributable to the exclusion of patients over age 65, other unique characteristics of the study population, or the relatively low yield (45%) of cultures grown and typed from MRSA nasal carriers identified by PCR. methodology has been shown to be more sensitive than selective media, which might account for some of the higher detection [22]. However, these data could also raise concerns about false positives among the cases identified by PCR, which might have detected methicillin sensitive S. aureus containing SCCmec fragments that lack the mecA gene, non-viable bacteria, or DNA residue that remained following decolonization [23,32,33,34]. The percentage of PCR positive swabs that did not grow colonies was similar to that reported in some but not all studies. For example, in a neonatal intensive care unit, MRSA was detected by PCR in 21 of 435 (4.8%) nasal swabs, and 11 (52.4%) of the PCR positive swabs grew MRSA colonies when cultured [35]. Molecular typing ( %) Controls Cases OR 95% CI (n = 119) (n = 117) At least high school or general education development degree 106 89.1 93 79.5 1.00 - Less than high school or general education development degree 13 10.9 24 20.5 2.04 0.75–5.50 No cats or dogs inside the home 79 66.4 72 61.5 1.00 - Cats or dogs inside the home 40 33.6 45 38.5 1.79 0.88–3.63 Non-Hispanic white race or ethnicitya 58 48.7 54 46.2 1.00 - Hispanic and/or non-white race or ethnicity 61 51.3 63 53.9 1.48 0.70–3.11 Did not smoke tobacco cigarettes in the past 12 mo. 76 63.9 65 55.6 1.00 - Smoked tobacco cigarettes in the past 12 mo. 43 36.1 52 44.4 1.60 0.80–3.19 Did not visit a gym or participate in sports in the past 2 weeksb 114 95.8 108 92.3 1.00 - Visited a gym or participated in sports in the past 2 weeks 5 4.2 9 7.7 2.64 0.64–10.88 Prior hospitalization and MRSA nasal carriage in the past 12 mo.c Not hospitalized in the past 12 mo. 49 41.2 46 39.3 1.00 - Hospitalized and never screened positive for MRSA in the past 12 mo. 64 53.8 41 35.0 0.40 0.19–0.87 Hospitalized and screened positive for MRSA at least once in the past 12 mo. 6 5.0 30 25.6 3.21 1.12–9.23 Household members c No household members 23 19.3 15 12.8 1.00 - Household members did not use antibiotics in the past 4 weeks and not hospitalized in the past 12 mo. 71 59.7 58 49.6 1.42 0.59–3.45 Household members used antibiotics in the past 4 weeks and/or hospitalized in the past 12 mo. 25 21.0 44 37.6 3.27 1.24–8.57 Abbreviation: months, mo.; odds ratio, OR; confidence interval, CI. aNon-white or Hispanic includes non-Hispanic black, Hispanic/Latino, Asian, American Indian, or other race/ethnicities. bThe gym visitation/sports participation variable reflects the 2 weeks prior to the hospital admission, even though 2 cases and 1 control were screened for MRSA 9 or more days before their current hospital admission. cEntered into the model as a 3-level categorical variable. Table 2. Estimates of association of methicillin resistant Staphylococcus aureus (MRSA) nasal carriage identified by polymerase chain reaction with medical and household exposures from a multivariable logistic model conditioned on age and gender. Table 2. References 1. Deurenberg RH, Stobberingh EE (2008) The evolution of Staphylococcus aureus. Infect Genet Evol 8: 747–763. 12. Casey JA, Cosgrove SE, Stewart WF, Pollak J, Schwartz BS (2013) A population-based study of the epidemiology and clinical features of methicil- lin-resistant Staphylococcus aureus infection in Pennsylvania, 2001–2010. Epidemiol Infect 141: 1166–1179. 1. Deurenberg RH, Stobberingh EE (2008) The evolution of Staphylococcus aureus. Infect Genet Evol 8: 747–763. 2. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, et al. (1998) Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 279: 593–598. 2. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, et al. (1998) Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 279: 593–598. 13. Bhattacharya D, Carleton H, Tsai CJ, Baron EJ, Perdreau-Remington F (2007) Differences in clinical and molecular characteristics of skin and soft tissue methicillin-resistant Staphylococcus aureus isolates between two hospitals in Northern California. J Clin Microbiol 45: 1798–1803. 3. McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, et al. (2003) Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococ- cus aureus isolates from the United States: establishing a national database. J Clin Microbiol 41: 5113–5120. 14. Stenhem M, Ortqvist A, Ringberg H, Larsson L, Olsson-Liljequist B, et al. (2006) Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Sweden 2000–2003, increasing incidence and regional differences. BMC Infect Dis 6: 30. 4. Seybold U, Kourbatova EV, Johnson JG, Halvosa SJ, Wang YF, et al. (2006) Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care-associated blood stream infections. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 42: 647–656. 15. Hidron AI, Kourbatova EV, Halvosa JS, Terrell BJ, McDougal LK, et al. (2005) Risk factors for colonization with methicillin-resistant Staphylococcus aureus (MRSA) in patients admitted to an urban hospital: emergence of community- associated MRSA nasal carriage. Clin Infect Dis 41: 159–166. 5. Peterson AE, Davis MF, Julian KG, Awantang G, Greene WH, et al. (2012) Molecular and Phenotypic Characteristics of Healthcare- and Community- Associated Methicillin-Resistant Staphylococcus aureus at a Rural Hospital. PLoS One 7: e38354. associated MRSA nasal carriage. Clin Infect Dis 41: 159–166. 16. Popovich KJ, Smith KY, Khawcharoenporn T, Thurlow CJ, Lough J, et al. (2012) Community-Associated Methicillin-Resistant Staphylococcus aureus Colonization in High-Risk Groups of HIV-Infected Patients. Clin Infect Dis 54: 1296–1303. 6. Author Contributions Conceived and designed the experiments: LS SW PDMM DR JS KMR DLN. Performed the experiments: LS KLA. Analyzed the data: LS KLA. Contributed reagents/materials/analysis tools: KMR LS. Wrote the paper: LS SW PDMM DR JS KLA DLN KMR. Analysis interpretation: DR JS SW LS KLA KMR. Conceived and designed the experiments: LS SW PDMM DR JS KMR DLN. Performed the experiments: LS KLA. Analyzed the data: LS KLA. Contributed reagents/materials/analysis tools: KMR LS. Wrote the paper: LS SW PDMM DR JS KLA DLN KMR. Analysis interpretation: DR JS SW LS KLA KMR. Conceived and designed the experiments: LS SW PDMM DR JS KMR DLN. Performed the experiments: LS KLA. Analyzed the data: LS KLA. Contributed reagents/materials/analysis tools: KMR LS. Wrote the paper: Acknowledgments We thank Ruth Ethridge (Vidant Medical Center Clinical Microbiology Department), who helped collect duplicate nasal swabs for culturing and typing. We thank Meghan Davis, Amy Peterson, Ellen Silbergeld, and Christopher Heaney for their recommendations during the preliminary planning and designing of this study. In addition, we are grateful to Devon Hall, Dothula Baron, Jennifer Hall, Clifton Smith, and Norma Mejia for their advice when we designed the structured interview questions. Community and household exposures may influence MRSA carriage and VMC’s hospital-based screening program may be detecting carriage among those exposed to MRSA. Further study of the relationship between MRSA carriage and community exposures, and continued surveillance for strains entering hospitals will help elucidate the dynamic epidemiology of MRSA. Molecular typing Therefore, results from this study might not be generalizable to members of other North Carolina communities, or to non-hospitalized populations. Because VMC only screens patients’ anterior nares, people with MRSA on other regions of the body could have been misidentified as controls. Additionally, although cases could have been carrying multiple MRSA strains [38], DNA of only 1 bacterial colony per culture-positive swabs was extracted and typed. Finally, partici- pants’ knowledge of their screening results could have influenced their interview responses. Table S2 Estimates of association of methicillin resis- tant Staphylococcus aureus (MRSA) nasal carriage identified by culture with medical and household exposures from a multivariable logistic model condi- tioned on age and gender. Table S3 Estimates of association of health care and community associated methicillin resistant Staphylococ- cus aureus (MRSA) nasal carriage with medical and household exposures from conditional logistic regres- sion models. This study has several strengths. One author (L.S.) conducted all interviews and abstracted data from medical records, which helped maintain internal consistency. By conducting in- hospital interviews, we collected information that would be unavailable by record review alone. By using information in patients’ medical records, the quality of the interview data was improved. Overall, there were small amounts of missing data and participation rates were high. Using the rapid PCR testing methodology to screen patients for MRSA allowed the interviewer to identify and approach eligible patients soon after they were admitted [22]. (DOCX) Molecular typing In another study, 123 of 599 nasal swabs were positive by PCR, of which 77 (62.6%) were positive by culture without enrichment [36]. In a third study, 606 nose, throat, and groin/perineum swabs were collected from 202 patients; 120 MRSA specimens were detected by PCR, of which 85 (70.8%) were detected by direct culture [33]. In our study, the delay from the time when the duplicate swabs from PCR positives were transported and plated onto selective media varied from 24 – 48 hours, which could have reduced subsequent culture yields. Additionally, a higher proportion of cases might have grown colonies on selective media had overnight enrichment been used, which has been shown to improve the sensitivity of cultures [37]. This study was non-randomized. In recent decades, emphasis on p-values and statistical tests of significance has been strongly discouraged in non-randomized study for several reasons [31]. Therefore, we have interpreted ORs based on their magnitude and biological plausibility, using 95% CIs as measures of precision rather than tests of significance. The 236 participants of this study were residents of 22 North Carolina counties. Based on United States Census data, percent- ages of Black residents in these counties ranged from 15% to 62% [18]. By comparison, 21% of all North Carolina residents are The 236 participants of this study were residents of 22 North Carolina counties. Based on United States Census data, percent- ages of Black residents in these counties ranged from 15% to 62% [18]. By comparison, 21% of all North Carolina residents are These results must be interpreted cautiously. The BD GenOhmH real-time PCR identified the 117 cases. After subculturing on selective media, MRSA was recovered from 49 of 108 available duplicate swabs of the anterior nares. The PCR August 2013 \| Volume 8 \| Issue 8 \| e73595 August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org 5 MRSA and Medical Household Exposures discharge summaries of methicillin resistant Staphylo- coccus aureus (MRSA) nasal carriers and controls. (DOCX) Black [18]. Therefore, results from this study might not be generalizable to members of other North Carolina communities, or to non-hospitalized populations. Black [18]. Therefore, results from this study might not be generalizable to members of other North Carolina communities, or to non-hospitalized populations. discharge summaries of methicillin resistant Staphylo- coccus aureus (MRSA) nasal carriers and controls. (DOCX) Black [18]. MRSA and Medical Household Exposures 23. French GL (2009) Methods for screening for methicillin-resistant Staphylococcus aureus carriage. Clin Microbiol Infect 15 Suppl 7: 10–16. 32. Huletsky A, Giroux R, Rossbach V, Gagnon M, Vaillancourt M, et al. (2004) New real-time PCR assay for rapid detection of methicillin-resistant Staphylo- coccus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol 42: 1875–1884. 24. Wacholder S, Silverman DT, McLaughlin JK, Mandel JS (1992) Selection of controls in case-control studies. III. Design options. Am J Epidemiol 135:1042– 50. 33. Rossney AS, Herra CM, Fitzgibbon MM, Morgan PM, Lawrence MJ, et al. (2007) Evaluation of the IDI-MRSA assay on the SmartCycler real-time PCR platform for rapid detection of MRSA from screening specimens. Eur J Clin Microbiol Infect Dis 26: 459–466. 25. Grisold AJ, Zarfel G, Hoenigl M, Krziwanek K, Feierl G, et al. (2010) Occurrence and genotyping using automated repetitive-sequence-based PCR of methicillin-resistant Staphylococcus aureus ST398 in Southeast Austria. Diagnostic Microbiology and Infectious Disease 66: 217–221. 34. Lucke K, Hombach M, Hug M, Pfyffer GE (2010) Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) in diverse clinical specimens by the BD GeneOhm MRSA assay and comparison with culture. J Clin Microbiol 48: 981–984. 26. Muto CA, Jernigan JA, Ostrowsky BE, Richet HM, Jarvis WR, et al. (2003) SHEA guideline for preventing nosocomial transmission of multidrug-resistant strains of Staphylococcus aureus and enterococcus. Infect Control Hosp Epidemiol 24: 362–386. 35. Sarda V, Molloy A, Kadkol S, Janda WM, Hershow R, et al. (2009) Active surveillance for methicillin-resistant Staphylococcus aureus in the neonatal intensive care unit. Infect Control Hosp Epidemiol 30: 854–860. 27. Gorwitz RJ, Kruszon-Moran D, McAllister SK, McQuillan G, McDougal LK, et al. (2008) Changes in the prevalence of nasal colonization with Staphylococcus aureus in the United States, 2001-2004. J Infect Dis 197: 1226–1234. 36. Farley JE, Stamper PD, Ross T, Cai M, Speser S, et al. (2008) Comparison of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture by use of BBL CHROMagar MRSA for detection of MRSA in nasal surveillance cultures from an at-risk community population. J Clin Microbiol 46: 743–746. 28. Gupta K, Martinello RA, Young M, Strymish J, Cho K, et al. (2013) MRSA nasal carriage patterns and the subsequent risk of conversion between patterns, infection, and death. PLoS One 8: e53674. , 29. References Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, et al. (2007) Invasive methicillin-resistant Staphylococcus aureus infections in the United States. Jama 298: 1763–1771. 17. Wing S, Cole D, Grant G (2000) Environmental injustice in North Carolina’s hog industry. Environ Health Perspect 108: 225–231. 7. Davis MF, Iverson SA, Baron P, Vasse A, Silbergeld EK, et al. (2012) Household transmission of meticillin-resistant Staphylococcus aureus and other staphylo- cocci. Lancet Infect Dis 12: 703–716. 18. The North Carolina Rural Center (2012) Rural Data Bank Custom Data Search. Available: http://www.ncruralcenter.org/Accessed March 2013. 19. Pofahl WE, Goettler CE, Ramsey KM, Cochran MK, Nobles DL, et al. (2009) Active Surveillance Screening of MRSA and Eradication of the Carrier State Decreases Surgical-Site Infections Caused by MRSA. J Am Coll Surg 208: 981– 988. 8. Uhlemann AC, Knox J, Miller M, Hafer C, Vasquez G, et al. (2011) The environment as an unrecognized reservoir for community-associated methicillin resistant Staphylococcus aureus USA300: a case-control study. PLoS One 6: e22407. 20. Tenover FC, Gay EA, Frye S, Eells SJ, Healy M, et al. (2009) Comparison of typing results obtained for methicillin-resistant Staphylococcus aureus isolates with the DiversiLab system and pulsed-field gel electrophoresis. J Clin Microbiol 47: 2452–2457. 9. Ferreira JP, Anderson KL, Correa MT, Lyman R, Ruffin F, et al. (2011) Transmission of MRSA between companion animals and infected human patients presenting to outpatient medical care facilities. PLoS One 6: e26978. 10. Weese JS, Dick H, Willey BM, McGeer A, Kreiswirth BN, et al. (2006) Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household. Vet Microbiol 115: 148–155. 21. Statistical Analysis Software (2012) Maps Online Zipcode Dataset. January 2012 ed. Available: http://support.sas.com/rnd/datavisualization/mapsonline/html/ misc2012.html. Accessed 2012 January. 22. Boyce JM, Havill NL (2008) Comparison of BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR versus the CHROMagar MRSA assay for screening patients for the presence of MRSA strains. J Clin Microbiol 46: 350– 351. 11. Cohen PR (2008) The skin in the gym: a comprehensive review of the cutaneous manifestations of community-acquired methicillin-resistant Staphylococcus aureus infection in athletes. Clin Dermatol 26: 16–26. August 2013 \| Volume 8 \| Issue 8 \| e73595 August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org 6 MRSA and Medical Household Exposures Miller LG, Diep BA (2008) Clinical practice: colonization, fomites, and virulence: rethinking the pathogenesis of community-associated methicillin- resistant Staphylococcus aureus infection. Clin Infect Dis 46: 752–760. 37. Van Heirstraeten L, Cortinas Abrahantes J, Lammens C, Lee A, Harbarth S, et al. (2009) Impact of a short period of pre-enrichment on detection and bacterial loads of methicillin-resistant Staphylococcus aureus from screening specimens. J Clin Microbiol 47: 3326–3328. p y 30. Lautenbach E, Tolomeo P, Nachamkin I, Hu B, Zaoutis TE (2010) The impact of household transmission on duration of outpatient colonization with methicillin-resistant Staphylococcus aureus. Epidemiol Infect 138: 683–685. 38. Mongkolrattanothai K, Gray BM, Mankin P, Stanfill AB, Pearl RH, et al. (2011) Simultaneous carriage of multiple genotypes of Staphylococcus aureus in children. J Med Microbiol 60: 317–322. 31. Greenland S (1990) Randomization, statistics, and causal inference. Epidemi- ology 1: 421–429. August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 7 7
https://openalex.org/W2275660181	https://ejournal.undip.ac.id/index.php/medianers/article/download/10187/8105	English	null	Prevention Strategies for HIV in Youth: A Concept Analysis	Nurse Media : Journal of Nursing/Nurse Media Journal of Nursing	2,015	cc-by-sa	4,435	1 Lecture at Sint Carolus School of Health Sciences, JL.Salemba Raya No.41, Jakarta Pusat 10440. Email: sondangrsianturi@gmail.com Prevention Strategies for HIV in Youth: A Concept Analysis Sondang Ratnauli Sianturi1 Nurse Media Journal of Nursing, 5 (1), 2015, 15 - 23 Available Online at http://ejournal.undip.ac.id/index.php/medianers Nurse Media Journal of Nursing, 5 (1), 2015, 15 - 23 Available Online at http://ejournal.undip.ac.id/index.php/medianers BACKGROUND HIV/AIDS is a disease that causes negative impact on all aspects of life, especially in young people. Young people are important to society as they are the future and are needed to assist in the development of Indonesia. Today, many young people in Indonesia have been diagnosed with HIV/AIDS. This rising number of young people contracting HIV/AIDS is due to many factors, such as the lack of knowledge and stigma about the disease itself, the discrimination of people suffering from this disease, free association with sexual practices, and the lack of attention from their family. Indonesia has many people who are living with HIV/AIDS, especially in Jakarta. Statistics from the Ministry of Health Republic of Indonesia (2014) notes that Jakarta is the city with the most people living with HIV/AIDS with 69,1 % of the people identified as living with the disease.. In addition, 577 have died from HIV/AIDS in 2011. Cumulative AIDS cases are 13,109 cases in young people between ages 15 – 29 years old. Children & Youth Unit, the World Bank (2006) stated that the numbers speak for themselves. Everyday 5000 to 6000 young people’s age 15-24 contracts HIV. With youth at the heart of the pandemic, HIV prevention must focus on young people. The Millennium Development Goal stated that increasing the knowledge of 15-24 year olds with HIV/AIDS is the primary goal and indicator of health. Although today’s youth are much more informed about HIV/AIDS than previous generations, even in countries with a general pandemic, more than 80% of young women still do not have “sufficient understanding” about how to avoid HIV ( World Bank, 2006). In addition, there are problems with the prevention of HIV among young people because there is still a high stigma and discrimination related to HIV/AIDS. Sometimes, information about HIV is not appropriately delivered to the youth. The current method of education is giving some information about health education, sex education and gender issues. The young people in Indonesia have little information from doctors or nurses who visit schools. There is also limited teaching about sex education, STDs, and HIV/AIDS. In addition, there is a lack of curriculum that includes sex education and reproductive health in young children. Today, the youth of Indonesia are increasingly being influenced to try something new due to exposure to technology such as the internet, Facebook, Twitter, and other social networking. ABSTRACT Purpose: To provide nurse educators how to address the problem of HIV/AIDS in the youth through the concept of prevention strategies Methods: Review articles published during 2005 until 2014 that focused on HIV/AIDS prevention strategies. The databases used in this study were EBSCO, Medline, CINAHL, PubMed. Result: HIV knowledge is an important factor influencing HIV risk perceptions and risk behaviors especially among youth.The concept of prevention strategies designed and tested to address risk behavior on young people at every step along their developmental path. Strategies are very important ways for preventing HIV/AIDS, especially in youth. Conclusion: The attributes that characterize the concept of strategies are: plan, process, studying different ways, focusing on the greatest need, and analyzing and learning. Although strategies are used in prevention, they are not maximized in Indonesia. The best strategies for the different age groups have not yet been determined. With use of the best strategies, nurse educator can teach nursing students how best to reach youth and other people in the community to prevent HIV. Result: HIV knowledge is an important factor influencing HIV risk perceptions and risk behaviors especially among youth.The concept of prevention strategies designed and tested to address risk behavior on young people at every step along their developmental path. Strategies are very important ways for preventing HIV/AIDS, especially in youth. p g y p y p g p y y Conclusion: The attributes that characterize the concept of strategies are: plan, process, studying different ways, focusing on the greatest need, and analyzing and learning. Although strategies are used in prevention, they are not maximized in Indonesia. The best strategies for the different age groups have not yet been determined. With use of the best strategies, nurse educator can teach nursing students how best to reach youth and other people in the community to prevent HIV. Keywords: HIV/AIDS, Prevention, Youth, Strategies Copyright © 2015, NMJN, p-ISSN 2087-7811, e-ISSN 2406-8799 16 Nurse Media Journal of Nursing, 5 (1), 2015, 16 Nurse Media Journal of Nursing, 5 (1), 2015, BACKGROUND Youth are much more sexually active at a younger age. There is also a rising use in tobacco, alcohol, and other drugs. Therefore, there is a need for appropriate prevention strategies for young children in the education and knowledge of HIV/AIDS. This can lead to a positive impact for young people, so that the incidence and prevalence of HIV /AIDS on young children can be reduced. There is a need for strategies that can approach the various parties in contact with young people and HIV/AIDS. This means approaches to HIV/AIDS prevention must come from individual, peer, family, school, and community programs. Schools and families can be important partners for reaching youth before high risk behaviors are established, because school is a primary level for youth social development and family is a place that provides structure and guidance to youths about sexuality, because family has the social power to teach youth socially acceptable behavior. Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87 17 Nurse Media Journal of Nursing, 5 (1), 2015, 17 Nurse Media Journal of Nursing, 5 (1), 2015, 17 Nurse Media Journal of Nursing, 5 (1), 2015, The analysis of concepts is a longstanding issue, both in philosophy and nursing. Concepts are called “the building blocks of theory” (Walker & Avant, 2005). The Walker and Avant Method of concept analysis will be used to perform this concept analysis paper. Concept analysis is a process that identifies unique attributes of a concept, provide precise operational definitions of the concept, and improve communication regarding the concept (Walker & Avant, 2005). As nurse educators, it is important to teach health in different places, one of which is in the school. Programs run by a nurse educator at a school, teach children prevention and promotion of health. Prevention and promotion strategies are used to improve community health status. By providing education to children in school, the younger generation of Indonesia can be healthier and contribute to the future development of the country. The concept of “Prevention Strategies for HIV in Youth” was chosen for this concept analysis because there is a need to explore and define what this concept is, so it can be applied to the rising rates of HIV/AIDS in the young people in Indonesia. For this concept analysis the focus of the literature review is the word “strategy”.The long range goal of this work is to provide information, raise awareness, and encourage the development of safe behavior to minimize the risk of infection, change in perception among youth about HIV/AIDS, and develop better prevention strategies for youth. Finally, if these goals can be reached, there will be better solutions to the current problems associated with HIV/AIDS in Indonesia. METHODS The researcher search materials and data by doing the literature review based on journals from EBSCO, Medline, CINAHL and Pubmed with 19 articles. These articles were published during 2005 until 2014 about HIV/AIDS prevention and HIV/AIDS strategies in youth. Strategy in Education gy A strategic imperative in low-income countries is a more explicit focus on the issue of dropouts, repetition, and completion rates. This relates directly to upgrading the quality and the relevance of the basic education delivery system, which in turn depends on the quality of teaching staff, the pedagogies used, and the participation and involvement of local communities. The most obvious strategic area of focus is expanding the capacity of education systems to widen access toward universal primary education. Included would be identification of optimum areas for additional classrooms and physical facilities, expansion of budgets for additional teachers, and provision of textbooks and other learning materials. (Asian Development Bank, 2008). Prakash Nair (2008) defines the strategy in education that Civitas Academica in school has their own strategy in teaching students. The teacher must find different ways to teach the student. Additionally, some strategies are more relevant to younger students, while others apply only to older students. Few innovation strategies can succeed without real leadership. However, in the world of education, good leadership at the top alone is not enough to improve student achievement. Strategy in Mathematics The theory called “Games of Strategy” may be described as a mathematical theory of decision-making by participants, in a competitive environment. A game of strategy is described by its set of rules. These rules specify clearly what each person called a “player” is allowed or required to do under all possible circumstances. Dresher (1961) defines strategy as a concept where a person makes a decision at every move. Each player formulates in advance of the play, a plan for executing the game from beginning to end. A player using a strategy loses no freedom of action since the strategy specifies the player’s actions in terms of the information that might become available. Every pair of strategies, consisting of one strategy for each player, determines a play of the game to consist of each player’s making one decision, namely the selection of a strategy (Dresher, 1961). Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87 18 Nurse Media Journal of Nursing, 5 (1), 2015, 18 Nurse Media Journal of Nursing, 5 (1), 2015, Strategy in Nursing There are many variations to Wilson’s method (Avant, 2000) in nursing. An example is analysis of pain management. Pain management is accepted in nursing as an integral component of the nurses’ mission in providing nursing care to clients. Davis (1992) used a concept analysis strategy to identify the role of patient involvement in managing their own pain. As a process that nurses experience frequently, understanding the properties and outcomes of interruption could lead to an effective program of research related to its outcomes on nurses and patients (Brixey, Robinson, Johnson, et al., 2007). This strategy is based on collaboration, critical thinking, and expertise of participants, complementarity, mutual trust building, and mutual consensus building (Meleis, 2007). O’Neill (2000) said that strategy is the importance of bringing together the ‘hard’ elements (structure, rewards) of strategic management and the ‘soft’ elements of organizational development (processes, culture, interpersonal relationship) is relevant to nurses today as they attempt to grapple with the need for commitment to professional ‘hands on’ caring expected by the public and the pressure to be more politically aware and involved in management. Strategy in Management USAID (2005) defines the heart of the strategy as basic education: facilitating the acquisition of basic skills, such as literacy, numeracy, and critical thinking that enable people and nations to thrive in a changing economic environment. USAID’s strategies emphasize the following strategic management principles are focused on countries with the greatest need and commitment, enhance host-country efficiency; emphasize project sustainability. Mc. Donald reports that strategic management is a process through which organizations analyze and learn from their internal and external environments, establish strategic direction, create strategies that are intended to move the organization in that direction, and implement those strategies, all in an effort to satisfy key stakeholders. Larger companies tend to use the strategic management process, this process is also a vital part of decision making in smaller companies. The strategic planning process tends to be a rather rigid and unimaginative process in many organizations. In other words, strategy should be both deliberate and emergent, and firms should both adapt to and enact with their environments. The situation determines which option to choose. In recent years, another perspective on strategy development has gained wide acceptance. A strategy can be thought of in either of two ways, as a pattern that emerges in a sequence of decisions over time or as an organizational plan of action that is intended to move a company toward the achievement of its shorter-term goals and, ultimately, its fundamental purposes. In some organizations, particularly those in rapidly changing environments and in small businesses, strategies are not planned in the formal sense of the word. In those cases, the strategy reflects the insight and intuition of the strategies or business owner, and it becomes clear over time as a pattern in a stream of decisions. Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87 19 Nurse Media Journal of Nursing, 5 (1), 2015, 19 Nurse Media Journal of Nursing, 5 (1), 2015, 20 Nurse Media Journal of Nursing, 5 (1), 2015, The third attribute is “focusing on the greatest need”. In developing a strategy there is a significant focus on where the greatest need is or where resources can be used to have the best outcome. Every event or activity has a goal, and the goal is to focus on where there is the greatest need. The fourth attribute is “analyzing and learning”. When working in a strategic manner it is important to always be analyzing and learning from what is being studied and implemented. This is one of the only ways in which strategies can be improved. This attribute can be actualized when the program is being formed, when it is implemented and when it has been evaluated. Analyzing and learning is important in evaluation in order to effectively assess the impact of youth programs on the change in HIV- prevention behaviors. The last attribute is a “process”. Strategy development is an ongoing process. King (2007) said analysis of collaborative learning interactions have demonstrated that what happens on the process level is important for predicting what cognitive benefits participants in a conversation take away from it. Those attributes can help understand better what is meant by the concept “strategies”. By employing better strategies in the prevention of HIV/AIDS young people will hopefully make more responsible choices about sexual behavior and practices. Working with communities, teachers, school councils, parent-teacher associations and learners themselves to assess, plan, implement, monitor, and evaluate education interventions will help improve program quality and ensure cultural sensitivity. This is an example of the concept. A nursing student is in her “community health clinical studies” assignment. The first step is “community assessment”. In this step the student must try to approach the people in that community. The assessment result is “The people’s need more information on HIV, particularly on young people”. They need more information about HIV because the behavior of young people in that community is at high risk for developing HIV. This request was forwarded by the student to the faculty. After receiving the faculty’s approval, the students create a plan about educational activities for the young people in the targeted community. At the first stage, the student hold a meeting with the local government to receive some information about HIV cases and condition related to it, and also discuss about the convenience date of the proposed activity to be held. DISCUSSION Researchers found that the characteristics relevant to strategy are integrated in many disciplines, including nursing, games, and management. The attributes identified from the literature review are: plan, process, studying different ways, focus on greatest need, along with analyzing and learning. Therefore, primary prevention strategies require a plan with an identified process based on the study of different ways to focus on the greatest needs with a continuous analyzing and learning of how to best address HIV/AIDS. The first attribute on this concept is “plan”. A plan is an important ingredient in any strategy. Without a plan efforts can be scattered and there is not a clear direction on what comes first, second and third. A plan is a scheme or method of acting, doing, proceeding, making, or advancing (dictionary, 2011). The plan must be effective, written, and clear, based on the real current situation. There also must be sufficient time for implementation and evaluation of a plan before moving onto another strategy. It should not be rushed. Rushing the plan will cause problems. The plan is used to guide policy, planning, and design decisions during implementation (Litman, 2011). The second attribute on this concept is “studying different ways”. In developing a strategy it is important to identify all the different ways in which the goal can be achieved. At the same time it is important to understand what is successful in different settings for different age groups and people. Young people think in accordance with the needs, interest, expectations, motives, and psychology of youth. Young people can learn in different ways. It is important to stimulate the various parties to provide information in different ways. Studying different ways refers to the concept that individuals differ in regard to what mode of instruction or study is most effective for them, so it means that different people learn information in different ways (Pashler, McDaniel, Rohrer, and Bjork, 2011). Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87 20 Nurse Media Journal of Nursing, 5 (1), 2015, 20 Nurse Media Journal of Nursing, 5 (1), 2015, 21 Nurse Media Journal of Nursing, 5 (1), 2015, ongoing education of students in the community is a process and more and different ideas will need to be presented so that students can gain a clear understanding of the problem of HIV/AIDS. ongoing education of students in the community is a process and more and different ideas will need to be presented so that students can gain a clear understanding of the problem of HIV/AIDS. Developing strategies that are effective in educating youth on HIV can affect their understanding of the disease. Understanding the disease of HIV and what actions prevent the spread of the disease can have significant long range impact on the productivity of the country and wellbeing of the citizens of the country. Included in these consequences would be less utilization of health care resources in caring for those afflicted with the illness and more compassion toward those that have contracted the illness. The long range goal of this work is to provide information, create a better understanding, raise awareness, and encourage the development of safe behaviors to minimize the risk of infection. A change in perception among youth about HIV/AIDS, and development of better prevention strategies for youth can be achieved if there will be better strategies to address the current problems associated with HIV/AIDS in Indonesia. In this concept, the strategy used to assess any change in the behavior of young people about HIV / AIDS prevention. Effective strategies are used for preventing all routes of HIV/AIDS transmission. The most effective prevention programs are those that use a combination of strategies to achieve maximum impact. One of the strategies that found in the literature review is to create a group of young people who are trained periodically related to HIV prevention, so that effective strategies can be seen and behavioral changes can be assessed easily. Before implementing a strategy that has been made, then that person should give approach to the relevant organizations or to the community. Also in the literature review, those strategies are also applied and implemented in the areas of business and management to find ways and different activities with others so that organizational goals can be achieved. In management, the strategy reflects the insight and intuition of the strategies or business owner, and it becomes clear over time as a pattern in a stream of decisions. 21 Nurse Media Journal of Nursing, 5 (1), 2015, The strategies that must reflect people made the thoughts, feelings, ideas, and wants youth and mold them along with the purpose. Strategies are effective ways to achieve goals made in a different way. To make the strategy will require a good planning is to look at the needs and goals of the interests of society and especially young people in HIV prevention. In addition, with the analysis and evaluation of the activity or activities that have been done then this strategy is practiced in different ways and methods than usual. So that young people can change their behavior and can help prevent HIV among young people. 20 Nurse Media Journal of Nursing, 5 (1), 2015, After that, the student will study further about the selected topic and discuss different and creative ways that the education can be provided. Finally, the students provide education and information about HIV to the young people in the community by using different media to explain the reproductive organs and using video animation to explain the causes of HIV. This was determined as the subjects where the students had the greatest need for education. The nursing students develop a test for the young people to be given before and after the educational program in order to estimate the level of knowledge about HIV. The results are very significant and allowed the students to analyze and learn from the people who participated in the education program. The nursing students learned from this activity that the young people have a lack of understanding about HIV. The nursing students learned that the Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87 21 Nurse Media Journal of Nursing, 5 (1), 2015, 21 Nurse Media Journal of Nursing, 5 (1), 2015, 22 Nurse Media Journal of Nursing, 5 (1), 2015, communities. The HIV prevention for youth should include education for behavior change, information about risk and side effect, condom promotion and provision, STI treatment, and harm reduction. To conduct prevention and promotion programs, nurses must perform the assessment, data collection and see the program was run in youth has not been successful, and then the nurse should plan an effective and better strategy to address the identified needs. This is a process where the community health nurse is always evaluating the plan so that the best ideas are identified for working with this population. Concept analysis is the basis for theory development. Although the identified attributes may enhance understanding of strategy, further work is needed to operationalize the concept. If nurse educators are to view “strategies” as more than resource provision and negotiation, they must develop a process which addresses the development, implementation, and evaluation of their work in the community.. With use of the best strategies, nurse educator can teach nursing students how best to reach youth and other people in the community to prevent HIV. CONCLUSION HIV knowledge is an important factor influencing HIV risk perceptions and risk behaviors especially among youth. Understanding the unique dynamics of the HIV epidemic among youth is critical to developing and implementing effective prevention and treatment programs. This program can be done by a nurse educator, where the rising HIV caseload increases the demand for care and support in health facilities and Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87 22 Nurse Media Journal of Nursing, 5 (1), 2015, 22 Nurse Media Journal of Nursing, 5 (1), 2015, REFERENCES Amfar AIDS Research. (2010). Youth and HIV/AIDS in the United States: Challenges and Opportunities for Prevention. Washington, DC: Amfar, The Foundation for AIDS Research. Asian Development Bank. (2008). Education and Skills: Strategies for Accelerate Development in Asia and the Pacific. Asian Development Bank, 1-123. p p CDC HIV/AIDS. (2008). HIV/AIDS among Youth. CDC HIV/AIDS Resources, 1-5. Cherie, A., Mitkie, G., Ismail, S., & Berhane, Y. (2005, April). Perceived Suddiciency and Usefulness of IEC Materials and Methods Related to HIV/AIDS among High School Youth in Addis Ababa, Ethiopia. African Journal of Reproductive Health, 9, 66-77. Children & Youth Unit. (2006). HIV/AIDS Prevention among Youth: What Works Washington DC: World Bank. Dimbuene, Z. T., & Defo, B. K. (2011). Fostering Accurate HIV/AIDS Knowledge among Unmarried Youths in Cameroon: Do Family Environment and Peers Matter? BMC Public Health, 1-12. Foster, C. B., McLaughlin, N., Gray, A., Ogedegbe, A., Hageman, I., Knowlton, C., . . . Beeder, A. (2010). Reducing HIV and AIDS through Prevention (RHAP): A Theoretically Based Approach for Teaching HIV Prevention to Adolescents through an Exploration of PopularMusic. Journal of Urban Health, 87, 440-451. Hamza, K. M., & Wickman, P.-o. (2008). Describing and Analyzing Learning in Action: An Empirical Study of the Importance of Misconceptions in Learning Science. Wiley Periodicals, Inc, 141-164. y Hunger, D., & Wheelen, T. L. (2011). Essentials of Strategic Management. US: HR Folka International. Mccoy, H., Malow, R., Edwards, R. W., Thurland, A., & Rosenberg, R. (2007). A Strategy for Improving Community Effectiveness of HIV/AIDS Intervention Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87 23 Nurse Media Journal of Nursing, 5 (1), 2015, 23 Nurse Media Journal of Nursing, 5 (1), 2015, Design: The Community Readiness Modelin the Caribbean. Informa Healthcare USA, Inc, 1579-1592. doi:10.1080/1086080701212535 Design: The Community Readiness Modelin the Caribbean. Informa Healthcare USA, Inc, 1579-1592. doi:10.1080/1086080701212535 Miller, K. S., Forehand, R., Wiegand, R., Fasula, A. M., Armistead, L., Long, N., & Wyckoff, S. C. (2011). Making HIV Prevention Programming Count: Identifying Predictors of Success in a Parent-Based HIV Prevention Program for Youth. AIDS Education and Prevention, 38-53. Ministry of Health Republic of Indonesia. (2014). Laporan terakhir kemenkes. Retrieved from Ministry of Health Republic of Indonesia. (2014). Laporan terakhir kemenkes. Retrieved from http://spiritia.or.id/Stats/StatCurr.php?lang=id&gg=1 National HIV/AIDS Strategy. (2011). Implementing the National HIV/AIDS Strategy. US: US Department of Housing and Urban Development. Oblinger, D. (2006). Simulations, Games, and Learning. Educause, 1-6. Oladepo, O., & Fayemi, M. M. (2011). Perceptions about Sexual Abstinence and Knowledge of HIV/AIDS Prevention among In-School Adolescents in a Western Nigerian City. BMC Public Health, 1-10. g y Palekar, R., Pettifor, A., Behets, F., & MacPhail, C. (2008). Association Between Knowing Someone Who Died of AIDS and Behavior Change Among South African Youth. Springer Science+Business Media, 903-912. Romero, L. M., Galbraith, J. S., Williams, L. W., & Gloppen, K. M. (2011). HIV Prevention Among African American Youth: How Well Have Evidence-Based Interventions Addressed Key Theoretical. AIDS Behavior, 976-991. Rose, C., Wang, Y.-C., Cui, Y., Arguello, J., Stegmann, K., Weinberger, A., & Fischer, F. (2008). Analyzing Collaborative Learning Processes Automatically: Exploiting the Advances of Computational Linguistics in Computer-Supported Collaborative Learning. International Society of the Learning Sciences, 237-271. Wang, S., & Keats, D. (2005). Developing an Innovative Cross-Cultural Strategy to Promote HIV/AIDS Prevention in Different Ethnic Cultural Groups of China. AIDS Care, 874-891. Wyatt, T. J., & Oswalt, S. B. (2011). Letting Students Be Innovative! Using Mini- Grants to Fund Student-Designed HIV/AIDS Education. Health Promotion Practice, 413-424. Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87 Copyright © 2015, NMJN,p-ISSN 2087-7811, e-ISSN 2406-87
https://openalex.org/W2149423092	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/505CD0DBE01F0EB23F1A84E580A53910/S0955603600089868a.pdf/div-class-title-a-guide-for-the-trainee-div.pdf	English	null	A guide for the trainee	Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin	1,993	cc-by	2,651	DANNYALLEN,Senior Registrar, Fromeside Clinic, Blackberry Hill, Stapleton, Bristol BS161 ED It is also far more interesting to many staff who, in my experience, feel that they are able to improve their practice, as opposed to quantitative audit which may easily be seen as a tool of management to improve productivity. The next decision is who is to be involved. Medical audit means just doctors; clinical audit can involve the whole team. In our case there was little debate, the nature of a day hospital clearly lends itself to a multidisciplinary approach but consideration may need to be given to whether this is always appropriate in other settings. One compromise might be to alter nate depending on the subject of the audit; this would be especially important if the discussion would tend to exclude other staff by its very nature-e.g. blood tests. Keeping staff interest is paramount. The model which I would recommend to the trainee or senior registrar embarking upon audit for the first time would be that developed by the team at the Royal South Hants Hospital (Edwards et al, 1987). They started off with case-note review and developed a system of audit meetings which stood the test of time. It is a similar system to that which is described by James (1992) as applied to a regional secure unit. p g p The next step is to have the inaugural meeting. As far as possible staff should be prepared to come with ideas for audit but as the concept will be new to some, do not expect instant success; it isenough that people show up and express interest. The facilitator's job at this meeting will be to explain what audit, as envisaged here, is about. It is just as important to state what it is not about, i.e. checking how much work people are doing or trying to analyse the nature of that work. Personal experience indicates that this sort of 'managerial' audit is excellent at getting people's backs up and achieves nothing of use. It depresses morale as opposed to raising it as qualitative audit can do. This article describes my experience in a day hospital but the methods described can easily be adapted to any setting. Psychiatric Bulletin (1993), 17, 534-536 Psychiatric Bulletin (1993), 17, 534-536 DANNYALLEN,Senior Registrar, Fromeside Clinic, Blackberry Hill, Stapleton, Bristol BS161 ED Audit is certainly the topic of the moment. It is much talked about and a lot of money is being made avail able for it, yet it seems that there are few real guide lines about how to do it. Many junior doctors who would like to do audit may be put off by the com puters and other paraphernalia which are deemed necessary for this purpose. While technology can help, it is by no means necessary and some of the best audit follows the same principles as research: keep it simple. The trainee interested in setting up an audit pro gramme will probably best act as a facilitator. I used the beginning of my time at the day hospital to talk about audit and gauge people's reactions to it. If you are keen enough, staff should be willing to donate the next vital component: time. There isno point in being over-ambitious; an hour a month, perhaps in a pre existing slot, occupied by a case-conference or similar meeting, is all that is needed Any more can lead to complaints, unfulfilled expectations and the demise of the programme although, of course, a little more time is needed by the facilitator. p The College (1989) has produced a preliminary report on audit in which the point ismade that quality is at least as important as quantity. It is also far more interesting to many staff who, in my experience, feel that they are able to improve their practice, as opposed to quantitative audit which may easily be seen as a tool of management to improve productivity. The model which I would recommend to the trainee or senior registrar embarking upon audit for the first time would be that developed by the team at the Royal South Hants Hospital (Edwards et al, 1987). They started off with case-note review and developed a system of audit meetings which stood the test of time. It is a similar system to that which is described by James (1992) as applied to a regional secure unit. p The College (1989) has produced a preliminary report on audit in which the point ismade that quality is at least as important as quantity. https://doi.org/10.1192/pb.17.9.534 Published online by Cambridge University Press The seventh audit meeting Assuming a six month review cycle as suggested above, it is now time to go back to reviewing the first month's audit aim. It is to be hoped your team will have now become adept at formulating aims within half an hour so in theory there should be plenty of time for review! In practice, the first one or two reviews may well be a bit traumatic as people have forgotten what it was all about so it may be wise to suspend the formulation section of your meeting and concentrate on review. It is important to establish that staff understand that they are not being criticised and care should be taken to avoid personalisation. Where standards fall short of the aims, as they invariably will, people should decide whether this is due to bad practice, poor recording or other errors or whether there is an inherent flaw in the aims and therefore whether the standard should be redesigned. g Obviously there is nothing special about these topics, but they were the result of what staff were interested in. Other topics fell by the wayside as it was felt there was little practical purpose in discuss ing them. Sometimes the topic audited bore little resemblance to the original idea. g The most important point when the subject has been discussed is to formulate the aims in an auditable fashion. This means two things: first, the aim should be 100% success. Knowing one will reach it does not allow one to aim at less. Second, the aims must be checkable. Information recording methods must be established clearly at the meeting or by named persons before the next meeting. To do less is to court failure. For example, we agreed that appointment notes, assessment letters, six monthly notes and outcome letters would be sent to GPs. In order to check this we had to institute a recording system; the original idea proved impractical so it was modified at the next meeting. g At this stage it is important that clear conclusions are reached otherwise the whole exercise may be seen as a waste of time. It is the job of the facilitator to attempt to draw together the threads of the argument and to try to salvage some modicum of logic from what can easily become a free-floating discussion. It is important not to become discouraged. The second audit meeting The second audit meeting The agenda for this meeting should contain three sections: audit topic of the month, revisions to last month's audit, and suggestions for next month's audit. When the ideas have run dry a tentative agenda for the second meeting is set and a definite date and time established. Again the agenda must go out in time to jog people's memories and get them thinking about the topics. The second section isimportant because often the practicalities of implementing last month's audit may not have been as straightforward as expected and modifications may be necessary. Thefirst audit meeting About a week before the meeting the facilitator should produce a brief agenda, he or she should then go about reminding people and enthusing them. Personal experience suggests that one simple topic per meeting is adequate. One usually gets through less than one sets out to at first and it is always better to finish a meeting early and postpone discussion than to beat out poor ideas in the last five minutes. This does not matter at all, because it is possible to have a rolling agenda and people know that they will have a chance to discuss things later. So what sort of things do people want to discuss? The topics which we started with were communication with GPs, pre scribing, recording attendance, client review, and handling information. The seventh audit meeting Audit ing is a skill which it takes a team some time to learn. There may well be low points, particularly at the re view, but it is important to plough on as staff will gradually understand the significance of 'closing the audit loop' and recording practice for future audit will gradually become part of normal working practice. Before closing it is important to make sure every one knows what is to be discussed next month and finally agree a time interval after which you will return to check that month's audit. Simplicity dic tates that this should be the same for all topics and a suggested interval is six months. After the meeting it is the facilitator's duty to A guide for the trainee forthcoming. If not, the facilitator can inject some of their own, perhaps using examples from this article! forthcoming. If not, the facilitator can inject some of their own, perhaps using examples from this article! How to get started The first thing that is needed is enthusiasm. In my case I was allocated a number of sessions at the day hospital without a particular brief. I was interested in audit and suggested to my consultant that Idevoted a proportion of my time to it. I was given the go-ahead but from then on all enquiries aimed at establishing what the audit should be about or what form it should take were met with obscure comments about someone in the library being an expert on computers! The good news for trainees who are not especially computer literate was that my audit was achieved without the use of computers, although they are a useful adjunct for those who want to use them. The point that needs to be made at the first meeting is that audit is a means by which professionals can set standards and see how well they perform by their own lights. This latter part is very important, as it gives a feeling of control to the staff, and rightly so. After this subject has been discussed more than half the meeting will probably have passed and it is time to brainstorm. Ideas for audit are likely to be 534 https://doi.org/10.1192/pb.17.9.534 Published online by Cambridge University Press 535 A guide for the trainee A guide for the trainee https://doi.org/10.1192/pb.17.9.534 Published online by Cambridge University Press JONSPEAR,Research Registrar, St Nicholas Hospital, Gosforth, Newcastle upon Tyne NE3 3XT The aim of medical audit is to improve the quality of medical care (Department of Health, 1989). There was concern that patients referred to a psychogeriatric service (Service X) did not have adequate access to computerised tomography. The nearest computerised tomography scanner was located in a neighbouring district and direct referrals were not accepted. Computerised tomography scans could be obtained indirectly by referral to neurosurgeons. Because of these difficulties "potentially treatable structural lesions" (such as cerebral tumours and subdural haematomas) may have been missed. We decided to compare the use of computerised tomography scans with a nearby service (Service Y) which had a com puterised tomography scanner on site. Service X had a catchment population of 33,000 aged over 65 and Service Y a catchment population of 23,420 aged over 65. The study All patients in service X and service Y referred in 1990 and who had computerised tomography head scans were included. Fifty controls were randomly selected from patients referred to service Y in 1990.Information from case-notes was recorded on a preforma. https://doi.org/10.1192/pb.17.9.534 Published online by Cambridge University Press 536 References EDWARDS,J. G., NUNN, C. M. H. & RICKETTS,B. S. (1987) Three years of medical audit in a psychiatric unit. Bulletin of the Royal College of Psychiatrists, 11, 154-155. Allen JAMES,A. ( 1992) Practical experience from a regional secure unit. Psychiatric Bulletin, 16, 84-85. THE ROYAL COLLEGE OF PSYCHIATRISTS(1989) Working Party from Council. Preliminary report on medical audit. Psychiatric Bulletin, 13, 577-580. Keeping going It is in the nature of the job that trainees move on. But it is important to give a thought to continuity. It is to be hoped that you will have generated enthusiasm among other staff and that one of them willbe keen to take on your mantle - perhaps another trainee, or, more permanently, a clinical assistant or member of the nursing staff. Whichever way, if it has been worth doing it will be worth continuing. After a while staff will hopefully look upon audit, not as some academic exercise imposed upon them by doctors, but as a tool which everyone can use to improve standards. After the meeting it is the facilitator s duty to produce a sheet of audit aims, to be circulated, posted and preferably placed in a central recording mechan ism for posterity. The sheet should explain the prin ciples of audit: aim for 100% success, review in x months, and alter standards or practice accordingly thereafter. https://doi.org/10.1192/pb.17.9.534 Published online by Cambridge University Press 536 Allen JAMES,A. ( 1992) Practical experience from a regional secure unit. Psychiatric Bulletin, 16, 84-85. References EDWARDS,J. G., NUNN, C. M. H. & RICKETTS,B. S. (1987) Three years of medical audit in a psychiatric unit. Bulletin of the Royal College of Psychiatrists, 11, 154-155. THE ROYAL COLLEGE OF PSYCHIATRISTS(1989) Working Party from Council. Preliminary report on medical audit. Psychiatric Bulletin, 13, 577-580. Psychiatric Bulletin (1993), 17, 536-537 Psychiatric Bulletin (1993), 17, 536-537 JAMES,A. ( 1992) Practical experience from a regional secure unit. Psychiatric Bulletin, 16, 84-85. JAMES,A. ( 1992) Practical experience from a regional secure unit. Psychiatric Bulletin, 16, 84-85. THE ROYAL COLLEGE OF PSYCHIATRISTS(1989) Working Party from Council. Preliminary report on medical audit. Psychiatric Bulletin, 13, 577-580. Findings Patients referred to Service Y were more likely to have a computerised tomography scan (21/222 v. 10/449; x=16; d.f. = l; P<0.001) than patients referred to Service Y. Patients who had computerised tomography were younger than controls (72.1 years v. 78.5 years; t = 2.4; /><0.05). Most patients who had computerised tomography were male (18/31) while most controls were female (38/50; P < 0.01). Weinberger (1984) has suggested the following indications for CT scans for elderly psychiatric patients: All patients who had computerised tomography scans had an indication. For Service X these were confusion (6) and dementia (4). For service Y the indications were confusion (4), dementia (11), first onset psychosis (5) and personality change (1). There was no difference in the frequency of risk factors between Service X (9/10) and Service Y (17/21). Nine patients (29%) had focal neurological signs; of these four had cerebral infarcts, two atrophy and three had potentially treatable lesions (a meningioma, a subdural haematoma and normal pressure hydroce- phalus). Focal neurological signs were a predictor of "potentially treatable lesions" (Fisher's Exact Test; P=0.008). Twenty-two patients did not have focal neurological signs; of these nine had infarcts, 11 atrophy and two were "normal". All three patients with "potentially treatable lesions" were from Service X. (a) confusion or dementia of unknown cause (a) confusion or dementia of unknown cause (b) first episode of psychosis of major depressive episode p (c) prolonged catatonia (d) lit h (d) personality change. (d) personality change. In a study of patients with confusion or dementia 36 out of 331 (10.9%) had potentially treatable lesions identified by CT scanning (Roberts & Caird, 1990). Risk factors which increase the probability of "potentially treatable lesions" in elderly patients with confusion or dementia are focal neurological signs, headaches, papilloedema, epilepsy (Bradshaw et al, 1983), reduced alertness and a duration of less than one year (Roberts & Caird, 1990). https://doi.org/10.1192/pb.17.9.534 Published online by Cambridge University Press
https://openalex.org/W2044418307	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0125331&type=printable	English	null	Prohibitin Is Involved in Patients with IgG4 Related Disease	PloS one	2,015	cc-by	5,268	Results One positive autoantigen was identified as prohibitin. ELISA analysis showed that a majority of patients with IgG4-RD have antibodies against prohibitin. Anti-prohibitin antibodies were present in the sera of patients with definite autoimmune pancreatitis (25/34; 73.5%), Miku- licz’s disease (8/15; 53.3%), retroperitoneal fibrosis (6/11; 54.5%), other probable IgG4-RD (26/29; 89.7%) and Sjögren’s syndrome (4/30; 13.3%) but not in apparently healthy donors (1/70; 1.4%). Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the Fundamental Research Funds for the Central Universities, Program for New Century Excellent Talents in University and the National Natural Science Foundation of China (No. 21105113; No. 31371203). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ImmunoHunt Corporation provided support in the form of a salary for author GYC, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS OPEN ACCESS Citation: Du H, Shi L, Chen P, Yang W, Xun Y, Yang C, et al. (2015) Prohibitin Is Involved in Patients with IgG4 Related Disease. PLoS ONE 10(5): e0125331. doi:10.1371/journal.pone.0125331 Academic Editor: Peter Szodoray, Institute of Immunology, Rikshospitalet, NORWAY Received: October 8, 2014 Accepted: March 11, 2015 Published: May 1, 2015 Copyright: © 2015 Du 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. Citation: Du H, Shi L, Chen P, Yang W, Xun Y, Yang C, et al. (2015) Prohibitin Is Involved in Patients with IgG4 Related Disease. PLoS ONE 10(5): e0125331. doi:10.1371/journal.pone.0125331 Objective IgG4-related disease (IgG4-RD) is a chronic systemic disease involved in many organs and tissues. As only limited autoantigens have been found since the beginning of this century, the aim of this study was to reveal new candidate autoantigens of IgG4-RD. RESEARCH ARTICLE RESEARCH ARTICLE Prohibitin Is Involved in Patients with IgG4 Related Disease Hongwu Du1, Lili Shi1, Peng Chen1, Weikang Yang1, Yiping Xun1, Chunhe Yang1, Lanqing Zhao1, Yabin Zhou1, Guangyu Chen2 1 112 Lab, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, China, 2 ImmunoHunt Corporation, Beijing, China hongwudu@ustb.edu.cn * hongwudu@ustb.edu.cn * hongwudu@ustb.edu.cn Methods Academic Editor: Peter Szodoray, Institute of Immunology, Rikshospitalet, NORWAY Academic Editor: Peter Szodoray, Institute of Immunology, Rikshospitalet, NORWAY Multiple cell lines including HT-29, EA.hy926, HEK 293 and HepG2 were used to test the binding ability of circulating autoantibodies from IgG4-RD sera. The amino-acid sequence was then analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem (MALDI-TOF/TOF) mass spectrometry. After the cloning and expression of recombinant pu- tative autoantigen in a bacterial expression system, the corresponding immuno assay was set up and utilized to observe the prevalence of serum autoantibodies in a large set of con- firmed clinical samples. Copyright: © 2015 Du 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. Cell lines HT-29, EA.hy926, HEK 293 and HepG2 cell lines were all purchased from American Type Cul- ture Collection (Rockville, MD). To cultivate the four cell lines above, DMEM (HyClone, UT) with 10% fetal bovine serum (HyClone, UT) was used as the culture medium. Because IgG4-RD can occur in various organs, which increases the complexity of the disease, HT-29 was selected as the representative of glandular epithelial cells; EA.hy926 as the representative of vein endo- thelial cells; and HEK 293 and HepG2 as the representatives of human organs involvement. Samples In this study, sera from 89 patients with IgG4-RD with an average age of 38 years (range 21 to 68, 36 female and 53 male) who conform to the criteria proposed by the Japan Pancreas Society [22], sera from 30 patients with Sjögren’s syndrome (SjS) (35 years old: range 15 to 58, 21 fe- male and 9 male) who were diagnosed according to the standard defined criteria [23], and sera from 70 matching healthy donors (36 years old: range 15 to 60, 46 female and 24 male) were enrolled as control. This study was approved by the ethical committee of the Peking Union Medical College Hospital, and each patient involved in this study provided written informed consent. Furthermore, written informed consent on behalf of the minors involved in the study was obtained from their guardians. Blood specimens were procured and medical record infor- mation including gender, age, height, weight and supplement use was collected. Sera were iso- lated, aliquoted and stored at -80°C until used. Prohibitin and IgG4-RD Conclusions An association between prohibitin and patients with some IgG4-RD was observed, although the results were quite heterogeneous among different individuals within autoimmune pan- creatitis, Mikulicz’s disease and retroperitoneal fibrosis. 1 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0125331 May 1, 2015 Introduction Competing Interests: The authors have declared that no competing interests exist. IgG4-RD has recently been recognized as a distinct clinical entity comprising a number of dis- orders, such as type 1 autoimmune pancreatitis (AIP) [1,2], sclerosing cholangitis [3], Miku- licz’s disease (MD) [4], tubulointerstitial nephritis [5] and retroperitoneal fibrosis (RPF) [6] etc. It is a chronic systemic disease with multi-organ involvement and IgG4-positive plasma cell infiltration [7]. The characteristics of IgG4-RD are high density of serum IgG4 and osmotic IgG4-positive plasma cells, which can infiltrate pancreas [8], salivary [9], lacrimal glands [10], lung [11], retroperitoneal space [6], kidneys [12], pituitary [13], eyes [14] and others [15]. Due to lack of specific clinical features in the early stage, current clinical practice greatly relies on the examination of histology, imaging and serology, which increases the difficulty of a clear di- agnosis. Autoimmunity has been considered the most probable pathogenesis of IgG4-RD [16] and several autoantigens have been suggested, including lactoferrin [17], carbonic anhydrase II (CAII) [18], amylase-alpha 2A [19], pancreatic secretory trypsin inhibitor (PSTI) [20] and plasminogen-binding protein peptide [21], with autoantibodies against these targets successful- ly examined in patients with IgG4-RD in laboratories. Furthermore, the molecular mechanism of IgG4-RD is not fully clarified yet, and it requires the discovery of more associated molecules through additional research. It is hoped that some of these molecules will be targeted in clinical applications, whereas others may be used to study the disease pathogenesis and how IgG4-RD relates to other autoimmune diseases. The aim of this study was to screen and identify new autoantigens which are closely related to the morbidi- ty of IgG4-RD. Immunoprecipitation Total extracts of HT-29 cells (500 μg) were incubated with 9 μL mixed sera (equal volumes from three positive IgG4-RD patients in western blotting detection) overnight at 4°C on a rota- tor. Subsequently, 50 μL of protein A-Sepharose beads (Sigma, MO) washed with PBS were added and incubated for 4 hours at 4°C. The immunoprecipitates were washed three times in 200 μL PBS. Then, the immunoprecipitates were suspended in a sample loading buffer and an- alyzed by 12% SDS-PAGE. Prohibitin and IgG4-RD on the slides overnight, fixed with 4% paraformaldehyde and followed by incubation with Tri- tonX-100. Sera from patients with IgG4-RD, SjS or healthy donors were diluted at 1: 20 in PBS, and incubated with the slides for 2 hours at 37°C. After extensive washing, the slides were incu- bated with fluorescein isothiocyanate (FITC)-conjugated goat-anti-human IgG (Bioss, Beijing, China) which had been diluted 1: 150 in PBS for 30 min. The slides were then examined under a fluorescence microscope (AMG, Bothell, WA). For the indirect immunofluorescence assay, the total cell fluorescence ratio was obtained by Image J software (NIH, MD). HT-29 cell-based ELISA HT-29 cells were plated in 96-well plates and cultured at 37°C. Cells were fixed with 4% para- formaldehyde and then washed with 5 ‰ PBST (Tween-20 in PBS, v/v). After fixation with 0.2% TritonX-100 for 10 min, cells were washed by using 5 ‰ PBST again, and allowed to dry. Then sera were diluted at 1: 100 with PBS and incubated with the cells. After incubation, horse- radish peroxidase-conjugated goat anti-human IgG at a dilution of 1: 10000 (ImmunoHunt, Beijing, China) was added. The binding of antibody was quantified by the addition of tetra- methylbenzidine (TMB, 1 mg/mL, Sigma). The chromogenic reaction was stopped with 2 M H2SO4 and the plates were obtained spectrophotometrically at 450 nm on a microplate reader (Tecan, Hombrechtikon, Switzerland). Western blotting The detailed procedure of Western blotting was performed as described elsewhere [25]. In brief, to screen the autoantibodies in the sera, HT-29 cells were lysed in RIPA buffer (Beyotime, Shanghai, China) with 1% protease inhibitor cocktail (Sigma-Aldrich, MO), combined with the loading buffer and boiled for 10 min. After removing the insoluble fraction by centrifuge, the eluted proteins were resolved by 12% SDS-PAGE and then transferred onto PVDF membrane (Merck Millipore, MA). Next the membrane was cut into 2-cm wide stripes. After blocking with PBS containing 5% nonfat milk for 1 hour at 37°C, the membrane was incubated with sera at a dilution of 1: 500. Horseradish peroxidase-conjugated goat anti-human IgG was used as the secondary antibody with a dilution of 1: 10,000 for 1 hour at 37°C. The positive bands were detected with enhanced chemiluminescence kit (Applygen, Beijing, China). Indirect immunofluorescence assays Indirect immunofluorescence assays were performed as previously described [24]. HT-29, EA. hy926, HEK 293 and HepG2 cell lines were plated on the slides. The cells were then cultured 2 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0125331 May 1, 2015 Expression and purification of target antigen The total RNA was isolated from HT-29 using TRIzol reagent (Invitrogen, CA). RT-PCR was performed according to the manufacturer’s instructions (Fermentas, MD). Human PHB pro- tein was over-expressed in the E.coli BL21, followed by purification with Ni-NTA resin (Qia- gen, Hilden, Germany). Protein concentrations were determined by BCA kit (Boisynthesis Biotechnology, Beijing, China). Purified recombinant protein was stored at -80°C. Prohibitin and IgG4-RD digestion buffer containing 50 mM NH4HCO3 and 12.5 ng/μL of trypsin in an ice-cold bath. After 1 hour ice-cold bath, the supernatant was removed and replaced with 50 mM NH4HCO3 to keep the gel pieces wet during enzymatic cleavage. MALDI-TOF/TOF MS analysis of the tryptic digestion solution was performed on 4700 Proteomics Analyzer mass spectrometer (Applied, Biosystems). CHCA (α-cyano-4-hydroxycinnamic acid) matrix was prepared by dis- solving 5 mg in 1 mL of 50:50 acetonitrile/water containing 0.1% tallow fatty acid. The mass spectrometric data were analyzed using the Mascot bioinformatics database search engine (Ma- trix Sciences, London, UK). Peptide sequences were interpreted from the MS/MS spectra by searching the Homo sapiens subset of the Swissprot protein database. Search parameters in- cluding carbamidomethylation of cysteines were set as a fixed modification, and then methio- nine oxidation was set as a variable modification, respectively. The peptide mass tolerance was set at ±100 ppm and the fragment mass tolerance at ±0.6 Da. Trypsin was specified as the pro- teolytic enzyme, and one missed cleavage was allowed. Human recombinant PHB based-ELISA ELISAs were performed as described previously [25]. Briefly, 0.1 μg/mL of recombinant protein was used to coat 96-well microtiter plates overnight at 4°C. After blocking with goat serum, sera from patients or controls were diluted at 1: 100 and added to the antigen-coated wells and incu- bated for 2 hours. Bound antibodies were incubated with horseradish peroxidase-conjugated mouse anti-human IgG4 (Abcam, MA) which had been diluted at 1:4,000 and reacted with TMB as substrate. Finally the reaction was stopped by adding 2 M H2SO4. The absorbance at 450 nm was measured with a microplate reader (Tecan, Hombrechtikon, Switzerland). Statistical analysis To determine whether the frequency of autoantibodies in each cohort of patients’ sera was sig- nificantly higher than that in sera from healthy donor, the statistical analysis was evaluated by SPSS software (Version 17, Chicago, IL), with results considered to be statistically significant if P< 0.05. The critical point for positive definition was the number with a higher value than that of the healthy donors (Mean + 3 SD). In-gel digestion and mass spectrometry analysis In-gel digestion and MALDI-TOF/TOF MS analysis were performed as previously described [24]. The gel band of interest was excised and detained by 25 mM NH4HCO3, containing 50% acetonitrile and dried with vacuum centrifugation. Then, 25 mM NH4HCO3 including 10 mM dithiotreitol (DTT) was added to cover the gel pieces and allowed to reduce for 2 hours at 37°C. After cooling to room temperature, the DTT solution was replaced by the same volume 25 mM NH4HCO3 containing 55 mM iodoacetamide to incubate for 45 min in the dark. After the gel pieces were washed and completely dried in a speed-vac, they were swollen in a 3 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0125331 May 1, 2015 HT-29 was selected as target for the antigen screening The autoantibody profile of IgG4-RD in HT-29, EA.hy926, HEK 293 and HepG2 cell lines was tested (Fig 1A–1D). The existences of autoantigens in the HT-29 cells were demonstrated by the presence of abundant fluorescence. Concurrently, by using different control sera to incu- bate with HT-29 cells, it was shown that the sera from SjS and HC have a relatively weaker or non-specific interaction (Fig 1E–1F). 4 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0125331 May 1, 2015 Prohibitin and IgG4-RD Fig 1. Immunofluorescence analysis. (A-D) Immunofluorescence was performed on HT-29 by confocal laser microscopy, then compared with other cells including EA.hy926, HEK 293 and HepG2. Total cell fluorescence was analyzed by Image J software and significant differences were found between HT-29 and three other cell lines (p<0.0001), indicating positive reactions in HT-29 cells with IgG4-RD sera. (E-F) Contro samples including SjS and HC were then tested on HT-29 cells, and the sera of patients with SjS were found to have a weaker specific reaction and no positive signal on HC. doi:10.1371/journal.pone.0125331.g001 Fig 1. Immunofluorescence analysis. (A-D) Immunofluorescence was performed on HT-29 by confocal laser microscopy, then compared with other cells including EA.hy926, HEK 293 and HepG2. Total cell fluorescence was analyzed by Image J software and significant differences were found between HT-29 and three other cell lines (p<0.0001), indicating positive reactions in HT-29 cells with IgG4-RD sera. (E-F) Control samples including SjS and HC were then tested on HT-29 cells, and the sera of patients with SjS were found to have a weaker specific reaction and no positive signal on HC. doi:10.1371/journal.pone.0125331.g001 doi:10.1371/journal.pone.0125331.g001 5 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0125331 May 1, 2015 Prohibitin and IgG4-RD Antibody titer Total fluorescent differences among four different cells lines were quantified by Image J soft- ware and significant differences were observed (Fig 2A). ELISA of HT-29 with the sera from 20 IgG4-RD patients (the first batch of samples in the lab) was performed. The sera from 5 IgG4-RD patients which presented relatively higher optical density in ELISA were selected to perform the subsequent Western blotting experiment (Fig 2B). PHB was identified as a target protein in IgG4-RD patients Through Western blotting analysis, a specific positive band near 30 kDa of molecular weight was observed from the reaction between 3 IgG4-RD sera and HT-29 cells (Fig 2C). However, no positive reaction was observed with healthy controls. Fig 2. Identification of target antigen. (A) The value of cell fluorescence was analyzed by Image J software. * indicates P<0.0001. (B) Five of 20 patients with IgG4-RD presenting relatively higher optical density values on ELISA for HT-29 cell were selected for Western blotting. (C) Western blotting of HT-29 cell extracts with the sera from 5 IgG4-RD patients showed a positive band with a molecular weight of approximately 30 kDa in 3 patients but not in healthy controls. (D) Immunoprecipitation was performed by incubating the extracts of HT-29 with IgG4-RD patient sera and an approximately 30 kDa protein band reacted with antibodies from IgG4-RD patients. doi:10.1371/journal.pone.0125331.g002 Fig 2. Identification of target antigen. (A) The value of cell fluorescence was analyzed by Image J software. * indicates P<0.0001. (B) Five of 20 patients with IgG4-RD presenting relatively higher optical density values on ELISA for HT-29 cell were selected for Western blotting. (C) Western blotting of HT-29 cell extracts with the sera from 5 IgG4-RD patients showed a positive band with a molecular weight of approximately 30 kDa in 3 patients but not in healthy controls. (D) Immunoprecipitation was performed by incubating the extracts of HT-29 with IgG4-RD patient sera and an approximately 30 kDa protein band reacted with antibodies from IgG4-RD patients. doi:10.1371/journal.pone.0125331.g002 6 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0125331 May 1, 2015 Prohibitin and IgG4-RD Immunoprecipitation was further carried out by incubating the extract of HT-29 cells with mixed sera, which resulted in an approximately 30 kDa protein band (Fig 2D). This protein was found to share approximately 40% sequence similarity with human PHB (NCBI accession number, gi\|4505773; Mascot score, 692), and 8 unique peptides of PHB were matched. Expression and purification of recombinant human PHB The recombinant expression plasmid pET-28a (+)-PHB was transformed into E.coli BL21 (DE). After 6 hours of induction by isopropyl-b-D-thiogalactopyranoside, the cells were lysed by ultrasonic and the extracts were analyzed by SDS-PAGE. High levels of expression of re- combinant PHB protein were observed (Fig 3A). Western blotting and immunoprecipitation To further demonstrate the immunoreactivity of PHB protein against the patients’ sera, puri- fied PHB was analyzed with western blotting using mixed sera. It was shown that the sera from patients with IgG4-RD but not from healthy donors could recognize the recombinant PHB protein. (Fig 3B). Fig 3. Verification of prohibitin. (A) The cloning, expression and purification of recombinant PHB protein. M, protein markers; lane 1, cell extracts of pET- 28a (+)-PHB/BL21 after IPTG induction for 6 hour at 37°C; lane 2, cell extracts of pET-28a (+)-PHB/BL21 before IPTG induction; lane 3, cell extracts of pET- 28a (+)-BL21 after IPTG induction. lane 4, cell extracts of BL21 after IPTG induction. (B) Western blot using purified PHB protein showed that only the sera from patients with IgG4-RD (lane 1) rather than HC (lane 2) contain antibodies against a 30 kDa cellular protein. (C) The expressed protein was purified and further identified by MS, which revealed its identity as PHB. (D) PHB protein was also identified in immunoprecipitates; lane 1, supernatant of immunoprecipitation; lane 2, immunoprecipitates; lane 3, control sample (the purified rhPHB). (E) The protein band on lane 2 was excised and identified by MALDI-TOF/TOF MS, which again revealed its identity as PHB. doi:10.1371/journal.pone.0125331.g003 Fig 3. Verification of prohibitin. (A) The cloning, expression and purification of recombinant PHB protein. M, protein markers; lane 1, cell extracts of pET- 28a (+)-PHB/BL21 after IPTG induction for 6 hour at 37°C; lane 2, cell extracts of pET-28a (+)-PHB/BL21 before IPTG induction; lane 3, cell extracts of pET- 28a (+)-BL21 after IPTG induction. lane 4, cell extracts of BL21 after IPTG induction. (B) Western blot using purified PHB protein showed that only the sera from patients with IgG4-RD (lane 1) rather than HC (lane 2) contain antibodies against a 30 kDa cellular protein. (C) The expressed protein was purified and further identified by MS, which revealed its identity as PHB. (D) PHB protein was also identified in immunoprecipitates; lane 1, supernatant of immunoprecipitation; lane 2, immunoprecipitates; lane 3, control sample (the purified rhPHB). (E) The protein band on lane 2 was excised and identified by MALDI-TOF/TOF MS, which again revealed its identity as PHB. doi:10.1371/journal.pone.0125331.g003 7 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0125331 May 1, 2015 Prohibitin and IgG4-RD After the purification of recombinant PHB protein with Ni-NTA resin, the eluted fractions were separated on SDS-PAGE and further confirmed by mass spectrometry (Fig 3C). doi:10.1371/journal.pone.0125331.g004 The prevalence of anti-PHB antibodies in IgG4-RD patients The prevalence of autoantibodies against human PHB was examined in various patients by ELISA. A total of 189 samples were tested in this study. Of the 89 sera with IgG4-RD analyzed, 65 (73%) were found to be reactive with PHB, but only 1 (1.4%) of the 70 healthy donors was positive (Fig 4A). Anti-PHB antibodies were present in the sera of patients with a different sub- type of IgG4-RD (Fig 4B) including definite AIP (25/34; 73.5%), MD (8/15; 53.3%), RPF (6/11; 54.5%), and other probable IgG4-RD (26/29; 89.7%). Discussion and Conclusions One advantage of this study is that the indirect immunofluorescence analysis for multiple cell lines was performed at the beginning to screen out enrichment of candidate autoantigen targets. Actually, dozens of cell lines of different origin were used in the pre-screening pro- cess (data not shown). The fluorescence signal intensity indicated the binding ability of auto- antigens with the sera from IgG4-RD, in which the promising cell lines with clear positive signals were selected and used for the next screening circle for putative target autoantigens. After the in-gel digestion and mass spectrometry analysis, the potential autoantigen was re- vealed to be PHB. PHB is involved in the developing process of many diseases [26] and may act as a tumor suppressor that can activate anti-proliferative activity by inhibiting cell cycle and the synthesis of DNA. Using ELISA, the levels of anti-PHB antibodies were clearly higher in sera from IgG4-RD than in healthy controls, leading us to hypothesize that the existence of anti-PHB au- toantibodies can accelerate cell proliferation which can originally inhibit the biological function of PHB, resulting in tissue enlargement. Moreover, MHC binding prediction tool from IEDB Analysis Resource was used to determine potential MHC-I binding motif in PHB. A consensus result by both Artificial neural network (ANN) and Stabilized matrix method (SMM) indicated peptide VSDDLTERA (from Val150 to Gla158) with low IC50 (nM) being a good MHC-I binder [27–31]. By investigating rat models of unilateral ureteral obstruction and renal tubule interstitial fi- brosis, researchers discovered that the expression of PHB was significantly lower in renal tis- sues, leading them to assume that PHB may play a role in interstitial fibrosis [32]. Besides, Low expression of PHB has also been observed in inflammatory bowel disease and may help to reduce the pain of colonic inflammation [33]. It has been hypothesized that PHB may be re- lated to innate immunity and may participate in the mediation of immune function and in- flammation. However, more research is needed to clarify the exact role that PHB plays in IgG4-RD. In summary, this study has revealed that PHB is associated with some IgG4-RD patients, in- cluding those in different IgG4-RD subtypes such as AIP, MD, and RPF. However, the patholo- gy role and clinical value of PHB remains unclear and need further investigations. Western blotting and immunoprecipitation Moreover, immunoprecipitation was further employed to confirm whether the PHB protein was a real IgG4-RD autoantigen. It was shown that the band of PHB protein was clearly present in the im- munoprecipitates, further indicating that the PHB protein was a target of IgG4-RD (Fig 3D). The protein bands were excised and also identified by mass spectrometry (Fig 3E). munoprecipitates, further indicating that the PHB protein was a target of IgG4-RD (Fig 3D). The protein bands were excised and also identified by mass spectrometry (Fig 3E). Fig 4. Anti-PHB autoantibodies induced in IgG4-RD patients. (A) The prevalence of autoantibodies against human PHB in sera from patients was observed. ELISA was used to detect the reactivity of serum IgG4 against recombinant human PHB protein. The anti-PHB antibodies were detected in 65 of 89 RA patients (73%), 4 of 30 SjS patients (13.3%) and 1 of 70 healthy donors (1.4%). The reactivity of anti-PHB antibodies was significantly higher than HC (*P<0.0001). (B) The patients with IgG4-RD were then divided into the following confirmed subtypes: AIP, definite autoimmune pancreatitis (25/34, 73.5%); MD, Mikulicz’s disease (8/15, 53.3%); RPF, retroperitoneal fibrosis (6/11, 54.5%); MIX, affect multiply organs (26/29, 89.7%). d i 10 1371/j l 0125331 004 Fig 4. Anti-PHB autoantibodies induced in IgG4-RD patients. (A) The prevalence of autoa Fig 4. Anti-PHB autoantibodies induced in IgG4-RD patients. (A) The prevalence of autoantibodies against human PHB in sera from patients was observed. ELISA was used to detect the reactivity of serum IgG4 against recombinant human PHB protein. The anti-PHB antibodies were detected in 65 of 89 RA patients (73%), 4 of 30 SjS patients (13.3%) and 1 of 70 healthy donors (1.4%). The reactivity of anti-PHB antibodies was significantly higher than HC (*P<0.0001). (B) The patients with IgG4-RD were then divided into the following confirmed subtypes: AIP, definite autoimmune pancreatitis (25/34, 73.5%); MD, Mikulicz’s disease (8/15, 53.3%); RPF, retroperitoneal fibrosis (6/11, 54.5%); MIX, affect multiply organs (26/29, 89.7%). doi:10.1371/journal.pone.0125331.g004 8 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0125331 May 1, 2015 Prohibitin and IgG4-RD References 1. Deshpande V, Chicano S, Finkelberg D, Selig MK, Mino-Kenudson M, Brugge WR, et al. Autoimmune pancreatitis: a systemic immune complex mediated disease. Am J Surg Pathol. 2006; 30: 1537–1545. PMID: 17122509 2. Okazaki K, Uchida K, Miyoshi H, Ikeura T, Takaoka M, Nishio A. Recent concepts of autoimmune pan- creatitis and IgG4-related disease. Clin Rev Allerg Immu. 2011; 41: 126–138. doi: 10.1007/s12016- 010-8214-2 PMID: 21170607 3. Kawaguchi K, Koike M, Tsuruta K, Okamoto A, Tabata I, Fujita N. 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https://openalex.org/W2883147371	https://europepmc.org/articles/pmc6081539?pdf=render	English	null	Epigallocatechin Gallate Attenuates Bladder Dysfunction via Suppression of Oxidative Stress in a Rat Model of Partial Bladder Outlet Obstruction	Oxidative medicine and cellular longevity	2,018	cc-by	7,651	Hindawi Oxidative Medicine and Cellular Longevity Volume 2018, Article ID 1393641, 10 pages https://doi.org/10.1155/2018/1393641 Hindawi Oxidative Medicine and Cellular Longevity Volume 2018, Article ID 1393641, 10 pages https://doi.org/10.1155/2018/1393641 Meng Gu ,1 Chong Liu ,1 Xiang Wan,1 Tianye Yang,2 Yanbo Chen,1 Juan Zhou,1 Qi Chen ,1 and Zhong Wang 1 Meng Gu ,1 Chong Liu ,1 Xiang Wan,1 Tianye Yang,2 Yanbo Chen,1 Juan Zhou,1 Qi Chen ,1 and Zhong Wang 1 1Department of Urology, Shanghai Ninth People’s Hospital Aﬃliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China 2Department of Emergency, Shanghai Ninth People’s Hospital Aﬃliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China ment of Emergency, Shanghai Ninth People’s Hospital Aﬃliated to Shanghai Jiao Tong University School of Medici i 200011, China Correspondence should be addressed to Qi Chen; qiqi_chenqi@163.com and Zhong Wang; zhongwang2000@ Received 19 December 2017; Revised 29 April 2018; Accepted 14 May 2018; Published 22 July 2018 Academic Editor: Claudio Cabello-Verrugio Academic Editor: Claudio Cabello-Verrugio Copyright © 2018 Meng Gu 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. Purpose. To investigate the protective eﬀect of epigallocatechin gallate (EGCG), a green tea extract, and its underlying mechanism on bladder dysfunction in a rat model of bladder outlet obstruction (BOO). Materials and Methods. Sprague-Dawley rats of BOO were surgically induced and followed by treatment with EGCG (5 mg/kg/day) or saline (control) via intraperitoneal injection. Cystometry was performed on four weeks postoperatively in conscious rats. H&E, Masson trichrome, and TUNEL staining were performed to observe tissue alterations. Oxidative stress markers were measured, and protein expression of Nrf2-ARE pathway was examined by immunohistochemistry and Western blotting. Results. Our data showed that EGCG could increase the peak voiding pressure and bladder compliance and prolong micturition interval of BOO rats compared with control and ﬁnally reduce the frequency of urinary. EGCG could ameliorate the increase of collagen ﬁbers and ROS induced by obstruction and increase the activity of SOD, GSH-Px, and CAT. The level of cell apoptosis was decreased in BOO rats treated with EGCG compared with control, and caspase-3 expression was reduced as well. Moreover, EGCG could activate the Nrf2 expression with elevation of its target antioxidant proteins. Conclusions. EGCG alleviates BOO-induced bladder dysfunction via suppression of oxidative stress and activation of the protein expression of Nrf2-ARE pathway. Oxidative Medicine and Cellular Longevity 2 cell death but at lower levels induce subtle changes in intracellular signaling pathways. An increase in ROS may contribute to alter bladder function in aging [6]. Superoxide dismutase (SOD) is one of the cell’s chief defenses against activated oxygen free radicals. Presenting in the peroxisomes of nearly all aerobic cells, catalase (CAT) act in association with SOD protects cells against free radical damage [7, 8]. SOD and CAT activities are associated with the shift from compensated to decompensated function of the bladder [7]. As a transcription factor, nuclear erythroid-related factor 2 (Nrf2) could promote expression of antioxidative genes through the antioxidant response element (ARE) to regulate cellular antioxidative responses and redox status [9]. It has been established in the literature that activation of the Nrf2-ARE pathway may ameliorate bladder dysfunction caused by bladder outlet obstruction [10]. around it after which the needle was removed and incision was closed. Sham operations were performed in an identical manner without tying the silk thread. From the ﬁrst day after operation, rats of the BOO + EGCG group received daily intraperitoneally injections of EGCG (5 mg/kg). EGCG was dissolved in PBS, and rats of the sham group and BOO group were given the same volume of PBS. 2.2. Cystometry Preparation and Cystometric Analysis. The cystometry preparation and cystometric analysis were per- formed four weeks after operation. A catheter was placed in the bladder of rats of all groups three days before cystometric analysis. As described in previous study, ﬂaring the end of the polyethylene tubing 50 (PE-50) to become a balloon serves as an anchor to maintain the tube within the bladder [7, 11]. A 1 cm incision was made on the dorsum between the scapulas in anesthetized rats followed by developing a plane between the skin and the underlying muscle to create a tunnel around the ventral abdomen. Cystometry preparation was carried out as followed that rats were anesthetized and a 1 cm incision was made on the dorsum between the scapulas. Then, we developed a plane between the skin and the under- lying muscle to create a tunnel around the ventral abdomen. After an abdominal midline incision was made to expose the bladder, we grasped the smooth end of the PE-50 tubing with the clamp and pull it back through the dorsum incision. Oxidative Medicine and Cellular Longevity Following the bulbed end was placed in the bladder dome and the purse string suture around the tubing was pulled tight, the dorsal and abdominal incisions were closed for cystometric analysis. As a major component of green tea, epigallocatechin-3- gallate (EGCG) has been studied for its antioxidative and anti-inﬂammatory properties. Reports revealed that pretreat- ment with EGCG could induce Nrf2 activation in cells as demonstrated by increasing expression and nuclear translo- cation of Nrf2 [11, 12]. Mechanical stretch and hypoxia have been reported to contribute to the functional and structural changes in the bladder after BOO, and previous studies have indicated that the activation of Nrf2 pathway by EGCG shows eﬀective protection in various diseases. However, whether EGCG could protect bladder tissue by activating the Nrf2 pathway in the BOO model has not been well deﬁned. Based on our previous study [10] that demonstrated the decrease of oxidative stress and activation of the Nrf2- ARE pathway may ameliorate bladder dysfunction induced by BOO, we explored the ability of EGCG to ameliorate bladder dysfunction by inhibiting oxidative stress via the regulation of the Nrf2-ARE pathway in a rat model of BOO in the present study. For cystometry, the conscious rats were placed in a metabolic cage, and the indwelling tubing was attached to a two-way valve that was connected to a pressure transducer as well as an infusion pump. Saline was infused into the bladder at a rate of 12 ml/h at room temperature in all groups. The cystometric parameters of maximal pressure, bladder capacity, and others were measured. The rats were euthanized after the experiment, and the bladder tissue was collected for further study. 1. Introduction major public health problem that seriously aﬀects the quality of life of patients and their partners. Benign prostatic hyperplasia (BPH) characterized by gradu- ally nonmalignant enlargement of prostate gland [1, 2] is a very common chronic disease in elderly men. As people age, most BPH patient will induce bladder outlet obstruction (BOO) with high bladder pressure and low ﬂow [3]. BOO is a common disorder of the urinary tract that could aﬀect quality of life and is a very rarely life-threatening disease but can induce signiﬁcant structural and functional changes of the bladder, which in turn brings lower urinary tract symptoms (LUTS) including urinary frequency, urgency, nocturia, and urge incontinence [4]. BOO caused by BPH has become increasingly prevalent with age and been considered as the As showed in previous studies, oxidative stress is consid- ered to be one of the mechanisms that triggers reactions chain involved in the development and progression of BPH and leads to injured function of the bladder [5]. Oxidative stress occurs in the cellular environment when there is an imbalance between the production of reactive oxygen species (ROS) and the ability of biological systems to repair oxidative damage or neutralize the eﬀects of reactive intermediates including peroxides and free radicals. Production of high levels of ROS causes a signiﬁcant decrease in antioxidant defense mechanisms leading to protein, lipid, and DNA damage and subsequent disruption of cellular functions and Oxidative Medicine and Cellular Longevity 2. Materials and Methods 2.1. BOO Model and Drug Treatment. BOO model of SD was established in adult male Sprague-Dawley rats (SD) weighing 240 to 260 g (Animal Center of the Shanghai Ninth People’s Hospital of Shanghai Jiao Tong University School of Medi- cine, Shanghai, China) according to our previous method [10]. All animals were housed by two per cage in a room under controlled temperature, humidity, and 12-hour light/ 12-hour dark cycles with free access to food and water. The rats were allowed to acclimatize for at least ﬁve days before the experiment. All experimental procedures were approved by the Ethics Committee of Shanghai Jiao Tong University School of Medicine. The SD rats were randomly divided into three groups: the sham-operated group, the BOO group, and the BOO treated with EGCG (purity>98%, Biotech Co. Ltd, China) group, and 8 rats were included in each group. BOO surgical operation was applied according to the method described previously in detail [13]. Brieﬂy, the rats were anesthetized and then abdominal midline incision (about 1.0 cm) was made to expose the bladder and proximal urethra. The proximal urethra was loosely tied with the needle using 3–0 silk thread after a 19-G needle was placed 2.3. Histological Examination and Immunohistochemical Staining. 5 μm sections were made after bladders being ﬁxed in 4% paraformaldehyde and embedded in paraﬃn. Hema- toxylin and eosin (H&E) staining was performed to observe general morphology of the bladders, and Masson trichrome staining was used to evaluate the level of tissue ﬁbrosis. Immunohistochemical staining was also conducted as previously described in our study. Brieﬂy, the sections were subjected with 10mM sodium citrate buﬀer (pH 6.0) to heat for antigen retrieval. The primary antibodies of anti-Nrf2 (Abcam, Cambridge, UK), HO-1 (Abcam, Cambridge, UK), PCNA (CST, Danvers, MA, USA), secondary antibodies of goat-anti-rabbit IgG-HRP (DAKO, Denmark), goat-anti- mouse IgG-HRP (DAKO, Denmark), and DAB detection kit (DAKO, Denmark) were used according to the manufac- turer’s instructions. Histological analysis was performed by a pathologist in a blinded manner. Oxidative Medicine and Cellular Longevity 3 Table 1: Outcomes of cystometric parameters in conscious rats. 2. Materials and Methods Sham BOO BOO + EGCG Peak voiding pressure 23.54 ± 2.13 42.4 ± 1.44∗ 55.16 ± 2.6∗# Capacity (ml) 1.15 ± 0.31 3.33 ± 0.38∗ 4.12 ± 0.49∗ Compliance (μl/cm H2O) 24.37 ± 3.12 13.17 ± 3.26∗ 21.36 ± 2.75# Micturition interval (min) 3.67 ± 0.64 2.07 ± 0.47∗ 3.03 ± 1.41∗# ∗Signiﬁcantly diﬀerent versus the sham group (P < 0 05). #Signiﬁcantly diﬀerent versus the BOO group (P < 0 05). Table 1: Outcomes of cystometric parameters in conscious rats. according to the manufacturer’s instructions of nuclear protein extraction kit (Beyotime Institute of Biotechnology, Shanghai, China). Following the steps outlined below, blad- der tissues were cut in pieces and homogenized in cytoplasm protein extraction reagent containing protease inhibitors, the lysates were centrifuged (10000 ×g/min) at 4°C for 5 min, and then the precipitation was collected and mixed with nucleo- protein extraction reagent for 30min on ice; after the lysates were centrifuged (10000 ×g/min) at 4°C for 10min, the supernatant was collected and used as nuclear protein extracts. Protein concentrations were measured using BCA protein assay kit (Thermo Scientiﬁc, MA, USA). The samples were run on 10% SDS-polyacrylamide gels (20 μg/lane), and then, proteins were transferred to PVDF membranes by elec- troblotting (200 mA). PVDF membranes were incubated in 5% BSA for 2 hours at room temperature, followed by three 5 minutes washing in TBST. The PVDF membranes were then incubated overnight at 4°C with anti-Nrf2 (1: 1000), HO-1 (1: 1000), NQO1 (1 : 1000), Caspase-3 (CST, Danvers, MA, USA) (1 : 1000), GAPDH (CST, Danvers, MA, USA) (1 : 2000), and Lamin-B (CST, Danvers, MA, USA) (1 : 2000) antibodies. GAPDH and Lamin-B were used as internal normalizer. Then, the membranes were washed for 10min three times in TBST and incubated with IgG- HRP antibody (1 : 2000) for 2 hours and ﬁnally examined by chemiluminescence. 2.4. Apoptosis Assay by TUNEL. The apoptosis level in the bladder smooth muscle was measured by the one-step TUNEL apoptosis assay kit (KeyGen Biotech, Nanjing, China) according to the manufacturer’s instructions. In brief, the sections were regularly hydrated and immersed in 1% Triton X-100 followed by being incubated with proteinase K solution for 30 minutes. And the sections were reacted with TdT solution for 1 hour and then streptavidin- TRITC solution for 30 minutes in a humidiﬁed and dark chamber, respectively. 2. Materials and Methods Finally, DAB detection kit was used to stain the sections to observe and analyze apoptotic cells by a pathologist in a blinded manner. 2.5. MDA, GSH-Px, Total SOD (tSOD), ROS, and CAT Determination. According to our previous study [10], we choose some marker routine laboratory indexes of oxidative stress as malondialdehyde (MDA), glutathione peroxidase (GSH-Px), total superoxide dismutase (tSOD), and catalase activity (CAT) were conducted. The content of MDA, GSH-Px, total tSOD, and CAT in the bladder tissues was measured according to the manufacturer’s protocols of the assay kit of MDA, tSOD, GSH-Px, and CAT (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Brieﬂy, the maximum absorbance was at 532 nm based on thiobarbi- turic acid (TAB) method to detect MDA level, the tSOD activity was measured based on the combination of xanthine and xanthine oxidase and the absorbance was read at 550 nm, and the GSH-Px activity assay was performed using the enzyme-catalyzed reaction product (reduced glutathione) and the absorbance was recorded at 412 nm. To measure the CAT activity, hydrogen peroxide reacted with ammo- nium molybdate, and the absorbance at 405 nm was then detected. Considering accumulation of reactive oxygen spe- cies (ROS) occurs and leads to functional alterations and pathological conditions such as a bladder dysfunction devel- oped with age [6, 14, 15]; ROS was also detected in our study. According to the procedure of the ROS assay kit (Sigma- Aldrich, MO, USA), DCFDA was used as ﬂuorescent probe following by washing using assay buﬀer. Immediately after the excitation at a wavelength of 485 nm, the absorbance is read at 535 nm. 2.7. Statistical Analysis. The values were presented as mean ±SD. SPSS 17.0 was used to evaluate the diﬀerence. Diﬀerences between groups were analyzed using one-way ANOVA with P < 0 05 considered signiﬁcant. 3. Results H&E (a) and Masson trichrome (b) staining in the sham, BOO, and BOO + EGCG bladders and (c) the percentage of collagen ﬁbers in muscular layer in the sham, BOO, and BOO + EGCG bladders; ∗P < 0 05 n 6 versus the sham group; #P < 0 05 n 6 versus the BOO group 4 Oxidative Medicine and Cellular Longevity Sham 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (a) BOO 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (b) BOO + EGCG 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (c) Figure 1: Eﬀect of EGCG on urodynamic changes in conscious rats. Sham 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (a) BOO 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (b) BOO + EGCG 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (c) Figure 1: Eﬀect of EGCG on urodynamic changes in conscious rats. (b) (c) Figure 1: Eﬀect of EGCG on urodynamic changes in conscious rats. Sham BOO BOO + EGCG (a) Masson Sham BOO BOO + EGCG (b) Sham BOO BOO + EGCG (a) Masson Sham BOO BOO + EGCG (b) 40 30 20 10 The percentage of collagen fibers 0 Sham BOO BOO + EGCG ⁎ ⁎# (c) Figure 2: Eﬀect of EGCG on bladder histological changes in BOO rats. Original magniﬁcation ×100. H&E (a) and Masson trichrome (b) staining in the sham, BOO, and BOO + EGCG bladders and (c) the percentage of collagen ﬁbers in muscular layer in the sham, BOO, and BOO + EGCG bladders; ∗P < 0 05, n = 6 versus the sham group; #P < 0 05, n = 6 versus the BOO group. (b) (b) 40 30 20 10 The percentage of collagen fibers 0 Sham BOO BOO + EGCG ⁎ ⁎# (c) (c) Figure 2: Eﬀect of EGCG on bladder histological changes in BOO rats. Original magniﬁcation ×100. 3. Results 3.1. EGCG Ameliorated Voiding Dysfunction Induced by BOO. The cystometry results in each group are showed in Table 1 which were obtained according to the urodynamic curve (Figure 1). The voiding pressure was signiﬁcantly increased in obstructed rats compared with sham rats. The peak voiding pressure in obstructed rats with EGCG treatment is higher than obstructed rats at the 4-week time point. Bladder capacity was signiﬁcantly higher in BOO rats compared with sham rats. Rats in the BOO +EGCG group showed the highest bladder capacity among all groups. Bladder compliance decreased signiﬁcantly at the 4-week time point after BOO. EGCG may rescue the compliance deterioration through increasing bladder capacity. It was found that the interval of micturition was shorter in BOO 2.6. Western Blotting. Total protein was extracted from frozen bladder tissues selected randomly from each group (n = 3) by trypan blue in RIPA buﬀer containing protease inhibitors. Muscle lysates were centrifuged at 10000 ×g/min at 4°C for 10 min, and the supernatant was collected and used as total protein extracts. The nuclear protein was extracted Oxidative Medicine and Cellular Longevity 4 Sham 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (a) BOO 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (b) BOO + EGCG 60 40 20 0 0 2 4 6 Time (min) 8 10 Vesical pressure (cm H2O) (c) Figure 1: Eﬀect of EGCG on urodynamic changes in conscious rats. Sham BOO BOO + EGCG (a) Masson Sham BOO BOO + EGCG (b) 40 30 20 10 The percentage of collagen fibers 0 Sham BOO BOO + EGCG ⁎ ⁎# (c) Figure 2: Eﬀect of EGCG on bladder histological changes in BOO rats. Original magniﬁcation ×100. 3. Results (d) The activity of CAT in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. (e) The relative activity of ROS in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. 5 Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity 5 0 50 100 150 200 # The activity of total SOD (U/mg protein) ⁎ ⁎ Sham BOO BOO + EGCG (b) 0 1 2 3 4 # The content of MDA (nmol/ml) ⁎ Sham BOO BOO + EGCG (a) 0 50 100 150 200 # The activity of total SOD (U/mg protein) ⁎ ⁎ Sham BOO BOO + EGCG (b) 0 1 2 3 4 # The content of MDA (nmol/ml) ⁎ Sham BOO BOO + EGCG (a) (b) (a) (a) (b) 0 100 200 300 # The activity of GSH-Px (U/mg protein) ⁎ ⁎ Sham BOO BOO + EGCG (c) 0 2 4 6 8 # Catalase activity CAT (U/mg protein) ⁎ ⁎ Sham BOO BOO + EGCG (d) (c) (d) 0 100 200 300 # ROS level (% sham) ⁎ Sham BOO BOO + EGCG (e) (c) (d) 0 100 200 300 # ROS level (% sham) ⁎ Sham BOO BOO + EGCG (e) (e) Figure 3: Eﬀect of EGCG on oxidative stress in the bladder of BOO rats. (a) MDA level in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. (b) The activity of total SOD in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. (c) The activity of GSH-Px in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. (d) The activity of CAT in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. 3. Results H&E (a) and Masson trichrome (b) staining in the sham, BOO, and BOO + EGCG bladders and (c) the percentage of collagen ﬁbers in muscular layer in the sham, BOO, and BOO + EGCG bladders; ∗P < 0 05, n = 6 versus the sham group; #P < 0 05, n = 6 versus the BOO group. rats than in sham rats, and the micturition interval in BOO +EGCG rats was signiﬁcantly increased compared to BOO rats. staining. BOO caused obvious histological changes, such as the structural damage of detrusor smooth muscle while treat- ment with EGCG signiﬁcantly alleviated these histological changes in the bladders of BOO rats. The area ratio of colla- gen ﬁbers was 10.77 ±2.39, 32.53 ±4.15, and 23.41 ± 3.53 in the sham group, BOO group, and BOO+ EGCG group, respectively (Figure 2(c)). EGCG suppressed collagen ﬁbers 3.2. Eﬀect of EGCG in Bladder Detrusor of BOO Rats on Histological Changes. As showed in Figure 2(a), EGCG had some protective eﬀect on BOO bladder based on H&E 0 1 2 3 4 # The content of MDA (nmol/ml) ⁎ Sham BOO BOO + EGCG (a) 0 50 100 150 200 # The activity of total SOD (U/mg protein) ⁎ ⁎ Sham BOO BOO + EGCG (b) 0 100 200 300 # The activity of GSH-Px (U/mg protein) ⁎ ⁎ Sham BOO BOO + EGCG (c) 0 2 4 6 8 # Catalase activity CAT (U/mg protein) ⁎ ⁎ Sham BOO BOO + EGCG (d) 0 100 200 300 # ROS level (% sham) ⁎ Sham BOO BOO + EGCG (e) Figure 3: Eﬀect of EGCG on oxidative stress in the bladder of BOO rats. (a) MDA level in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. (b) The activity of total SOD in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. (c) The activity of GSH-Px in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. with BOO rats, EGCG treatments signiﬁcantly inhibited MDA level increase induced by BOO (1.81 ± 0.37 versus 2.81 ± 0.58). The activities of tSOD and GSH-Px had some content of decrease in BOO rats compared with sham rats (89.65 ±10.70 versus 108.06 ± 11.88; 174.71 ±11.68 versus 218.18 ± 12.44), respectively. However, EGCG treatment could signiﬁcantly increase the tSOD and GSH-Px activities in BOO rats (Figures 3(b) and 3(c)). As compared with sham rats, CAT activity signiﬁcantly decreased in the BOO group Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity 6 (P < 0 05), which was upregulated by EGCG, even com- pared with the sham group (Figure 3(d)). In contrast to CAT, ROS was increased in the BOO group as compared with the sham group, and EGCG decreased it signiﬁcantly (P < 0 05) (Figure 3(e)). [16, 17]. It is also suggested that oxidative stress plays a critical role in bladder outlet obstruction-mediated bladder dysfunction [16, 17]. BOO-induced bladder remodeling in a rat model is similar to that in patients with BPH who suﬀered from bladder outlet obstruction. It was showed in our previous study that rats of BOO model were useful models to explore structural and functional alterations in the bladder [10]. Therefore, exploring good ways to reduce oxidative stress of bladder caused by BOO has been considerably attractive. 3.4. EGCG Inhibited Cell Apoptosis and Prompted Proliferation in the Bladder of BOO Rats. TUNEL staining was performed to explore the eﬀect of EGCG on the cell apoptosis of the bladder in BOO rats. The number of apopto- tic cells in the bladder of BOO rats was markedly increased compared with sham rats (p < 0 01), whereas EGCG treat- ment signiﬁcantly decreased the number of apoptotic cells in the bladder of BOO rats (p < 0 05) (Figures 4(a) and 4(b)). Western blotting showed that the caspase-3 expression was signiﬁcantly increased in the bladder of BOO rats; however, the expression of caspase-3 was decreased in BOO +EGCG rats (Figure 4(c)) which was in accordance with the result of TUNEL staining. Moreover, the expres- sion of PCNA measured by the immunohistochemical staining showed that EGCG caused signiﬁcantly increase on cell proliferation in the BOO + EGCG group compared with the BOO group (Figure 4(d)). EGCG is a major catechin in green tea with functions of antioxidant, antiproliferative, anti-inﬂammatory, and attenuating metabolic syndrome, without adverse eﬀects [18, 19]. Hsieh et al. reported that EGCG can attenuate the prostate enlargement in BPH rats accompanied with meta- bolic syndrome induced by high-fat diet combined with testosterone injection [10]. For human, according to reports in the literature, ten days’ repeated administration of oral doses of EGCG of up to 800 mg per day was found to be safe and very well tolerated. The content of EGCG in green tea is about 10%. Oxidative Medicine and Cellular Longevity No obvious side eﬀects occur in the amount of 20 cups of green tea per day [20]. In the present study, we investigated the eﬀect of EGCG on BOO-induced bladder dysfunction in vivo and the daily intraperitoneal injection dose (5mg/kg EGCG) was used which was similar to the dose that has been proved eﬀective in other’s study [10, 21]. 3.5. EGCG Aﬀected Protein Expression of the Nrf2-ARE Pathway. Considering that Nrf2 is a vital mediator in regulat- ing cellular antioxidative response, we explored the eﬀect of EGCG on Nrf2 and its downstream target proteins in our study. The expression of Nrf2 measured by immunohisto- chemical staining was signiﬁcantly higher in the muscular layers of the bladder in the BOO + EGCG group compared with the BOO group (Figure 5(a)). The expression of Nrf2 in the cell (t-Nrf2) and nucleus (n-Nrf2) was also measured by Western blotting. It is shown in Figures 5(b) and 5(c) that Nrf2 expression (t-Nrf2 and n-Nrf2) was signiﬁcantly increased in the bladder of the BOO +EGCG group com- pared with the BOO group, and the t-Nrf2 was increased in the bladder of the BOO group compared with the sham group. It was worth noting that our results demonstrated the expression of Nrf2 was mainly located in the nucleus of the bladder cells while the expression of Nrf2 in the nucleus showed no signiﬁcant diﬀerence between the BOO group and the sham group. We then detected the expression of Nrf2’s downstream target proteins as HO-1 and NOQ1 to investigate its antioxidative function. Our data indicated that the expression of HO-1 was increased in the BOO + EGCG group compared with the BOO group, which was consistent with the result of n-Nrf2 expression (Figures 5(d) and 5(e)). The level of NQO1 in the BOO group was higher than that in the sham group, but lower than that in the BOO +EGCG group (Figure 5(f)). We performed urodynamic studies in conscious rats of BOO to accurately reﬂect the real situation. The cystometric parameters such as bladder capacity, maximal pressure, compliance, and micturition interval were measured; we found that the micturition interval, capacity, and compliance were signiﬁcantly increased, and the peak voiding pressure had some extent of increase by treatment with EGCG in BOO rats. The histological staining showed that bladder smooth muscle bundles were severely damaged and collagen deposition was increased in BOO rats. Oxidative Medicine and Cellular Longevity This increase of collagen deposition was suppressed by EGCG treatment for 4 weeks in BOO model rats. Our results indicated that there is an inverse relationship between bladder compliance and collagen ﬁbers, and EGCG could protect bladder from BOO-induced dysfunction and morphological damage. y p g g In order to investigate antioxidant activity of EGCG in the bladder of BOO rats, we measured MDA level of bladder tissue in the three groups. MDA level is used as an indicator of lipid peroxidation, as lipids in the cell membrane are destroyed to generate MDA in an amount proportional to the degree of tissue destruction. Our results showed that BOO signiﬁcantly increased the level of MDA in the bladder. It was also showed in our results that the increase in the level of MDA was signiﬁcantly reduced in BOO rats treated with EGCG which corresponds with other antioxidants [9]. We found that the activities of some redox status markers, such as GSH-Px and total SOD, were signiﬁcantly decreased in the BOO group compared with the sham group which is similar to the forecast result and other literature [10], and EGCG signiﬁcantly enhanced the activities of these markers in BOO rats. Our data also showed that EGCG could upreg- ulate CAT activity and downregulate ROS induced by BOO 3. Results (e) The relative activity of ROS in the bladder of the three groups, ∗P < 0 05 versus the sham group, n = 6; #P < 0 05 versus the BOO group, n = 6. in the BOO + EGCG group as compared with that in the BOO group (Figure 2(b)). with BOO rats, EGCG treatments signiﬁcantly inhibited MDA level increase induced by BOO (1.81 ± 0.37 versus 2.81 ± 0.58). The activities of tSOD and GSH-Px had some content of decrease in BOO rats compared with sham rats (89.65 ±10.70 versus 108.06 ± 11.88; 174.71 ±11.68 versus 218.18 ± 12.44), respectively. However, EGCG treatment could signiﬁcantly increase the tSOD and GSH-Px activities in BOO rats (Figures 3(b) and 3(c)). As compared with sham rats, CAT activity signiﬁcantly decreased in the BOO group 3.3. EGCG Attenuated Oxidative Stress in the Bladder of BOO Rats. To evaluate the level of oxidative stress, we measured the content of MDA, SOD, GSH-Px, CAT activity, and ROS. As showed in Figure 3(a), the content of MDA in the bladder was signiﬁcantly increased in BOO rats compared with sham rats (2.81 ± 0.58 versus 1.52 ± 0.33). Compared 4. Discussion As investigated in animal models, chronic bladder ischemia might produce oxidative leading to denervation of the blad- der and the expression of tissue-damaging molecules in the bladder wall, which could be responsible for the development of bladder hyperactivity progressing to bladder underactivity 7 Oxidative Medicine and Cellular Longevity TUNEL BOO BOO + EGCG Sham (a) 0 10 20 30 40 # The percentage of apoptotic cells Sham BOO BOO + EGCG ⁎ ⁎ (b) 0 2 4 6 8 10 # Relative caspase-3 expression Sham BOO BOO + EGCG ⁎ ⁎ (c) 0 10 20 30 40 50 # Relative PCNA expression GAPDH Caspase-3 Sham BOO BOO + EGCG Sham BOO BOO + EGCG ⁎ ⁎ (d) BOO BOO + EGCG PCNA Sham (e) gure 4: Eﬀect of EGCG on cell apoptosis and proliferation in BOO rats. (a) TUNEL staining showed the cell apoptosis level of the bladde l the three groups. (b) The statistical results of TUNEL staining in the three groups. ∗P < 0 01, n = 6 versus the sham group; #P < 0 01, n rsus the BOO group. (c) The protein expression of caspase-3 in the bladder of the three groups. (d-e) The statistical results of PCNA in adder of the three groups using immunohistochemical staining to show cell proliferation. ∗P < 0 05 versus the sham group; #P < 0 05 ver e BOO group. Original magniﬁcation ×200 for TUNEL and immunohistochemical staining. TUNEL Sham BOO + EGCG BOO (a) Sham BOO BOO + EGCG a) 0 2 4 6 8 10 # Relative caspase-3 expression Sham BOO BOO + EGCG ⁎ ⁎ (c) (a 0 10 20 30 40 # The percentage of apoptotic cells Sham BOO BOO + EGCG ⁎ ⁎ (b) (b) (c) 0 10 20 30 40 50 # Relative PCNA expression GAPDH Caspase-3 Sham BOO BOO + EGCG Sham BOO BOO + EGCG ⁎ ⁎ (d) PCNA (c) (b) ( ) 0 10 20 30 40 50 # Relative PCNA expression Sham BOO BOO + EGCG ⁎ ⁎ GAPDH Caspase-3 Sham BOO BOO + EGCG (d) BOO (e) (d) BOO + EGCG PCNA Sham BOO BOO + EGCG Sham (e) (e) Figure 4: Eﬀect of EGCG on cell apoptosis and proliferation in BOO rats. (a) TUNEL staining showed the cell apoptosis level of the bladder in all the three groups. 4. Discussion (b) The statistical results of TUNEL staining in the three groups. ∗P < 0 01, n = 6 versus the sham group; #P < 0 01, n = 6 versus the BOO group. (c) The protein expression of caspase-3 in the bladder of the three groups. (d-e) The statistical results of PCNA in the bladder of the three groups using immunohistochemical staining to show cell proliferation. ∗P < 0 05 versus the sham group; #P < 0 05 versus the BOO group. Original magniﬁcation ×200 for TUNEL and immunohistochemical staining. Nrf2 Sham BOO BOO + EGCG (a) 0 1 2 3 4 # Relative total Nrf2 expression GAPDH t-Nrf2 Sham BOO BOO + EGCG Sham BOO BOO + EGCG ⁎ ⁎ (b) 0 1 2 3 4 # Relative nuclear Nrf2 expression Lamin-B n-Nrf2 Sham BOO BOO + EGCG Sham BOO BOO + EGCG ⁎ (c) HO-1 Sham BOO BOO + EGCG (d) 0 2 4 6 8 10 # Relative HO-1 expression Sham BOO HO-1 BOO + EGCG GAPDH Sham BOO BOO + EGCG ⁎ ⁎ (e) 0 5 10 15 # Relative NQO1 expression Sham BOO + EGCG GAPDH BOO NQO1 Sham BOO BOO + EGCG ⁎ ⁎ (f) ure 5: Eﬀect of EGCG on protein expression of the Nrf2-ARE pathway. (a) The immunohistochemical staining of Nrf2. Pr ession of total Nrf2 (t-Nrf2) and nuclear Nrf2 (n-Nrf2) in the bladder of the three groups measured by Western blotting is show and (c). The protein expression of HO-1 measured by immunohistochemical staining and Western blotting in the bladder of the ps is shown in (d) and (e). (e) The protein expression of NQO1 in the bladder of the three groups. ∗P < 0 05 versus the sham g 0 05 versus the BOO group. Acknowledgments This research was supported by the Program of Shanghai City Committee of Science and Technology (15DZ1941502 and 15DZ1941503), Shanghai Pudong New Area Health and Family Planning Project (no. PW2013D-3), Key Disci- plines Group Construction Project of Pudong Health Bureau of Shanghai (PWZxq2014-11), and Program for Outstanding Medical Academic Leader and Speciality Construction Pro- ject of Pudong Health and Family Planning Commission of Shanghai (no. PWZz2013-16). Authors’ Contributions Meng Gu and Chong Liu contributed equally to this study. 4. Discussion Oxidative Medicine and Cellular Long Oxidative Medicine and Cellular Longevity 8 Nrf2 Sham BOO BOO + EGCG (a) 0 1 2 3 4 # Relative total Nrf2 expression GAPDH t-Nrf2 Sham BOO BOO + EGCG Sham BOO BOO + EGCG ⁎ ⁎ (b) 0 1 2 3 4 # Relative nuclear Nrf2 expression Lamin-B n-Nrf2 Sham BOO BOO + EGCG Sham BOO BOO + EGCG ⁎ (c) Nrf2 Sham BOO + EGCG BOO (a) BOO BOO + EGCG (a) G 0 1 2 3 4 # Relative nuclear Nrf2 expression Lamin-B n-Nrf2 Sham BOO BOO + EGCG Sham BOO BOO + EGCG ⁎ (c) Sham ( 0 1 2 3 4 # Relative total Nrf2 expression GAPDH t-Nrf2 Sham BOO BOO + EGCG Sham BOO BOO + EGCG ⁎ ⁎ (b) (b) (c) BOO (d) BOO + EGCG HO-1 Sham Sham 0 2 4 6 8 10 # Relative HO-1 expression Sham BOO HO-1 BOO + EGCG GAPDH Sham BOO BOO + EGCG ⁎ ⁎ (e) 0 5 10 15 # Relative NQO1 expression Sham BOO BOO + EGCG ⁎ ⁎ (f) (f) (e) Figure 5: Eﬀect of EGCG on protein expression of the Nrf2-ARE pathway. (a) The immunohistochemical staining of Nrf2. Protein expression of total Nrf2 (t-Nrf2) and nuclear Nrf2 (n-Nrf2) in the bladder of the three groups measured by Western blotting is shown in (b) and (c). The protein expression of HO-1 measured by immunohistochemical staining and Western blotting in the bladder of the three groups is shown in (d) and (e). (e) The protein expression of NQO1 in the bladder of the three groups. ∗P < 0 05 versus the sham group; #P < 0 05 versus the BOO group. Oxidative Medicine and Cellular Longevity 9 the Nrf2-ARE pathway and suppressing cellular apoptosis in the bladder of BOO rats. compared with the sham group. It was worth noting that increase of CAT activity in bladder tissue of BOO rats after 4 weeks was similar to our previous study [22], while not all match the other report that CAT activity increased after 2–4 weeks of obstruction, but it decreased markedly in the rabbit bladder with decompensation 8 weeks after partial bladder outlet obstruction (PBOO) [7], which need us to further explore. All the results may indicate that EGCG could alleviate oxidative stress by increasing the activities of antioxidative enzymes in BOO rats. Conflicts of Interest EGCG reported recently can exert a protective eﬀect on rats with obstructive nephropathy by activating the Nrf2 signaling pathway. The H2O2-exposed hMSCs showed cellu- lar senescence with signiﬁcantly increased protein levels of acetyl-p53 and acetyl-p21 in comparison with the control hMSCs which was prevented by pretreatment with EGCG [21, 23]. By contrast, in Nrf2-knockdown hMSCs, EGCG lost its antioxidant eﬀect, exhibiting high levels of acetyl-p53 and acetyl-p21 following EGCG pretreatment and H2O2 expo- sure. This indicates that Nrf2 may be involved in the antise- nescent eﬀect of EGCG in hMSCs [10]. Taken together, these ﬁndings suggested the important role of EGCG in preventing oxidative stress-induced cellular senescence and apoptosis through Nrf2 activation. Antioxidants could ameliorate bladder dysfunction in BOO rats according to our previous study [10]; the eﬀect of EGCG on ameliorating bladder dysfunction in BOO model and changing the Nrf2-ARE sig- naling pathway was investigated in present study. By immu- nohistochemical staining and Western blotting, we found that the expression of Nrf2 protein in the bladder of BOO rats with EGCG treatment was signiﬁcantly increased compared to BOO rats. In addition, the level of Nrf2 in the nucleus was markedly increased in the BOO rats when treated with EGCG. The result indicated that EGCG could promote the transportation of Nrf2 into nucleus and the transcription of its target antioxidant genes. We next mea- sured the expression level of HO-1 and NQO1, the down- stream genes of Nrf2-ARE pathway by Western blotting. The levels of HO-1 and NQO1 increased signiﬁcantly in BOO rats after EGCG treatment. Above all, our study sug- gested that EGCG could promote Nrf2’s translocation into the nucleus and the expression of its target antioxidant genes, which ameliorated the oxidative stress in BOO rats. The authors declare that they have no competing interests. Abbreviations ARE: Antioxidant response element BOO: Bladder outlet obstruction BPH: Benign prostatic hyperplasia GSH-Px: Glutathione peroxidase CAT: Catalase activity H&E: Hematoxylin and eosin HO-1: Heme oxygenase-1 LUTS: Lower urinary tract symptoms MDA: Malondialdehyde NQO1: NAD(P)H : quinone oxidoreductase 1 Nrf2: Nuclear erythroid-related factor 2 PE-50: Polyethylene tubing 50 ROS: Reactive oxygen species EGCG: Epigallocatechin-3-gallate SOD: Superoxide dismutase TUNEL: TdT-mediated dUTP nick end labeling. It is well known that cell apoptosis plays a vital role in leading to bladder dysfunction in BOO model. In our study, TUNEL staining results manifested that a signiﬁcant reduc- tion of the number of apoptotic cells was found in the treatment of EGCG in BOO rats; it is also found that the expression of caspase-3 in the bladder was decreased in BOO rats when treated with EGCG. These results suggested that EGCG as an antioxidant might protect bladder against BOO-induced apoptosis via decreasing the expression of caspase-3. Meanwhile, the eﬀect of EGCG on cell prolifera- tion by PCNA staining was also measured. Results showed that the cell proliferation level was signiﬁcantly higher in the bladder of BOO rats with EGCG treatment. Oxidative Medicine and Cellular Longevity 10 2011,” Neurourology and Urodynamics, vol. 31, no. 3, pp. 322– 326, 2012. [18] R. A. Isbrucker, J. A. Edwards, E. Wolz, A. Davidovich, and J. Bausch, “Safety studies on epigallocatechin gallate (EGCG) preparations. 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https://openalex.org/W1557239369	https://journals.abcjournal.aosis.co.za/index.php/abc/article/download/827/778	English	null	An overview of <i>Penicillium</ (Hyphomycetes) and associated teleomorphs in southern Africa	Bothalia	1,992	cc-by	16,454	INTRODUCTION activity, correlated with penicillus types, as well as growth rates at 5°C and 37°C, as differential criteria. This concept was later fully developed in a monograph (Pitt 1979). Shortly afterwards, a well-illustrated atlas of penicillia by Ramirez (1982) was published. However, the value of Pitt’s (1979) guide to the taxonomy of Penicillium was confirmed at the First international Penicillium and Aspergillus workshop (Samson & Pitt 1985), when Pitt’s species concept and methods were incorporated in the recommen dations for future taxonomic practice in this genus. ‘Species of Penicillium are so abundant and so con spicuous in all sorts of stale or decaying organic matter that they constitute a part of the common conception of mould, and are loosely referred to as ‘blue’ or ‘green’ mould' (Raper & Thom 1949). Representatives of this multi-faceted genus are of ecological importance because they are abundant and widespread in the environment; they are fruit deteriorators and contribute greatly to post-harvest decay; they have industrial applications such as in cheese- making; and they produce secondary metabolites and mycotoxins, including the indispensable antibiotics. Previously, the name Penicillium was applied to both the hyphomycetous and ascomycetous states. However, separation of the teleomorphic states of Penicillium from the anamorph, as implemented by Pitt (1979), is in accor dance with Art. 59 of the International Code of Botanical Nomenclature and is of practical value for the taxonomist. Stolk & Scott (1967) re-introduced the use of the teleo- morph name Eupenicillium Ludwig for a portion of the genus Penicillium. Monographic contributions to the genus Eupenicillium were made by Scott (1968a, b) and Stolk & Samson (1983). The teleomorphic genus Talaromyces C.R. Benjamin is separated from Eupenicillium on the basis of ascocarp morphology. The former genus is charac terised by the production of gymnothecia composed of loosely intertwined hyphae, as opposed to cleistothecia. Stolk & Samson (1972) as well as Pitt (1979) have contributed to the taxonomy of this group. The generic name Penicillium (Latin, penicillus = little brush) was first introduced in 1809 by Link who very briefly described the genus with three species, namely P. candidum Link, P expansum Link and P. glaucum Link. The true identity of these fungi has been difficult to determine, but an apple-rotting fungus was linked to P. expansum by Thom (1910). Bothalia 22,1: 7 7 -9 1 (1992) Bothalia 22,1: 7 7 -9 1 (1992) An overview of Penicillium (Hyphomycetes) and associated teleomorphs in southern Africa A.L. SCHUTTE* Keywords: Eupenicillium, fungi, Hyphomycetes, Penicillium, overview, southern Africa, Talaromyces, taxonomy * Mycology Unit, Plant Protection Research Institute, Private Bag X134, Pretoria 0001. MS. received: 1991-09-26. UITTREKSEL Literatuur aangaande die hifomiseetgenus Penicillium Link en sy teleomorwe Eupenicillium Ludwig en Talaromyces C.R. Benjamin in die Republiek van Suid-Afrika, Lesotho, Mosambiek, Namibie, Swaziland en Transkei is nagegaan tot 1990. Die verwysings word gegroepeer onder die opskrifte mikologie, plantpatologie, industriele toepassing. mediese belang. mikotoksiene en chemiese werk. 'n Alfabetiese lys van die spesies wat in suidelike Afrika aangeteken is, asook die gasheer en/of substraat waarop elke spesie aangemeld is, word met die toepaslike verwysings gegee; eksemplare in verskeie fungus- versamelings word ook ingesluit. Alhoewel die meeste van die bekende PenicilUum-spcsies reeds in suidelike Afrika aangeteken is, is diepgaande werk op alle navorsingsgebiede random hierdie genus steeds nodig. ABSTRACT Literature on the hyphomycete genus Penicillium Link and its teleomorphs, Eupenicillium Ludwig and Talaromyces C.R. Benjamin, is surveyed in the Republic of South Africa, Lesotho, Mozambique, Namibia, Swaziland and Transkei up to 1990. References are grouped under the headings, general mycology, plant pathology, industrial application, medical importance, mycotoxins and chemical work. An alphabetical list of the species recorded in southern Africa as well as the host and/or substrate from which each species has been reported is presented with relevant literature references; specimens in various culture collections are also incorporated. Although most of the known Penicillium species have already been reported from southern Africa, in-depth work is still required in all fields of research concerning this genus. INTRODUCTION Although the validity of the generic name has been questioned over the years, Hawksworth (1985) concluded that Penicillium Link should be considered correct and indicated that he had previously designated a neotype of P. expansum Link as the type species of the genus. Succeeding the works of Thom (1910, 1930), the manual by Raper & Thom (1949) has been the standard work on Penicillium for nearly 30 years. Subsequently, a new era in Penicillium identification was heralded by Pitt (1973), who used the ability of isolates to grow at reduced water A multidisciplinary approach to the identification of Penicillium is becoming more prevalent (Bridge et al. 1985). Protein electrophoresis (Bent 1967), the API ZYM testing system (Bridge & Hawksworth 1984), pyrolysis gas chromatography (Soderstrom & Frisvad 1984), physio logical and biochemical methods (Bridge 1985), enzyme electrophoresis (Cruickshank & Pitt 1987), studies on Bothalia 22,1 (1992) Bothalia 22,1 (1992) 78 however, as the name is no longer in use and Raper & Thom (1949) referred to P. gratioti only as: ‘apparently some member of the P. janthinellum series’. thermal denaturation of DNA (Paterson et al. 1990), elec tron microscopy (Ramirez 1982; Kozakiewicz 1989) and the production of secondary metabolites and mycotoxins (Frisvad & Filtenborg 1983; Frisvad et al. 1990) have recently been used to supplement traditional methods of identification. Numerous penicillia have been reported subsequently in general surveys of fungi on various substrates. Cohen (1950) conducted the first survey of soil fungi in South Africa, comparing the effect of different burning and grazing treatments, and he recorded nine Penicillium species. Scott (1968a) described eight new Eupenicillium species from soil and included these in a more extensive monograph of the genus (Scott 1968b). Penicillium was found to be the genus of Fungi Imperfecti with the largest number of species represented in Zululand soil (Eicker 1969). The same locality yielded P. olsonii Bain. & Sartory throughout the soil profile, whereas P. javanicum Van Beyma showed a marked decrease with increasing soil depth (Eicker 1970). Eicker (1973) found the penicillia to have an even distribution in different litter layers of Eucalyptus maculata Hook. f. and later found the genus to be common on Panicum coloratum L. litter (Eicker 1976). P. cyclopium Westling was isolated from angora goat dung, but Mitchel (1970) indicated that it was probably an aerial contaminant. INTRODUCTION Members of this genus identified at the Mycology Unit in recent years were often found to differ somewhat from the descriptions given by Pitt (1979). This raised the ques tion of whether these variations are consistent for all South African isolates. In addition, preliminary investigations indicated that Penicillium species are frequently only briefly mentioned in publications or included in lists of fungi from surveys. It was therefore considered advan tageous to gather this scattered information in order to compile a list of Penicillium species recorded in southern Africa, to bring this information in line with modem taxonomic systems, and to indicate areas requiring further research. This paper is an overview of publications dealing with all aspects of the Penicillium species reported in South Africa, Lesotho, Mozambique, Namibia, Swaziland and Transkei up to 1990. Literature is grouped according to various fields of research and presented in chronological order. The National Collection of Fungi, including the dried collection and the culture collection, collections donated to the Mycology Unit, the collection of the Medical Research Council as well as catalogues of inter national culture collections, served as additional sources of information. Foreign isolates used for chemical work, have been mentioned but not listed. No attempt has been made to verify published data, the identity of Penicillium isolates, or any other information. High quality stored maize obtained from six localities, studied by Van der Westhuizen & Bredell (1972) was found to have a high percentage of Penicillium spp., with P. oxalicum Currie & Thom often comprising 30% of the fungi recorded. On stored lucerne seed, species of this genus did not increase during an increased period of storage (Marasas & Bredell 1973). The composition and distribution of soil fungi in the western Transvaal was studied by Papendorf (1976) and one of his isolates, described as the new species P. striatosporum Stolk (Stolk 1969), was later re-identified by Pitt (1979) as P. restric- tum Gilman & Abbott. Penicillium spp. were found to be scarce on leaves and litter of Cenchrus ciliaris L. (Bezuidenhout 1977), in aerospora of an Eragrostis curvula (Schrad.) Nees pasture (Van der Merwe et al. 1979) and in the soil of Kaokoland, Namibia (Eicker et al. 1982). Many of the above-mentioned species are included in the checklist and bibliography of South African fungi com piled by Gorter (1979) for the period 1947-1977. Industrial applications Penicillia encountered in industry were first reported by Van der Bijl (1920) in his study of the deterioration of cane sugar crystals and solutions in storage. This record is also of taxonomic interest as two of these Penicillium isolates had been sent to Thom, whose comments accom panying the identifications are included. One of these isolates was deposited in PREM: 14262 P. luteum- purpurogenum group. P. digitatum on citrus was reported by Pole Evans (1911) who stressed the importance of good sanitation in orchards to combat this fungus. To determine the presence of patho genic fungal spores at the Cape harbour, Pole Evans (1920) exposed agar plates in the railway trucks and in cold storage rooms on the docks and on the ships. Davel & Neethling (1930) dealt with fungi in dairies and mentioned the use of P. camembertii Thom, P. glaucum and P. roquefortii Thom in cheese factories, indicating that members of this group can be troublesome in these surroundings. Coles (1925) recorded P. glaucum on Stilton and Wenslydale cheese and Radmore (1986) did a micro biological study of air in dairies. Other work done on penicillia in the dairy industry is discussed under the heading ‘Mycotoxins’. These pathogens were later listed by Verwoerd (1929). Doidge & Van der Plank (1936) subsequently conducted a survey on the fungi causing rot of oranges and lemons, indicating P. digitatum as the most important, with P. italicum and P. verrucosum Dierckx also present. They (Doidge & Van Der Plank 1936) remarked that although a large number of additional Penicillium spp. were isolated during the survey, no attempt was made to identify these species which were apparently saprophytic and growing on decaying tissues. Van der Plank (1945) did experimental work with hypochlorous acid as a bleach and disinfectant of citrus fruit, finding it effective against P. digitatum conidia. Martin (1960) listed seven saprophytic Penicillium species in citrus soil and found five species in adjacent virgin soil. Other Penicillium species of plant pathologi cal interest were mentioned by Doidge et al. (1953), Roth (1967), Wager (1972) and Gorter (1977). The bulb pathogen P. corymbiferum Westling, isolated by Wager, was de posited in the IMI culture collection where it was examined by Pitt (1979). An interesting use for Penicillium was found in reducing the stickiness of molasses meal (Roth 1968), for which P. notatum Westling was used on a commercial scale. Plant pathology During the early 1900’s the deteriorators, P. digitatum, P. expansum Link and P. italicum Wehmer, became a major problem for the fruit producing industry by hampering exports to Europe (Pole Evans 1920). Most of the South African isolates mentioned by Thom (1930) had been sent to the USA for identification by V.A. Putterill. Putterill was in charge of a mycological laboratory in Cape Town in 1918, and later worked at the fruit inspection service (Doidge 1950). These first South African Penicil lium records probably concerned fruit rot, although they are listed as having an undetermined host. General mycology 79 Bothalia 22,1 (1992) Apparently the type material of this fungus has been lost (C. Ramirez pers. comm.). Apparently the type material of this fungus has been lost (C. Ramirez pers. comm.). 1988). This fungus was able to decompose filter paper as well as detached groundnut pods. Surface disinfected roots of Medicago spp. yielded eight different Penicillium spp. (Lamprecht et al. 1988). P. spinulosum Thom was found to be pathogenic on onions (Naude & Jooste 1989) and P. hirsutum Dierckx on bulbs of flowering plants (Schutte 1990). 1988). This fungus was able to decompose filter paper as well as detached groundnut pods. Surface disinfected roots of Medicago spp. yielded eight different Penicillium spp. (Lamprecht et al. 1988). P. spinulosum Thom was found to be pathogenic on onions (Naude & Jooste 1989) and P. hirsutum Dierckx on bulbs of flowering plants (Schutte 1990). Additional reports of South African isolates may be found in the monographs on Penicillium by Thom (1930), Raper & Thom (1949), Pitt (1979) and Stolk & Samson (1983), as well as in catalogues of international culture collections. Unidentified members of the genus were reported on Japanese radish seed (Holtzhausen 1978), groundnuts (Ferreira & Lutchman 1989), recalcitrant seed (Berjak et al. 1989; Mycock & Berjak 1990), barley seed (Liibben et al. 1989) and maize cultivars (Rheeder et al. 1990). Industrial applications Although photographs of eight different Penicillium spp. are included, only the series to which they belong are given. The wine industry noted various identified and unidentified Penicillium spp. on grapes (Le Roux et al. 1973), their incidence on healthy grapes being 60% and on Botrytis infected fruit 70%. Heat resistant fungi posing problems for apple juice canners, turned out to be teleomorphs of P. vermiculatum Dangeard and P. brefeldianum Dodge (Van der Spuy et al. 1975). This work is referred to world-wide in connec tion with heat resistance of fungal spores. The thermophilic Talaromyces dupontii Griffen & Maublanc. was isolated during a study of fungi in mushroom compost (Eicker 1977). Penicillium species encountered later when various casings for mushroom production were tested, were indicated as potentially harmful (Smit 1984). Martin & Keen (1978) found P. crustosum to be common in home made beer as well as on sorghum malt used for brewing. A low incidence of Penicillium spp. on commercial and industrial sorghum malt was reported by Rabie & Liibben (1984). Matthee (1968) studied P. expansum, the pathogen and deteriorator of stored pome fruits, and indicated that older or bruised fruit was more susceptible. Holtzhausen & Knox-Davies (1974) used this fungus as an experimental organism in chemical seed treatments. Combrink et al. (1980) found that a longer exposure time of apples to a sodium hypochlorite solution had a better fungicidal effect on P. expansum conidia than a stronger solution. P. funiculosum Thom reportedly caused a core rot of apples and formed a moist infection (Combrink et al. 1985). Members of the genus were also isolated from litchi fruit (Roth 1963), bananas (Roth & Loest 1965) and mangoes (Wehner et al. 1981). P. pinophilum Hedgcock apparently enhances disease symptoms of groundnut pods in the presence of Chalara elegans Nag Raj & Kendrick (Baard General mycology The first published record of the genus Penicillium in southern Africa appears to be that of P. digitatum (Pers. ex Fr.) Sacc. on citrus (Pole Evans 1911). In this publica tion Pole Evans mentioned that in 1903, the Government Entomologist for Natal reported great losses to the orange crop, due to a mould. He noted that he had collected the causative fungus, P. digitatum, from fallen oranges in the Northern Transvaal five years before (i.e in 1906). Doidge (1950) listed all Penicillium species recorded up to 1945, including specimens in the Collection of the Timber Research Laboratories, Chamber of Mines, Johannesburg, as well as those mentioned by Thom (1930). Allsop et al. (1987) found a more varied fungal flora present in the rhizosphere than in the non-rhizosphere area of a fynbos site; several Penicillium species were reported, including P. novae-zeelandiae Van Beyma and Eupenicil lium pinetorum Stolk, reported in South Africa for the first time. McLean & Berjak (1987) studied the mycoflora of maize and indicated P. variabile Sopp as the most frequent internal contaminant of maize seed, while P. brexicom- pactum Dierckx was isolated from 15% of the seedlings. Wittaker et al. (1989) reported a decline in Penicillium species after hot water treatment of stored maize seed, penicillia were found to be present on Eucalyptus (Lund- quist & Baxter 1985), Pinus in the Transvaal (Lundquist 1986), Pinus in the Cape (Lundquist 1987) and common on stored seed of indigenous plants (Isaacs & Benic 1990). P. crustosum Thom and P. purpurescens (Sopp) Biourge have been indicated as endophytes of grass species (De Villiers 1989). Ramirez (1990) based the description of P. krugeri Ramirez on 26 isolates collected from soil at different localities in the Kruger National Park in 1987. The Penicillium specimen accessioned in the National Collection of Fungi first was lP armeniacum Berk’ (PREM 187— see checklist), recorded by the Government Laboratories Johannesburg, on Zea mays on 12 September 1906. This fungus was not a Penicillium, however, but probably belongs in Monilia (Thom 1930). The second Penicillium entry, ‘P. gratioti Sartory’ (PREM 5587— see checklist), was recorded by PA. van der Bijl from the City Deep Mine in Johannesburg, on 7 December 1912. Thom (1930) provided more data about this isolate, recording its optimum temperature and utili zation of various sugars. Its true identity is not clear. Mycotoxins The discovery in the 1960’s of aflatoxin and its carcino genic effects created renewed interest in fungal contamina tion. In the search for members of the aflatoxin-producing Aspergillus flavus group, numerous species of the closely related genus Penicillium were also encountered and details of their distribution recorded. Scott (1965), the first South African to test fungi for toxicity by feeding day old ducklings with infected meal, found P. islandicum Sopp, P. oxalicum, P. rubrum Stoll and P. urticae to be acutely toxic, whereas P. piceum Raper & Fennell had a less severe effect. This paper subsequently became a citation classic. The fungal flora of stock feeds, and the incidence of toxicity, was investigated by Van Warmelo (1967), who found that Penicillium had a low incidence on these substrates. Wehner & Rabie (1970) did toxicity tests with micro-organisms from nuts and dried fruit, including P. frequentans Westling, P. notatum and three unidentified Penicillium spp., none of which turned out to be toxic. Holzapfel (1968), Steyn et al. (1975), McGrath et al. (1976) and Neethling & McGrath (1977) studied various aspects of cyclopiazonic acid (e.g. biosynthesis, structure and production), a toxic metabolite of P. cyclopium. However, Frisvad (1989) stated that the isolate used for all the above-mentioned cyclopiazonic acid work, namely CSIR 1085, was not P. cyclopium but P. griseofulvum Dierckx. Pitt came to the same conclusion as indicated by De Jesus et al. (1981). Frisvad (1989) stated that P. viridicatum (CSIR 1029) used by Hutchison et al. (1973) had also been misidentified. Martin (1974) compiled a table of all information avail able on mycotoxin-producing fungi, dividing them into field and storage fungi. Mutagenicity of Penicillium mycotoxins to Salmonella typhimurium was studied by Wehner et al. (1978) and negative results were reported for griseofulvin, patulin and penicillic acid. In a similar study, the mycotoxin emodin, produced by P. rugulosum Thom, was found to be a frameshift mutagen (Wehner et al. 1979). As no local isolates were mentioned in the above- mentioned work, the species concerned have not been included in the appended list. Various mycotoxins other than the above-mentioned were studied locally. Oxalin produced by P. oxalicum received attention from Nagel et al. (1976), Vleggaar & Wessels (1980) and Steyn & Vleggaar (1983), while PR toxin produced by P. roquefortii was studied by Gorst- Allman & Steyn (1982). Certain isolates of P. Chemical work Chemical work A variety of Penicillium mycotoxins have been extracted and characterized in South Africa. Steyn (1969) described a new, rapid and sensitive system for the separation and detection of eleven different mycotoxins, followed by work on secalonic acid D, a toxic metabolite of P. oxalicum (Steyn 1970). The isolation of viridicatum toxin from P. viridicatum Westling was reported by Hutchison et al. (1973). Nagel et al. (1972) reported on the production of the highly toxic citreoviridin and made a study of the morphological characteristics of various isolates of its producer, P. puhillorum Turfitt. Steyn et al. (1982) studied the biosynthesis of the above-mentioned citreoviridin. Medical importance Marasas & Van Rensburg (1986) found this genus most prevalent on crops in the area where Mseleni joint disease occurs in Kwazulu. Some of the work mentioned under the heading “Mycotoxins1 also has a medical application. milk curd. Kriek & Wehner (1981) proved the toxicity of P. i tali cum, isolated from an orange, to laboratory animals. The effect of maize meal infected with this fungus was not as detrimental to ducklings as to rats. The nature of the lesions observed in rats was similar to those caused by the toxic P. islandicum. Dutton & Westlake (1985) found the incidence of Penicillium spp. as well as contamination by its mycotoxins to be low on cereal and animal feedstuffs. Kellerman et al. (1988) implicated Penicillium as a mycotoxin producer but gave no examples. The Medical Research Council tested various isolates of 30 Penicillium spp. for toxicity to ducklings and found most to have a detrimental effect (C.J. Rabie pers. comm.). All isolates were identified by J.I. Pitt and are listed under the abbreviation MRC. These authors all studied the relation ship between fungi and mycotoxins, but the mycotoxins themselves called for more detailed chemical studies. milk curd. Kriek & Wehner (1981) proved the toxicity of P. i tali cum, isolated from an orange, to laboratory animals. The effect of maize meal infected with this fungus was not as detrimental to ducklings as to rats. The nature of the lesions observed in rats was similar to those caused by the toxic P. islandicum. Dutton & Westlake (1985) found the incidence of Penicillium spp. as well as contamination by its mycotoxins to be low on cereal and animal feedstuffs. Kellerman et al. (1988) implicated Penicillium as a mycotoxin producer but gave no examples. The Medical Research Council tested various isolates of 30 Penicillium spp. for toxicity to ducklings and found most to have a detrimental effect (C.J. Rabie pers. comm.). All isolates were identified by J.I. Pitt and are listed under the abbreviation MRC. These authors all studied the relation ship between fungi and mycotoxins, but the mycotoxins themselves called for more detailed chemical studies. Medical importance Although members of the genus are known to cause allergies and to produce mycotoxins, Penicillium is men tioned infrequently in literature on medical mycology. Bothalia 22.1 (1992) 80 Bothalia 22.1 (1992) Fungal allergy was the motivation for three five-year surveys of aerospora, two done in Johannesburg (Ordman & Etter 1956; Ordman 1963) and one in Windhoek (Ordman 1970). Penicillium made up about 10% of the fungi isolated and showed no seasonal prevalence. Fungal contamination of food was investigated by Gilman (1972), in an attempt to correlate diet and liver cancer in man and a variety of identified penicillia were listed. Antimycotic and antibacterial activity of soil fungi was studied by Eicker (1975) who found positive effects against both organisms, by P. chrysogenum and P. cyclopium. Horwitz & Wehner (1977) warned that the presence of antibiotics produced by P. chrysogenum Thom used in salami curing may pose a health hazard for persons sensitive to penicil lin. Penicillium was also amongst the fungi present on com believed to be the cause of oesophageal cancer in Transkei and in the high rate area of the disease, 43 % of the samples were infected with this organism (Marasas et al. 1981). Marasas & Van Rensburg (1986) found this genus most prevalent on crops in the area where Mseleni joint disease occurs in Kwazulu. Some of the work mentioned under the heading “Mycotoxins1 also has a medical application. Fungal allergy was the motivation for three five-year surveys of aerospora, two done in Johannesburg (Ordman & Etter 1956; Ordman 1963) and one in Windhoek (Ordman 1970). Penicillium made up about 10% of the fungi isolated and showed no seasonal prevalence. Fungal contamination of food was investigated by Gilman (1972), in an attempt to correlate diet and liver cancer in man and a variety of identified penicillia were listed. Antimycotic and antibacterial activity of soil fungi was studied by Eicker (1975) who found positive effects against both organisms, by P. chrysogenum and P. cyclopium. Horwitz & Wehner (1977) warned that the presence of antibiotics produced by P. chrysogenum Thom used in salami curing may pose a health hazard for persons sensitive to penicil lin. Penicillium was also amongst the fungi present on com believed to be the cause of oesophageal cancer in Transkei and in the high rate area of the disease, 43 % of the samples were infected with this organism (Marasas et al. 1981). DISCUSSION Union of South Africa Department of Agriculture Reprint No. 20 : 1- 10. COMBRINK, J.C., KOTZE, J.M .. WEHNER, F.C. & GROBBELAAR, C.J. 1985. Fungi associated with core rot of Starking apples in South Africa. Phytophylactica 17: 81-83. COMBRINK, J.C.. MATTHEE. F.N. & KOCK. J.S. 1980. Stability of sodium hypochlorite solutions and their effectiveness against conidia of Penicillium expansum Thom. Phytophylactica 12: 85-87. The role that penicillia play in the ecology of natural ecosystems as well as in cultivated areas, has not been investigated in this country. Certain Penicillium species have antimycotic as well as antibacterial activities (Eicker 1975). Others are strongly antagonistic to soil-borne plant pathogens such as Gaeumarmomyces, Pythium and Rhizoc- tonia, whereas some members of Talaromyces have antifungal as well as antiprotozoal capacities (Domsch et al. 1980). Biological control of plant pathogens by Penicil lium species deserves attention, as it may well be of economic importance. CRUICKSHANK. R.H. & PITT. J.I. 1987. Identification of species in Penicillium subgenus Penicillium by enzyme electrophoresis. Mycologia 79: 614—620. DAVEL. H.B. & NEETHLING. H.L. 1930. Skimmels en hid bestryding in die melkery. Union of South Africa Department of Agriculture Science Bulletin No. 80: 1—18. DE JESUS, A.E., GORST-ALLMAN, C .P, STEYN, PS., VAN HEERDEN, F.R.. VLEGGAAR. R & WESSELS. PL. 1983a. Tremorgenic mycotoxins from Penicillium crustosum. Biosynthesis of Penitrem A. Journal of the Chemical Society Perkin Trans 1 8: 1863-1983. DE JESUS. A.E., STEYN, P.S., VAN HEERDEN. F.R. & VLEG GAAR. R. 1984. Structure elucidation of the janthitrems. novel tremorgenic mycotoxins from Penicillium janthinellum. Journal of the Chemical Society Perkin Trans 1 4: 697-701. The successful use in Penicillium taxonomy of physio logical and various biochemical methods, mycotoxin profiles and electron microscopy, has been indicated. However, these techniques have not yet been applied to this genus in South Africa and may be of value in determining relationships between species and groups as well as indicating new species. DE JESUS, A.E., STEYN. PS'. VAN HEERDEN. F.R . VLEGGAAR, R. & WESSELS. PL. 1983b. Tremorgenic mycotoxins from Penicillium crustosum: isolation of pemtrems A-F and the structure elucidation and absolute configuration of penitrem A1. Journal of the Chemical Society Perkin Trans I 8: 1847-1983. DEJESUS. A.E.. STEYN. P.s’. VAN HEERDEN, F.R.. VLEGGAAR. R & WESSELS. PL. 1983c. Tremorgenic mycotoxins from Penicillium crustosum. Structure elucidation and absolute con figuration of pemtrems B-F1. Journal of the Chemical Society' Perkin Trans I 8: 1857-1861. DISCUSSION BENT, K.J. 1967. Electrophoresis of proteins of 3 Penicillium species on acrylamide gels. Journal of General Microbiology 49: 195-200. DISCUSSION The large number of undetermined Penicillium species in the literature cited is an indication that scientists in South Africa have a history of not attempting to identify mem bers of this genus. Other than that done by Scott (1968a, b), work published on Penicillium in South Africa is clearly fragmentary and many of the isolates obtained early this century were identified overseas. The use of correctly identified Penicillium isolates in any scientific research must be stressed. Mistaken identities have been reported for South African studies (Frisvad 1989); voucher speci mens deposited in recognized culture collections will assist in overcoming this problem and will also make isolates available to other scientists. BERJAK. P., FARRANT. J.M., MYCOCK, D.J. & PAMMENTER, N.W. 1989. Deterioration and pathology of recalcitrant seeds. Twenty-seventh annual congress of the South African Society for Plant Pathology. Phytophylactica 21: 106 (Abstract). Plant Pathology. Phytophylactica 21: 106 (Abstract). BEZUIDENHOUT, H. 1977. ’n Ondersoek van die Hyphomycetes ge- assosieer met Cenchrus ciliaris L. M.Sc. thesis, University of Pretoria. BRIDGE. PD. 1985. An evaluation of some physiological and biochem ical methods as an aid to the characterization of species of Penicillium subsection Fdsciculala. Journal of General Microbi ology 131: 1887-1895. BRIDGE, PD. & HAWKSWORTH. D.L. 1984. The API ZYM enzyme testing system as an aid to the rapid identification of Penicillium isolates. Microbiological Sciences 1: 232-234. BRIDGE, P D ., HAWKSWORTH, D.L., KOZAKIEWICZ, Z., ONIONS, A H.S., PATERSON, R.R.M. & SACKIN. M.J. 1985. An integrated approach to Penicillium systematics. In R.A. Samson & J.I.Pitt. Advances in Penicillium and Aspergillus systematics. Plenum Press. New York. With the exception of P. hordei Stolk, P. olivicolor Pitt and Talaromyces stipitatus (Thom) C. R. Benjamin, all the Penicillium species listed by Samson & Pitt (1985) as common, have been recorded in southern Africa. However, teleomorphic penicillia have been reported infrequently as they require special isolation techniques (Scott 1968b). Synnematous members of the genus appear to be scarce and most representatives in the National Collection of Fungi, PREM and PPRI are recent acquisi tions. COHEN, C. 1950. The occurrence of fungi in the soil, after different burning and grazing treatments of the veld in the Transvaal. South African Journal of Science 46 : 260—264. COLES, L. 1925. The manufacture of Stilton and Wensleydale cheese. DISCUSSION Much meaningful work on Penicillium. one of the more common and economically important genera of fungi, is therefore still to be done in the fields of taxonomy, ecology, biological control and chemotaxonomy. DE JESUS, A.E.. STEYN. PS.. VLEGGAAR. R , KIRBY. G.W., VARLEY. M.J. & FERREIRA. N.P 1981. Biosynthesis of a-cyclopiazonic acid. Steric course of proton removal during cyclisation of 0-cyclopiazonic acid in Penicillium griseofidvum. Journal of the Chemical Society Perkin Trans 1 12: 3292—3294. ACKNOWLEDGEMENTS DE VILLIERS. J.C. 1989. Endofitiese fungusse teenwoordig in n aantal Suid-Afrikaanse gnasspesies M.Sc. thesis. University of Potchef- stroom for C H E. The encouragement of members of staff and assistance of Alice Baxter of the Plant Protection Research Institute in preparing this manuscript, is gratefully acknowledged. DOIDGE, E M. 1950. The South African fungi and lichens to the end of 1945 Bothalia 5: 1-1094. DOIDGE. E M , BOTTOMLEY, A.M ., VAN DER PLANK. J.E. & PAUER. G.D 1953 A revised list of plant diseases in South Africa. Union of South Africa Department of Agriculture Science Bulletin No. 346: 1-125. Mycotoxins crustosum are able to produce tremorgenic mycotoxins and these were examined in detail by Maes et al. (1982) and De Jesus et al. (1983a, b, c). P. janthinellum Biourge, associated with rye grass staggers was found to produce janthitrems, tremorgenic mycotoxins studied by De Jesus et al. (1984). For most of these investigations the authors obtained authenticated isolates or had their fungal cultures verified, mostly by Pitt. The presence of mycotoxin-producing fungi on cheese was investigated by Liick et al. (1976) and unidentified Penicillium spp. were isolated from 33 out of 43 cheese samples. Some of the isolates tested had a toxic effect on ducklings. Seven isolates of P. roquefortii, isolated from blue cheese showed a variation in toxicity, whereas the four isolates of P. camembertii tested had a less pronounced effect (Liick et al. 1978). A noteworthy finding of Liick & Wehner (1979) was that Penicillium isolates grown on maize were more toxic to ducklings than those grown on In 1985, South Africa hosted the IUPAC Symposium on mycotoxins and phycotoxins (Steyn & Vleggaar 1986) where a paper concerning synthesis of the Penicillium mycotoxins cyclopiazonic acid and viridamine was presented by Holzapfel (1986). 81 Bothalia 22,1 (1992) Bothalia 22,1 (1992) BAARD, S.W 1988. 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SMIT, M. 1984. ’n Studie van verskillende deklaagmateriale in die kom- mersiele verbouing van Agaricus brunnescens Peck. M.Sc. thesis, University of Pretoria. NAUDE, S.P & JOOSTE, W.J. 1989. Five fungal pathogens of storage onions. Twenty-seventh annual congress of South African Society for Plant Pathology. Phytophylactica 21: 110 (Abstract). SODERSTROM. B. & FRISVAD, J.C. 1984. Separation of closely related asymmetric penicillia by pyrolysis gas chromatography and mycotoxin production. Mycologia 76: 408-419. STEYN, P.S. 1969. The separation and detection of several mycotoxins by thin-layer chromatography. Journal of Chromatography 45: 473-475. NEETHL1NG, DC. & McGRATH, R.M. 1977. Metabolic development and mitochondrial changes during cyclopiazonic acid production in Penicillium cyclopium. Canadian Journal of Microbiology 23: 856-872. STEYN, P.S. 1970. The isolation, structure and absolute configuration of secalonic acid D, the toxic metabolite of Penicillium oxalicum. Tetrahedron Letters 26: 51—57. ORDMAN, D. 1963. The air-borne fungi in Johannesburg. A second five-year survey: 1955—1959. South African Medical Journal 37: 325-328. STEYN, P.S. & VLEGGAAR, R. REFERENCES A microbiological study of the air in dairy' processing and packaging plants. M.Sc. thesis, University of Pretoria. THOM, C. 1910. Cultural studies of species of Penicillium. Bulletin for the Bureau of Animal Industry 118: 1—109. U.S. Department of Agriculture. RAMIREZ, C. 1982. Manual and atlas of the penicillia. Elsevier, Amsterdam. THOM, C. 1930. The Penicillia. Bailliere, Tindall & Cox, London. THOM, C. 1930. The Penicillia. Bailliere, Tindall & Cox, London. VAN DER BUL, PA. 1920. Studies on some fungi and the deterioration of sugar. Union of South Africa, Department of Agriculture Science Bulletin No. 18: 1-19. RAMIREZ, C. 1990. A new species of Penicillium isolated from soil at the Kruger National Park in South Africa. Mycopathologia 110: 23-29. RAPER, K.B. & THOM, C. 1949. A manual of the penicillia. Baltimore, Williams & Wilkins. VAN DER MERWE, W.J.J., EICKER. A.. MARASAS. W.F.O. & KELLERMAN, T.S. 1979. Aerospora of an Eragrostis curvula pasture in South Africa. Onderstepoort Journal of Veterinary Research 46: 19-25. RHEEDER, J P., MARASAS, W.F.O., VAN WYK. PS., DU TOIT, W., PRETORIUS, A.J. & VAN SCHALKWYK. D.J. 1990. Incidence of Fusarium and Diplodia species and other fungi in naturally infected grain of South African maize cultivars. Phytophylactica 22 : 97-102. VAN DER PLANK. J.E. 1945. The use of hypochlorous acid and its salts in citrus packhouses for bleaching sooty blotch and as disinfectants against mould. Union of South Africa. Department of Agriculture and Forestry Science Bulletin No. 241: 1-60. ROTH, G. 1963. Post harvest decay of litchi fruit Republic of South Africa, Department of Agricultural Technical Services Technical Communication No. 11: 1-16. VAN DER SPUY. J.E., MATTHEE, F.N. & CRAFFORD. D.J.A. 1975. The heat resistance of moulds Penicillium vemuculatum Dangeard and Penicillium brefeldianum Dodge in apple juice. Phytophylac tica 7: 105-107. ROTH, G. 1967. Citrus fruit decay in South Africa caused by Penicillium digitatum Sacc. Phytopathologische Zeitschrift 58: 383-3% . ROTH. G. 1968. Biological treatment of molasses meal to reduce stickiness. Proceedings of the South African Sugar Technologists’ Association, April 1968: 57-64. VAN DER WESTHUIZEN. G.C.A. & BREDELL, I .H .1972. The fungus flora of high quality stored maize in South Africa. 1966-1972. Final Report Republic of South Africa Department of Agricul tural Technical Services. Unpublished. ROTH. G. & LOEST, F.C. 1965 Collar rot of banana hands and its associated micro-organisms. Republic of South Africa, Depart ment of Agricultural Technical Services Technical Communica tion No. 44: 1-14. REFERENCES 1983. Roquefortine, an intermediate in the biosynthesis of oxaline in cultures of Penicillium oxalicum. Journal of Chemical Society Chemical Communications 1983: 560, 561. ORDMAN, D. 1970. Seasonal respiratory allergy in Windhoek: the pollen and fungus factor. South African Medical Journal 44: 250—253. ORDMAN, D., & ETTER, K.G. 1956. The air-borne fungi in Johan nesburg. A five-year survey as a basis for the study of fungus allergy in South Africa. South African Medical Journal 30: 1054-1058. STEYN, P.S. & VLEGGAAR, R. 1986. Mycotoxins and phycotoxins. Papers presented at the sixth International IUPAC Symposium. Elsevier, Amsterdam. PAPENDORF, M.C. 1976. The soil mycoflora of an Acacia karroo com munity in the western Transvaal. Bothalia 12: 123-127. STEYN, PS., VLEGGAAR, R., FERREIRA, N.P., KIRBY. G.W. & VARLEY, M.J. 1975. Steric course of proton removal during the cyclisation of /3-cyclopiazonic acid in Penicillium cyclopium. Journal of the Chemical Society Chemical Communications 459: 465. 466. PATERSON, R.R.M., KING, G.J. & BRIDGE, PD. 1990. High resolution thermal denaturation studies on DNA from 14 Penicil lium strains. Mycological Research 94: 152-156. PITT, J.I. 1973. An appraisal of identification methods for Penicillium species: novel taxonomic criteria based on temperature and water relations. Mycologia 65: 1135-1157. STEYN, P.S., VLEGGAAR, R., WESSELS, P.L. & WOUDENBERG, M. 1982. Biosynthesis of citreoviridin. A carbon-13 N.M.R. study. Journal of the Chemical Society Perkin Trans I 9 : 2175 -2178. PITT, J.I. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, London. STOLK, A.C. 1969. Four new species of Penicillium. Antonie van Leeuwenhoek 35 : 261-274. STOLK, A.C. & SAMSON, R.A. 1972. The genus Talaromyces. Studies on Talaromyces and related genera 2. Studies in Mycology 2: 1-65. POLE EVANS, I.B. 1911. The citrus fruit-rot, caused by the blue mould. Penicillium digitatum (Fr.), Sacc. Union of South Africa Department of Agriculture Farmers Bulletin No. 11: 1-7. STOLK, A.C. & SAMSON, R.A. 1983. The ascomycete genus Eupenicil lium and related Penicillium anamorphs. Studies in Mycology 23: 1-149. POLE EVANS, I.B. 1920. Report on cold storage conditions for export fruit at Cape Town. Union of South Africa Department of Agriculture No. 2: 1-11. STOLK, A.C. & SCOTT, D.B. 1967. Studies on the genus Eupenicillium Ludwig. 1. Taxonomy and nomenclature of penicillia in relation to their sclerotioid ascocarpic states. Persoonia 4: 391-405. RABIE, C.J. & LUBBEN, A. 1984. The mycoflora of sorghum malt. South African Journal of Botany 3: 251-255. RADMORE, K. 1986. CHECKLIST OF PENICILLIUM, EUPENICILUUM AND TALAROMYCES SPECIES RECORDED IN SOUTHERN AFRICA These isolates are listed under the substrate soil, but some isolates could have been isolated from Acacia karroo litter. C , e co ec o o ape do ( 9 6), ece ved o e U ve s y o o c e s oo o C. . . ese so a es a e s ed u de e subs a e soil, but some isolates could have been isolated from Acacia karroo litter. l soil, but some isolates could have been isolated from Acacia karroo litter. UCT, a collection obtained from the University of Cape Town which contained isolates of Alls , UCT, a collection obtained from the University of Cape Town which contained isolates of Allsopp et al. (1987). soil: Eicker (1969, 1973) = cyclopium Westling Allium cepa: PREM 44737 Arachis hypogaea: Gilman (1972) cereal and legume products: Scott (1965) cheese: Luck & Wehner (1979) dung: Mitchell (1970) natural gum: Roth (1968) soil: Eicker (1975); CSIR 409; MCP 378 Sorghum caffrorum: CSIR 519, 534, 542, 543 Vitis vinifera: Doidge (1950, et al. 1953) Zea mays: Van der Westhuizen & Bredell (1972) CSIR 258, 303, 358, 403, 461, 462, 543, 659, 719; PREM 43751, 44302, 44303 = johanniolii Zaleski undetermined host: Thom (1930) = lanosocoeruleum Thom Medicago spp.: Van Warmelo (1967) Vitis spp.; Le Roux et al. (1973) = martensii Biourge Arachis hypogaea: Gilman (1972) Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972); CSIR 660 = solitum Westling material: Doidge (1950) biforme (see P. camembertii) brefeldianum (see E. javanicum var javanicum) brevicompactum Dierckx aerospora: Roth (1968) apple puree: MRC 3137 Avena saliva: MRC 2824 brattice cloth: Doidge (1950) cereal and legume products: Scott (1965) compost: PPRI 3186 debris: PPRI 4068 fodder: PPRI 3631 Medicago saliva: PREM 44475, 44477, 44519 natural gum: Roth (1968) Prunus persica var. nucipersica: PPRI 3597 soil: Eicker (1975); CSIR 327; MCP 371 REFERENCES VAN WARMELO, K.T. 1967. The fungus flora of stock feeds in South Africa Onderstepoort Journal of Veterinary Research 34: 4 3 9 - 450. SAMSON, R.A. & PITT, J.I. 1985. Advances in Penicillium and Aspergillus systematics. Plenum Press, New York. VERWOERD. L. 1929. A preliminary checklist of diseases of cultivated plants in the winter rainfall area of the Cape Province. Union of South Africa. Department of Agricultural Science Bulletin No. 88: 1-18. SCHUTTE, A.L. 1990. A Penicillium bulb pathogen Twenty-eighth annual Congress of the South African Society for Plant Pathology Phytophylactica 22: 150 (Abstract). VLEGGAAR. R & WESSELS, PL. 1980. Stereochemistry of the dehydrogenation of (2S)-histidine in the biosynthesis of roque fortine and oxaline Journal of the Chemical Society Chemical Communication 4: 160-162. SCOTT, D.B 1965. Toxigenic fungi isolated from cereal and legume products. Mycopathologia et Mycologia Applicata 25: 2 0 -2 2 2 . SCOTT, D B 1968a. Studies on the genus Eupenicillium Ludwig. 4. New species from soil. Mycopathologia el Mycologia Applicata 36: 1-27. WAGER. V.A. 1972. Records of diseases of ornamental plants not pre\iously recorded in South Africa. Republic of South Africa, Department of Agricultural Technical Services Technical Com munication No. 100: 1-14. SCOTT, D.B. 1968b. The genus Eupenicillium Ludwig. CSIR Research Report No. 272: 1-150. Pretoria SEIFERT, K.A & SAMSON, R.A 1985. The genus Coremium and the synnematous penicillia In R.A Samson & J.I Pitt, Advances in Penicillium and Aspergillus systematics Plenum Press, New York. WEHNER. F.C., BESTER. S.. KCTTZE. J M & BRODRICK. H.T. 1981 Fungi associated with post-harvest decay of mangoes in South Africa. South African Mango Grower's Association Research Report 2: 81-88 84 Bothalia 22,1 (1992) WEHNER, F.C. & RABIE, C.J. 1970. The micro-organisms in nuts and dried fruits. Phytophylactica 2: 165-170. Aspergillus and Penicillium mycotoxins. Mutation Research 58: 193-203. Aspergillus and Penicillium mycotoxins. Mutation Research 58: 193-203. Aspergillus and Penicillium mycotoxins. Mutation Research 58: 193-203. y p y WEHNER, F.C., THIEL, PG. & DU RAND, M. 1979. Mutagenicity of the mycotoxin emodin in the Salmonella!microsome system. Applied and Environmental Microbiology 37: 658—660. WITTAKER, A., MYCOCK, D.J. & BERJAK, P. 1989. Towards elimination of seed storage mycoflora: wet-heat trails with maize. Twenty-seventh Annual Congress of the South Afri can Society for Plant Pathology. Phytophylactica 21: 110 (Ab stract). WITTAKER, A., MYCOCK, D.J. & BERJAK, P. 1989. Towards elimination of seed storage mycoflora: wet-heat trails with maize. CHECKLIST OF PENICILLIUM, EUPENICILUUM AND TALAROMYCES SPECIES RECORDED IN SOUTHERN AFRICA Penicillium species recorded in southern Africa up to 1990 are arranged alphabetically and the host and/or substrate from which each species has been recorded is given with the relevant literature reference. Species names are listed as cited in the original publication in roman type, with a cross reference to the epithet currently accepted by Pitt (1979) in bold, except in the case of Eupenicillium, where the revision proposed by Stolk & Samson (1983) has been followed, or where older epithets have been traced (Seifert & Samson 1985). In the past, ascosporic fungi were included in the genus Penicillium, posing nomenclatural problems ( ) in designating the anamorph-teleomorph relationship. Consequently, species known to produce a teleomorphic state have been listed under Penicillium with a cross reference to either Eupenicillium or Talaromyces, which are listed separately. The following abbreviations are used in the list: CBS, South African isolates listed in the 1990 List of Cultures of the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. IMI, cultures in the 1988 Catalogue of the Culture Collection of CAB International Mycological Institute, Kew, United Kingdom. , g y g , , g MRC, isolates in the Culture Collection of the Medical Research Council, all identified by Pitt (C.J. Rabie pers. comm ). PPRI, isolates in the Culture Collection of the National Collection of Fungi. Several of these have been identified or verified by Pitt. PREM, isolates deposited in the National Collection of Fungi as dried material. The National Collection of Fungi recently acquired three additional fungal culture collections. Most of these cultures were and had scant accompanying data, but local isolates are listed with numbers under their appropriate abbreviations: CSIR, isolates listed in a collection obtained from the Council for Scientific and Industrial Research, which included some isolates of CSIR, isolates listed in a collection obtained from the Council for Scientific and Industrial Research, which included some isolates of Scott (1968a, b). MCP, the collection of Papendorf (1976), received from the University of Potchefstroom for C.H.E. These isolates are listed under the substrate soil, but some isolates could have been isolated from Acacia karroo litter. MCP, the collection of Papendorf (1976), received from the University of Potchefstroom for C.H. il b t i l t ld h b i l t d f A i k litt MCP, the collection of Papendorf (1976), received from the University of Potchefstroom for C.H.E. GENUS PENICILLIUM acidoferum (see P. canescens) aculeatum Raper & Fennell cereal and legume products: Scott (1965) soil: CSIR 348 adametzii Zaleski Allium cepa: PREM 44729 soil: Papendorf (1976); Allsopp et al. (1987); MCP 35, 221, 222, 1159 ventilation tubing: Doidge (1950) Zea mays. Van der Westhuizen & Bredell (1972) adametzioides Abe ex G. Smith Zea mays'. McLean & Berjak (1987); PREM 47619 alutaceum (see E. terrenum) arenicola Chalabuda mushroom casing: Smit (1984) asperum (see E. crustaceum) atramentosum Thom chicken feathers and droppings: PPRI 4086; PREM 48602 dung: PPRI 3703, 4049; PREM 49878, 50682 atrovenetum (see P. melinii) armeniacum Berk (Monilia, Thom 1930) Zea mays: PREM 187 aurantiobrunneum (see P. glabrum) aurantiocandidum (see P. aurantiogriseum) aurantiogriseum Dierckx Arachis hypogaea: MRC 330 Aristea major: PPRI 4302 cheese: PREM 49040. 49042 Hordeum vulgare: MRC 2670 Panicum miliaceum: MRC 245 Vigna subterranea: MRC 284 Zea mays: McLean & Berjak (1987); PREM 47622 = aurantiocandidum Dierckx REFERENCES Twenty-seventh Annual Congress of the South Afri can Society for Plant Pathology. Phytophylactica 21: 110 (Ab stract). pp gy WEHNER, F.C., THIEL, RG., VAN RENSBURG, S.J. & DEMASIUS, I.P.C. 1978. Mutagenicity to Salmonella typhimurium of some acidoferum (see P. canescens) aculeatum Raper & Fennell cereal and legume products: Scott (1965) soil: CSIR 348 aculeatum Raper & Fennell cereal and legume products: Scott (1965) soil: CSIR 348 cheese: Luck & Wehner (1979) dung: Mitchell (1970) natural gum: Roth (1968) adametzii Zaleski Allium cepa: PREM 44729 soil: Papendorf (1976); Allsopp et al. (1987); MCP 35, 221, 222, 1159 ventilation tubing: Doidge (1950) Zea mays. Van der Westhuizen & Bredell (1972) ( ); ; Sorghum caffrorum: CSIR 519, 534, 542, 543 g ff Vitis vinifera: Doidge (1950, et al. 1953) f g Zea mays: Van der Westhuizen & Bredell (1972) CSIR 258, 303, concentricum (see P. coprophilum) concentricum (see P. coprophilum) molasses meal: Roth (1968) citreonigrum Dierckx mushroom casing: Smit (1984) = citreoviride Biourge Zea mays: Van der Westhuizen & Bredell (1972); CBS 239.65; CSIR 138, 505, 568, 590 brefeldianum (see E. javanicum var javanicum) 85 Bothalia 22,1 (1992) Allium cepa: PREM 44777 Arachis hypogaea: Gilman (1972); MRC 241, 263, 283, 294, 2109 Avicennia spp.: PREM 47616, 47617 cereal and legume products: Scott (1965) dried leaves: MRC 320, 333, 334 fruit: Doidge (1950); Thom (1930) Ipomoea batatas: PPRI 3571 Manihot esculenta: MRC 212, 232, 249 Medicago spp. . Lamprecht (1988); PREM 48312 natural gum: Roth (1968) Ph l MRC 178 210 222 304 313 Allium cepa: PREM 44777 Arachis hypogaea: Gilman (1972); MRC 241, 263, 283, 294, 2109 Avicennia spp.: PREM 47616, 47617 cereal and legume products: Scott (1965) dried leaves: MRC 320, 333, 334 fruit: Doidge (1950); Thom (1930) Ipomoea batatas: PPRI 3571 Manihot esculenta: MRC 212, 232, 249 Medicago spp. . Lamprecht (1988); PREM 48312 natural gum: Roth (1968) Phaseolus spp.: MRC 178, 210, 222, 304, 313 soil: Allsopp et al (1987); Cohen (1950); Eicker (1969, 1970); Papendorf Sorghum caffrorum: CSIR 531, 547 Vitis spp.: Le Roux et al. (1973) pp ( ) Zea mays: McLean & Berjak (1987); Pitt (1979); Van der Westhuizen & Bredell (1972); CSIR 81, 95, 219, 330, 378, 459, 593, 623, 665, 675; PPRI 3630; PREM 43741, 43742 , 47537, 47831 undetermined host: CBS 287.53 (albino mutant) = stoloniferum Thom soil: Cohen (1950) Z V d W th i & B d ll (1972) CSIR 238 Zea mays: McLean & Berjak (1987); Pitt (1979); Van der Westhuizen & Bredell (1972); CSIR 81, 95, 219, 330, 378, 459, 593, 623, 665, 675; PPRI 3630; PREM 43741, 43742 , 47537, 47831 undetermined host: CBS 287.53 (albino mutant) Ipomoea batatas: PPRI 3571 Manihot esculenta: MRC 212, 232, 249 soil: Cohen (1950) Zea mays: Van der Westhuizen & Bredell (1972); CSIR 238 Sorghum caffrorum: MRC 2332 (1973) Zea mays: Van der Westhuizen & Bredell (1972); CSIR 265, 302, 328, 428, 434, 644 undetermined host: Doidge (1950) citreonigrum Dierckx mushroom casing: Smit (1984) = citreoviride Biourge Zea mays: Van der Westhuizen & Bredell (1972); CBS 239.65; CSIR 138, 505, 568, 590 citreoviride (see P. citreonigrum) citrinum Thom R h (1968) canescens Sopp Barleria obtusa: PPRI 3808 flannel: PREM 33287 Protea cynaroides: PPRI 3786 soil: Papendorf (1976) Zea mays: Van der Westhuizen & Bredell (1972) = acidoferum Sopp (near P. canescens. Raper & Thom 1949) Citrus sinensis: Doidge (1950) = kapuscinskii Zaleski soil: MCP 384 swine meal: Van Warmelo (1967) = steckii Zaleski Arachis hypogaea: Van Warmelo (1967) cereal and legume products: Scott (1965) soil: Eicker (1969, 1970); CSIR 346, 381, 382, 384. 385, 387 Zea mays: Van der Westhuizen & Bredell (1972); Van Warmelo (1967); CSIR 341, 383, 426, 444. 454, 595, 670: PREM 43752 Zea mays: Van der Westhuizen & Bredell (1972); Van Warmelo (1967); CSIR 341, 383, 426, 444. 454, 595, 670: PREM 43752 camembertii Thom Phaseolus spp.: MRC 178, 210, 222, 304, 313 cheese. Davel & Neethling (1930); Luck et al. (1978); PPRI 3122; PREM 47740 soil: Allsopp et al. (1987); Cohen (1950); Eicker (1969, 1970); Papendorf (1976); CSIR 370, 372 , 373, 374 = biforme Thom Sorghum caffrorum: MRC 2332 Sorghum caffrorum: MRC 2332 Arachis hypogaea: Van Warmelo (1967) Arachis hypogaea: Van Warmelo (1967) Vigna subterranea: MRC 224, 280 Medicago spp.: Van Warmelo (1967) g , Zea mays: Gilman (1972); McLean & Berjak (1987); Van der Westhuizen & Bredell (1972); CSIR 152, 352, 393, 394, 549, 661, 708; MRC 257, 258, 262, 266, 293, 294, 307, 437, 444; PREM 44304, 44305. 47620, 47621 = steckii Zaleski Arachis hypogaea: Van Warmelo (1967) cereal and legume products: Scott (1965) soil: Eicker (1969, 1970); CSIR 346, 381, 382, 384. 385, 387 Zea mays: Van der Westhuizen & Bredell (1972); Van Warmelo (1967); CSIR 341, 383, 426, 444. 454, 595, 670: PREM 43752 Zea mays: Gilman (1972); McLean & Berjak (1987); Van der Westhuizen & Bredell (1972); CSIR 152, 352, 393, 394, 549, 661, 708; MRC 257, 258, 262, 266, 293, 294, 307, 437, 444; PREM 44304, 44305. 47620, 47621 Medicago spp.: Van Warmelo (1967) canescens Sopp Barleria obtusa: PPRI 3808 flannel: PREM 33287 Protea cynaroides: PPRI 3786 soil: Papendorf (1976) Zea mays: Van der Westhuizen & Bredell (1972) = acidoferum Sopp (near P. canescens. Raper & Thom 1949) Citrus sinensis: Doidge (1950) = kapuscinskii Zaleski soil: MCP 384 swine meal: Van Warmelo (1967) capsulatum Raper & Fennell dried fish: Pitt (1979); IMI 140 284 Medicago saliva: PREM 44469 Zea mays: CSIR 181 casei (see P. roquefortii) charlesii (see P. fellutanum) chermesinum Biourge soil: Martin (1960) chrysogenum Thom aerospora: Roth (1968) Arachis hypogaea: Van Warmelo (1967); PPRI 3658; PREM 48261 cereal and legume products: Scott (1965) fishmoth gut: PREM 49016, 49017 grass: PPRI 4277 Hordeum vulgare: MRC 2807 Medicago spp.: Lamprecht (1988); PREM 48321 molasses meal: Roth (1968) mushroom casing: Smit (1984) natural gum: Roth (1968) nuts and dried fruit: Wehner & Rabie (1970) soil: Eicker (1975); Martin (1960); PREM 48767 Sorghum caffrorum: CSIR 427; MRC 1682 Zea mays: Gilman (1972); McLean & Berjak (1987); Van der Wssthuizen & Bredell (1972); Van Warmelo (1967); CSIR 436, 453, 477 = notatum Westling aerospora: Roth (1968) Allium cepa: PREM 44738 Cenchrus ciliaris: Bezuidenhout (1977) cereal and legume products: Scott (1965) Medicago saliva: PREM 44466, 44552 molasses meal: Roth (1968) natural gum: Roth (1968) nuts and dned fruit: Wehner & Rabie (1970) soil: CSIR 317, 318 Sorghum caffrorum CSIR 285, 286 Vitis spp. Le Roux et al. claviforme (see P. vulpinum) claviforme (see P. vulpinum) claviforme (see P. vulpinum) commune (see P. puberulum) citreoviride (see P. citreonigrum) 1953); Doidge & Van der Plank (1936); Van der Plank (1945); Venwerd (1929); CSIR 558, 561; PPRI 3737 soil: Eicker (1969, 1973) soil: Eicker (1969, 1973) = digitatum Sacc. var. califomicum Thom soil: Eicker (1969, 1973) = digitatum Sacc. var. califomicum Thom = digitatum Sacc. var. califomicum Thom Physalis peruviana: Doidge et al. (1953) Citrus sinensis: Doidge (1950); Doidge & Van der Plank (1936); PREM 30659 Zea mays: Van Warmelo (1967) fuscum (Sopp) Biourge (application uncertain, Pitt 1979) Medicago sativa: PREM 44401 expansum Link p Arachis hypogaea: Pitt (1979); MRC 199; PREM 48381 cereal and legume products: Scott (1965) Vigna subterranea: MRC 174 griseofulvum Dierckx birdseed: PPRI 3701 cereal and legume products: Scott (1965) cubed dogfood: PPRI 3306, 3679 Dalbergia obovata: PPRI 3702; PREM 49887 fishmoth gut: PPRI 3123 Manihot esculenta: Pitt (1979); MRC 270, 273 Medicago spp.: Lamprecht (1988); PREM 48317, 48318 silage: CBS 315.63 soil: De Jesus et al. (1981); Cohen (1950); PPRI 4281 Vigna subterranea: MRC 312 Watsonia marginata PPRI 3809 Zea mays: MRC 214 = urticae Bain, cereal and legume products: Scott (1965) soil: CSIR 391 fellutanum Biourge face cream: PPRI 4306 Protea spp.: PPRI 3980 soil: MCP 390, 391 Zea mays: Pitt (1979); CBS 268.65; IMI 162 083, 162 114; CSIR 284 = charlesii G. Smith cereal and legume products: Scott (1965) soil: Papendorf (1976); MCP 48, 117 Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972); CSIR 284, 389, 401, 476 citreoviride (see P. citreonigrum) citrinum Thom aerospora: Roth (1968) Bothalia 22,1 (1992) Bothalia 22,1 (1992) 86 frequentans (see P. glabrum) debris: PPRI 3782 fodder: PPRI 3887, 4225 Protea scolopendriifolia: PPRI 4014; PREM 47704 Watsonia marginata: PPRI 3724 funiculosum Thom Ananas comosus: Doidge (1950, et al. 1953); PPRI 4307 Arachis hypogaea: Baard (1988); Gilman (1972); Pitt (1979); PPRI 3634; PREM 48015 cereal and legume products: Scott (1965) Cyperaceae spp.: PPRI 3632; PREM 48604 Eucalyptus maculata: Eicker (1973) Malus sylvestris: Combrink et al. (1985) Medicago sativa: PREM 44513 Phaseolus spp.: MRC 281 soil: Allsopp et al. (1987); Doidge (1950); Eicker (1969, 1973) Martin (1960); Papendorf (1976); CSIR 141, 362, 365, 367, 368, 369; MCP 189, 336; PPRI 3504; UCT Zea mays: Gilman (1972); McLean & Berjak (1987); Van der Westhuizen & Bredell (1972); CSIR 23, 82, 83, 92, 93, 221, 242, 300, 613; PPRI 3633; PREM 43754, 43755, 43756, 43757, 47637 undetermined host: Thom (1930); Raper & Thom (1949) = varians G. Smith Zea mays: Van Warmelo (1967) funiculosum Thom Ananas comosus: Doidge (1950, et al. 1953); PPRI 4307 Arachis hypogaea: Baard (1988); Gilman (1972); Pitt (1979); PPRI 3634; PREM 48015 digitatum (Pers. ex Fr.) Sacc. aerospora: Pole Evans (1920) Carica papaya: Doidge (1950, et al. 1953) Citrus aurantium: Doidge et al. (1953) citrus fruit: Pole Evans (1911); Roth (1967) Citrus limonia: Doidge (1950, et al. 1953); CSIR 562, 563; PPRI 37 Citrus nobilis var deliciosa: Doidge (1950 et al 1953) digitatum (Pers. ex Fr.) Sacc. aerospora: Pole Evans (1920) Carica papaya: Doidge (1950, et al. 1953) Citrus aurantium: Doidge et al. (1953) citrus fruit: Pole Evans (1911); Roth (1967) yp pp ; Eucalyptus maculata: Eicker (1973) yp Malus sylvestris: Combrink et al. (1985) Citrus limonia: Doidge (1950, et al. 1953); CSIR 562, 563; PPRI 3740 Citrus limonia: Doidge (1950, et al. 1953); CSIR 562, 563; PPRI 3740 Citrus nobilis var. deliciosa: Doidge (1950, et al. 1953) g ( , ); , ; Citrus nobilis var. deliciosa: Doidge (1950, et al. 1953) Phaseolus spp.: MRC 281 soil: Allsopp et al. (1987); Doidge (1950); Eicker (1969, 1973) Martin (1960); Papendorf (1976); CSIR 141, 362, 365, 367, 368, 369; MCP 189, 336; PPRI 3504; UCT g ( , Citrus paradisi: PPRI 3319; PREM 48908 p Citrus sinensis: Doidge (1950, et al. digitatum var. califomicum (see P. digitatum) divaricatum Thom (Scopulariopsis, Raper & Thom 1949) sugar: Van der Bijl (1920) glabrum (Wehmer) Westling dung: PPRI 4308 Medicago sativa: PREM 44535, 44550 Melianthus comosus: PPRI 3807 soil: Allsopp et al. (1987) wine bottle cork: PPRI 3637; PREM 48406 = aurantiobrunneum Dierckx soil: Cohen (1950) = flavidorsum Biourge soil: Cohen (1950) = frequentans Westling Allium cepa: PREM 44767 Arachis hypogaea: Gilman (1972) cereal and legume products: Scott (1965) nuts and dried fruit: Wehner & Rabie (1970) soil: Papendorf (1976); MCP 122, 185, 190 Sorghum caffrorum: CSIR 546 Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972); Van Warmelo (1967); PREM 44300 l di li ( E t ) diversum Raper & Fennell Eucalyptus cloeziana: PPRI 3731; PREM 49865 Medicago sativa: PREM 44517 duclauxii Delacr. aerospora: Roth (1968) Asparagus officinalis: PPRI 4083 grass roots: PPRI 3B0; PREM 47754 mine timber: Doidge (1950); Pitt (1979); Raper & Thom (1949); IMI 200 309 molasses meal: Roth (1968) natural gum: Roth (1968) soil: PPRI 3983, 4305; PREM 48938 dupontii (see T. thermophilus) echinulatum Raper & Thom ex Fassatiova granadilla juice: PPRI 3585 elongatum (see P. expansum) erubescens (see E. terrenum) expansum Link aerospora: Pole Evans (1920) Arachis hypogaea: Pitt (1979); MRC 199; PREM 48381 cereal and legume products: Scott (1965) granadilla juice: PPRI 3584; PREM 49415 Malus sylvestris: Combrink et al. (1980); Doidge (1950, et al. 1953); PPRI 4215 molasses meal: Roth (1968) natural gum: Roth (1968) pome fruit: Matthee (1968) Psidium guajava: PREM 48383 Strelitzia reginae: PPRI 4278 soil: CSIR 398, 410; PPRI 4279 Vigna subterranea: MRC 174 Vitis vinifera: Doidge (1950, et al. 1953); Le Roux et al. (1973); MRC 101 Zea mays: Van der Westhuizen & Bredell (1972); CSIR 15, 71, 226, 326, 404, 443, 483, 527, 659, 717; MRC 177; PREM 47512 undetermined host: Holtzhausen & Knox-Davies (1974) = elongatum Dierckx Vitis vinifera: Doidge (1950, et al. 1953) fellutanum Biourge face cream: PPRI 4306 Protea spp.: PPRI 3980 soil: MCP 390, 391 Zea mays: Pitt (1979); CBS 268.65; IMI 162 083, 162 114; CSIR 284 = charlesii G. Smith cereal and legume products: Scott (1965) soil: Papendorf (1976); MCP 48, 117 Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972); CSIR 284, 389, 401, 476 erubescens (see E. terrenum) Venvoerd (1929) Citrus limonia: Doidge (1950, et al. 1953) Citrus maxima: Doidge (1950, et al. 1953) Citrus nobilis var. deliciosa: Doidge (1950); PPRI 3723; PREM 48607 islandieum Sopp cereal and legume products: Scott (1965) g p contaminant: PPRI 3124, 3714; PREM 47753, 49869 Sorghum caffrorum: Rabie & Liibben (1984) Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1 Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972) d i d h R & Th (1949) Th (1930) CBS 176 6 y ( ); ( ) ndetermined host: Raper & Thom (1949); Thom (1930); CBS 176.6 janczewskii Zaleski = soppii Zaleski Eucalyptus maculata: Eicker (1973) soil: Eicker (1969, 1970, 1973): PREM 44281 undetermined host: CSIR 1398 yp ( ) soil: Eicker (1969, 1970, 1973): PREM 44281 undetermined host: CSIR 1398 minioluteum Dierckx Dianthus caryophyllus: PPRI 3982 Hordeum vulgare: MRC 1756 paper: PPRI 3659; PREM 49874 soil: PREM 48586. 48587 Zea mays McLean & Berjak (1987); PPRI 3984. 4020: PREM 47533, 47538, 47539, 47544 . 47618 janthinellum Biourge Arachis hypogaea PREM 48262 brattice cloth: Doidge (1950) cereal and legume products: Scott (1965) Medicago spp. Lamprecht (1988); PREM 48319 PPRI 4309; PREM 48606 mushroom casing: Smit (1984) Prunus persica: Doidge (1950) Prunus salicina: Doidge (1950) = pedemontanum Mosca & Fontana janczewskii Zaleski Barleria obtusa: PREM 49890 contaminant: PREM 47702 Encephalartos laevifolius: PPRI 3179 Medicago spp.: Lamprecht (1988); PREM 48320 Pinus elliottii: PREM 48907 soil: Allsopp et al. (1987); Eicker (1969); PPRI 3586 undetermined host: CBS 384.67 = nigricans Bain, cereal and legume products: Scott (1965) Medicago saliva: PREM 44523 soil: Martin (1960); CSIR 325 Sorghum caffrvrum: MRC 1552 Zea mays Van der Westhuizen & Bredell (1972); Van Warmelo (1967) janthinellum Biourge Arachis hypogaea PREM 48262 brattice cloth: Doidge (1950) cereal and legume products: Scott (1965) Medicago spp Lamprecht (1988); PREM 48319 italicum Wehmer Citrus sinensis: Doidge et al. (1953); Doidge & Van der Plank (1936); Venvoerd (1929) hirayamae (see E. euglaucum) = corymbiferum Westling Omithogalum spp.: Wager (1972); IMI 068 414 cereal and legume products: Scott (1965) inusitatum (see E. inusitatum) flavidorsum (see P. glabrum) javanicum) jensenii Zaleski Allium cepa: PREM 44761 mushroom casing : Smit (1984) soil: Eicker (1969, 1970); PREM 44256 Zea mays: Van der Westhuizen & Bredell (1972), PREM 43740, 43753 = intricatum Thom soil: Cohen (1950) flannel: Doidge (1950) johannioli (see P. aurantiogriseum) kapuscinskii (see P. canescens) krugeri Ramirez soil: Ramirez (1990) lanosocoeruleum (see P. aurantiogriseum) lanosum (see P. puberulum) lapidosum (see E. lapidosum) lilacinum Thom (Paecilomyces lilacinus, Pitt 1979) soil: Martin (1960); Papendorf (1976) swine meal: Van Warmelo (1967) Zea mays: Van der Westhuizen & Bredell (1972) lividum Westling debris: PPRI 3707; PREM 49886 soil: PPRI 4043 luteum (see T. luteus) luteoviride Biourge (indeterminate, Pitt 1979) aerospora: Doidge (1950); Thom (1930) martensii (see P. aurantiogriseum) megasporum Orput & Fennell Encephalartos laevifolius: PREM 49069 melinii Thom debris: PREM 47699 Encephalartos laevifolius: PPRI 3178; PREM 49070 mouse nest material: PPRI 4223 mushroom casing: Smit (1984) soil: Allsopp et al. (1987); Pitt (1979); PPRI 4042; PREM 47699 = at rove ne turn G. Smith Arachis hypogaea: CBS 240.65 Zea mays: Van der Westhuizen & Bredell (1972); CSIR 323, 324 meridianum (See E. meridianumi miczvnskii Zaleski debris: PPRI 3710; PREM 49882 soil: Allsopp et al. (1987); PPRI 4040 Zea mays: MRC 426 undetermined host: Pitt (1979) = pedemontanum Mosca & Fontana soil: Papendorf (1976); MCP 127 Zea mays: PREM 44301 = soppii Zaleski Eucalyptus maculata: Eicker (1973) soil: Eicker (1969, 1970, 1973): PREM 44281 undetermined host: CSIR 1398 minioluteum Dierckx Dianthus caryophyllus: PPRI 3982 Hordeum vulgare: MRC 1756 paper: PPRI 3659; PREM 49874 soil: PREM 48586. 48587 Zea mays McLean & Berjak (1987); PPRI 3984. 4020: PREM 47533, 47538, 47539, 47544 . 47618 montanense Christensen & Bakus soil: PPRI 4041 multicolor (see P. sclerotiorum i nigncans (see P. janczewskii) javanicum (see E. javanicum var. javanicum) flavidorsum (see P. glabrum) flavidorsum (see P. glabrum) 87 Bothalia 22,1 (1992) = roseocitreum Biourge aerospora: Doidge (1950); Thom (1930) herquei Bain & Sartory cereal and legume products: Scott (1965) debris: PPRI 3904 soil: Eicker (1975); CSIR 359, 360, 361, 363; PPRI 4218; PREM 48559 Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972); Van Warmelo (1967); CSIR 364, 402, 474, 538, 731 = coralligerum Nicot & Pionnat soil: CSIR 1072 hirayamae (see E. euglaucum) hirsutum Dierckx Allium sativum: PPRI 3792; PREM 47862 Asparagus officinalis: PPRI 4219 Omithogalum spp.: Pitt (1979); Schutte (1990); CBS 502.75; PPRI 3795 Gladiolus spp.: PPRI 3598, 3600, 3601, 3602; PREM 49414 = corymbiferum Westling Omithogalum spp.: Wager (1972); IMI 068 414 humuli Van Beyma soil: Eicker (1969, 1973) Eucalyptus maculata: Eicker (1973) implicatum Biourge Allium cepa: PREM 44779 Arachis hypogaea: Van Warmelo (1967) cereal and legume products: Scott (1965) Zea mays: Van der Westhuizen & Bredell (1972); CSIR 139, 355, 506 inflatum Stolk & Malla soil: PPRI 3206; PREM 49071 intricatum (see P. jensenii) inusitatum (see E. inusitatum) islandieum Sopp Arachis hypogaea: Gilman (1972) cereal and legume products: Scott (1965) contaminant: PPRI 3124, 3714; PREM 47753, 49869 Sorghum caffrorum: Rabie & Liibben (1984) Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972) undetermined host: Raper & Thom (1949); Thom (1930); CBS 176.68 italicum Wehmer aerospora: Pole Evans (1920) Citrus sinensis: Doidge et al. (1953); Doidge & Van der Plank (1936); Venvoerd (1929) Citrus limonia: Doidge (1950, et al. 1953) Citrus maxima: Doidge (1950, et al. 1953) Citrus nobilis var. deliciosa: Doidge (1950); PPRI 3723; PREM 48607 Citrus sinensis: Doidge (1950); Kxiek & Wehner (1981); IMI 78 681; PPRI 4309; PREM 48606 fodder: PREM 48386, 48389 mushroom casing: Smit (1984) Prunus persica: Doidge (1950) Prunus salicina: Doidge (1950) janczewskii Zaleski Barleria obtusa: PREM 49890 contaminant: PREM 47702 Encephalartos laevifolius: PPRI 3179 Medicago spp.: Lamprecht (1988); PREM 48320 Pinus elliottii: PREM 48907 soil: Allsopp et al. (1987); Eicker (1969); PPRI 3586 undetermined host: CBS 384.67 = nigricans Bain, cereal and legume products: Scott (1965) Medicago saliva: PREM 44523 soil: Martin (1960); CSIR 325 Sorghum caffrvrum: MRC 1552 Zea mays Van der Westhuizen & Bredell (1972); Van Warmelo (1967) janthinellum Biourge Arachis hypogaea PREM 48262 brattice cloth: Doidge (1950) cereal and legume products: Scott (1965) Medicago spp. Lamprecht (1988); PREM 48319 y j (1972); PREM 47545 javanicum (see E. javanicum var. roquefortii Thom q cheese: Davel & Neethling (1930); Doidge (1950); Luck et al. (1978); CSIR 390, 392, 423, 447, 450, 455, 493, 497, 498, 499, 502, 503. 507, 509, 510, 512; PREM 49041, 49050; PPRI 3167, 3190, 3889; UCT puberulum Bain. Allium cepa: PREM 44765 Arachis hypogaea: Pitt (1979); MRC 335 Encephalartos laevifolius: PPRI 3205 soil: PPRI 3204 Zea mays: Van der Westhuizen & Bredell (1972) = commune Thom aerospora: Roth (1968) Cenchrus ciliaris: Bezuidenhout (1977) flannel: PREM 33289 molasses meal. Roth (1968) Zea mays: Van der Westhuizen & Bredell (1972) Zea mays: Van der Westhuizen & Bredell (1972); Van Warmelo (1967) casei St b Zea mays: Van der Westhuizen & Bredell (1972); Van Warmelo (1967) = casei Staub Zea mays: Van der Westhuizen & Bredell (1972); Van Warmelo (1967) i St b = casei Staub soil: Eicker (1969. 1970, 1973); PREM 44280 = rubrum Stoll Arachis hypogaea: CSIR 13 raistrickii (see P. raciborskii) restrietum Gilman & Abbott Acacia karroo: Pitt (1979) Helianthus annuus: PREM 47856 soil: Allsopp et al. (1987); Papendorf (1976); MCP 23 = striatisporum Stolk Acacia karroo: Stolk (1969); IMI 151 749 soil: Stolk (1969); Papendorf (1976); MCP 116, 213 = purpurogenum var. rubisclerotium Thom Acacia karroo: Stolk (1969); IMI 151 749 soil: Stolk (1969); Papendorf (1976); MC janthinellum Biourge multicolor (see P. sclerotiorum i nigncans (see P. janczewskii) Bothalia 22,1 (1992) 88 notatum (see P. chrysogenum) novae-zeelandiae Van Beyma Protea spp.: PPRI 3978 soil: Allsopp et al. (1987); PPRI 4222 ochrochloron Biourge soil: Eicker (1969) ochrosalmoneum (see E. ochrosalmoneum) olivinoviride (see P. viridicatum) olsonii Bain. & Sartory debris: PPRI 4038 Eucalyptus maculata: Eicker (1973) Gloxinia spp. PPRI 3706 mushroom casing: Smit (1984) soil: Eicker (1969, 1970, 1973) Tribulus terrestris: PPRI 3308; PREM 49207 Zea mays: PREM 47861, 47863 paraherquei (see P. simplicissimum) paraherquei (see P. simplicissimum) purpurogenum var. rubisclerotium (see P. pinophilum) paxilli Bain. Encephalartos laevifolius: PPRI 3183, 3184 mouse nest material: PPRI 4220 pusillum (see E. einnamopurpureum) putterillii Thom (Geosmithia putterillii, Pitt 1979) aerospora: Doidge (1950); Thom (1930) pedemontanum (see P. miczynskii) raciborskii Zaleski raciborskii Zaleski aerospora: PPRI 3712; PREM 49885 Arachis hypogaea: PPRI 3664 soil: Stolk & Samson (1983); PPRI 4217 Watsonia marginata: PPRI 3722 = raistrickii G. Smith Arachis hypogaea: Pitt (1979); MRC 197 cereal and legume products: Scott (1965) mushroom casing: Smit (1984) soil: Allsopp et al. (1987); CSIR 388 Sorghum caffrorum: CSIR 526, 528, 529, 545 Zea mays: McLean & Berjak (1987); Van der Westhuizen & Bredell (1972); CSIR 4; PREM 47636 piceum Raper & Fennell cereal and legume products: Scott (1965) debris: PPRI 4019; PREM 49864 soil: CSIR 345 palitans (see P. viridicatum) g Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972); PREM 43747 g Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972); PREM 43747 pulvillorum (see P. simplicissimum) purpurescens (Sopp) Biourge Fingerhuthia africana: De Villiers (1989); PREM 49278 mushroom casing: Smit (1984) Protea spp.: PPRI 4284 stored foods: IMI 141 658 Vitis vinifera: PPRI 3574 purpurescens (Sopp) Biourge Fingerhuthia africana: De Villiers (1989); PREM 49278 mushroom casing: Smit (1984) Protea spp.: PPRI 4284 stored foods: IMI 141 658 Vitis vinifera: PPRI 3574 purpurescens (Sopp) Biourge Fingerhuthia africana: De Villiers (1989); PREM 49278 mushroom casing: Smit (1984) Protea spp.: PPRI 4284 stored foods: IMI 141 658 Vitis vinifera: PPRI 3574 ochrosalmoneum (see E. ochrosalmoneum) olivinoviride (see P. viridicatum) olsonii Bain. & Sartory debris: PPRI 4038 Eucalyptus maculata: Eicker (1973) Gloxinia spp. PPRI 3706 mushroom casing: Smit (1984) soil: Eicker (1969, 1970, 1973) Tribulus terrestris: PPRI 3308; PREM 49207 Zea mays: PREM 47861, 47863 purpurogenum Stoll Arachis hypogaea: Gilman (1972); CSIR 13 Manihot esculenta: Pitt (1979); MRC 181 oxalicum Currie & Thom Aloe asperifolia: MCP 351 Arachis hypogaea: PREM 48260, 48567 cereal and legume products: Scott (1965) debris: PPRI 4039 dried fish: MRC 322 face cream: PPRI 3272 fodder: PREM 48584, 48585 soil: CSIR 331, 332, 333, 335, 338 Sorghum caffrorum: CSIR 296, 522, 523 Zea mays: Doidge (1950); McLean & Berjak (1987); Nagel et al. (1976); Steyn (1970); Van der Westhuizen & Bredell (1972); CSIR 210, 293, 368, 504, 555, 589, 615, 620, 643, 650, 676; PREM 47542 Vitis spp.: Le Roux et al. (1973) pp ( ) Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972); MRC y ( ); 315; PPRI 3120. 3783; PREM 49018 pedemontanum (see P. miczynskii) petchii Sartory & Bain, (indeterminate, Pitt 1979) Ananas comosus: Doidge (1950) sclerotiorum Van Beyma variabile Sopp Allium cepa: PREM 44757, 44764 Arachis hypogaea: Gilman (1972) cereal and legume products: Scott (1965) coconut matting: Raper & Thom (1949); IMI 040 040 Hordeum \ulgare: MRC 1755 leaves: MRC 319 Medicago saliva: PREM 44547, 44548 paper: PPRI 3657; PREM 49876 soil: CSIR 206, 353, 356, 467 Vitis spp.: Le Roux el al. (1973) Zea mays: Gilman (1972); McLean & Berjak (1987): Van der Westhuizen & Bredell (1972); Van Warmelo (1967); CSIR 70, 73, 232 , 269, 2%, 395, 464. 472, 548; PREM 43743, 43744 . 43745, 43746, 47540 47541 47543 variabile Sopp Allium cepa: PREM 44757, 44764 Arachis hypogaea: Gilman (1972) cereal and legume products: Scott (1965) coconut matting: Raper & Thom (1949); IMI 040 040 Hordeum \ulgare: MRC 1755 leaves: MRC 319 Medicago saliva: PREM 44547, 44548 paper: PPRI 3657; PREM 49876 soil: CSIR 206, 353, 356, 467 Vitis spp.: Le Roux el al. (1973) Zea mays: Gilman (1972); McLean & Berjak (1987): Van der Westhuizen & Bredell (1972); Van Warmelo (1967); CSIR 70, 73, 232 , 269, 2%, 395, 464. 472, 548; PREM 43743, 43744 . 43745, 43746, soil: Eicker (1969, 1973, 1975); Papendorf (1976) Zea mays: Van der Westhuizen & Bredell (1972); CSIR 208, 397; MCP. PREM 43748 Zea mays: Gilman (1972); McLean & Berjak (1987): Van der Westhuizen & Bredell (1972); Van Warmelo (1967); CSIR 70, 73, 232 , 269, 2%, 395, 464. 472, 548; PREM 43743, 43744 . 43745, 43746, 47540, 47541, 47543 varians (see P. funiculosum) velutinum Van Beyma Eucalyptus maculata: Eicker (1973) Medicago saliva: PREM 44522, 44549 soil: Eicker (1969. 1970. 1973); PREM 44260 Zea mays: Van der Westhuizen & Bredell (1972) PREM 44306. 47514 4067; PREM 48902, 48903, 48904 Sorghum caffrorum: MRC 2206 ventilation tubing: Doidge (1950) verrucosum Dierckx cheddar cheese: Pitt (1979) Citrus sinensis: Doidge (1950, et al. 1953); Doidge & Van der Plank (1936) debris: PPRI 3121 Phaseolus spp: MRC 220 soil: PPRI 3575 Zea mays: Van der Westhuizen & Bredell (1972); CSIR 77 soil: Papendorf (1976); MCP 39, 105, 188 = piscarium Westling soil: Papendorf (1976); MCP 187 verruculosum Peyronel Casuarina spp.: PREM 47707 Eucalyptus maculata: Eicker (1973) Orvza saliva: MRC 171 soil: Allsopp et al. (1987); Eicker (1969. 1970. 1973); CSIR 347; PPRI 3501; PREM 44265. 48017; UCT Zea mays: PPRI 3837 Zea mays: Nagel & Steyn (1972); CSIR 1405. 1406 terrenum (see E. terrenum) terrenum (see E. terrenum) terrestre Raper & Thom (application uncertain. Pitt 1979) Zea mays: Van der Westhuizen & Bredell (1972) thomii Maire aerospora: Roth (1968) cereal and legume products: Scott (1965) Cussonia paniculata : PPRI 3784 molasses meal: Roth (1968) soil: Eicker (1975); Papendorf (1976); CSIR 752; MCP 38; PPRI 3237, 4044; UCT aerospora: Doidge (1950); Thom (1930) Arachis hypogaea: Gilman (1972) Zea mays: Van der Westhuizen & Bredell (1972) Zea mays: Van der Westhuizen & Bredell (1972) soil: CSIR 344; MCP 372 trzebinskii (see P. spinulosum) timber: Doidge (1950) Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972) sclerotiorum Van Beyma fodder: PREM 48876 Kniphofia spp.: PREM 48877 soil: Pitt (1979); Raper & Thom (1949); PREM 48571; PPRI 3901. 4069. 4139 Zea mays: Stolk & Samson (1983); MRC 425 = multicolor Grigorieva-Manoilova & Poradielova (application un certain, Pitt 1979) cereal and legume products: Scott (1965) Eucalyptus maculata: Eicker (1973) soil: Eicker (1969, 1973, 1975); Papendorf (1976) Zea mays: Van der Westhuizen & Bredell (1972); CSIR 208, 397; MCP PREM 43748 roseocitreum (see P. griseoroseum) roseopurpureum Dierckx soil: Papendorf (1976) 89 Bothalia 22,1 (1992) Zea mays: CSIR 400 rubrum (see P. purpurogenum) rugulosum Thom aerospora: Roth (1968) Allium cepa: PREM 44775 Arachis hypogaea: PREM 48388 coconut matting: Doidge (1950) Gladiolus spp.: PPRI 3596; PREM 49413 natural gum: Roth (1968) Zea mays: Van der Westhuizen & Bredell (1972); PREM 43739 = tardum Thom aerospora: Doidge (1950); Thom (1930) Arachis hypogaea: Gilman (1972) soil: CSIR 344; MCP 372 timber: Doidge (1950) Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972) Bothalia 22,1 (1992) Zea mays: CSIR 400 rubrum (see P. purpurogenum) rugulosum Thom aerospora: Roth (1968) Allium cepa: PREM 44775 Arachis hypogaea: PREM 48388 coconut matting: Doidge (1950) Gladiolus spp.: PPRI 3596; PREM 49413 natural gum: Roth (1968) Zea mays: Van der Westhuizen & Bredell (1972); PREM 43739 = tardum Thom aerospora: Doidge (1950); Thom (1930) Arachis hypogaea: Gilman (1972) soil: CSIR 344; MCP 372 timber: Doidge (1950) Zea mays: Gilman (1972); Van der Westhuizen & Bredell (1972) Bothalia 22,1 (1992) terlikowskii (see P. spinulosum) Papendorf (1976); CSIR 312 Papendorf (1976); CSIR 312 Sorghum caffrorum: Rabie & Liibben (1984); CSIR 316, 521, 533 p ( ); Sorghum caffrorum: Rabie & Liibben (1984); CSIR 316, 521, 533 p ( ); Sorghum caffrorum: Rabie & Liibben (1984); CSIR 316, 521, 533 Sorghum caffrorum: Rabie & Liibben (1984); CSIR 3 g ff Tulipa spp.: Doidge et al. (1953) Tulipa spp.: Doidge et al. (1953) Vitis spp.: Le Roux et al. (1973); Verwoerd (1929) Vitis spp.: Le Roux et al. (1973); Verwoerd (1929) Zea mays: Gilman (1972); Marasas et al. (1981); Marasas & Van Rensburg (1986); McLean & Berjak (1987); Vkn Warmelo (1967); Wittaker et al. (1989); CSIR 218, 264, 414, 415 Zea mays: Van der Westhuizen & Bredell (1972) lapidosum Scott & Stolk soil: Scott (1968b); CBS 318.66, CSIR 1035; PREM 48880 Zea mays: CSIR 1093 unrecorded host: Stolk & Samson (1983) smoked shrimps: Gilman (1972) soil: Allsopp et al. (1987); Cohen (1950); Eicker (1975, et al. 1982); soil: Allsopp et al. (1987); Cohen (1950); Eicker (1975, et al. 1982); waksmanii Zaleski yp ( ) soil: Eicker (1969, 1970, 1973); PREM 44264 ( , , ); * P. gladioli McCulloch & Thom g ( ) soil: Eicker (1975); Papendorf (1976); MCP 40 g Gladiolus spp.: Doidge (1950, et al. 1953); PREM 30706 ( ); p ( ); Zea mays: Van der Westhuizen & Bredell (1972); MRC 203 GENUS EUPENICILLIUM * P. lapidosum Raper & Fennell soil: IMI 113 748; PREM 48880; UCT * P. lapidosum Raper & Fennell soil: IMI 113 748; PREM 48880; UCT alucateum (see E. terrenum) alucateum (see E. terrenum) molasses meal: Roth (1968) soil: CSIR 1088, 1089 Zea mays: CSIR 606 crustaceum Ludwig soil: Scott (1968b), CBS 214.71, 215.71, 216,71; CSIR 1026, 1027, 1057, 1102, 1105, 1124; PREM 48551 P. asperum (Shear) Raper & Thom Eucalyptus maculata: Eicker (1973) soil: Eicker (1969, 1970, 1973); PREM 44264 * P. gladioli McCulloch & Thom Gladiolus spp.: Doidge (1950, et al. 1953); PREM 30706 molasses meal: Roth (1968) soil: CSIR 1088, 1089 Zea mays: CSIR 606 crustaceum Ludwig soil: Scott (1968b), CBS 214.71, 215.71, 216,71; CSIR 1026, 1027, 1057, 1102, 1105, 1124; PREM 48551 P. asperum (Shear) Raper & Thom Eucalyptus maculata: Eicker (1973) soil: Eicker (1969, 1970, 1973); PREM 44264 * P. gladioli McCulloch & Thom Gladiolus spp.: Doidge (1950, et al. 1953); PREM 30706 ehrlichii (see E. javanicum var. javanicum) erubescens (see E. terrenum) euglaucum (Van Beyma) Stolk & Samson soil: Stolk & Samson (1983); CBS 467.67 Zea mays: Stolk & Samson (1983); CBS 238.65 = anatolicum Stolk soil: Scott (1968b); Stolk & Samson (1983) CSIR 1095, 1113 = hirayamae Scott & Stolk soil: Allsopp etal. (1987); Scott (1968b); CSIR 1112; PPRI 3264; PREM 49212 Zea mays: CBS 238.65; CSIR 445 * P. hiravamae Scott & Stolk Zea mays: CSIR 487, 554; IMI 136 205 hirayamae (see E. euglaucum) inusitatum Scott soil: Scott (1968a); Stolk & Samson (1983); CBS 351.67; CSIR 1096; PREM 48570 ^ P. inusitatum Scott soil: IMI 136 214 javanicum (Van Beyma) Stolk & Scott var. javanicum apple juice: Stolk & Samson (1983) soil: Stolk & Samson (1983); CBS 211.71 undetermined host: Stolk & Samson (1983) = brefeldianum (B. Dodge) Stolk & Scott apple juice: CBS 291.62 soil: Scott (1968b); Stolk & Samson (1984), CBS, CSIR 1002, 1010, 1011, 1012, 1013; 1028, 1029, 1030, 1068, 1069, 1108, 1109; PPRI 3260; PREM 48555 undetermined host: Stolk & Samson (1983) ^ P. brefeldianum B. Dodge apple juice: Van der Spuy et al. (1975) = ehrlichii (Klebahn) Stolk & Scott soil: Scott (1968b), CSIR 1025, 1026, 1027; MCP, ; PPRI 3262, 3695; PREM 49195, 49362 = javanicum (Van Beyma) Stolk & Scott Arachis hypogaea: CSIR 416, 417, 419, 420, 421, 424; PREM 48259 soil: Scott (1968b), CSIR 1004, 1005, 1006, 1007, 1008, 1009, 1015, 1018, 1019, 1025, 1026, 1027, 1110; 48382, 48550; * P. brefeldianum (see E. javanicum var. javanicum) cinnamopurpureum Scott & Stolk Pinus spp.: CBS 492.66; CSIR 946 soil: Scott (1968b); Stolk & Samson (1983); CBS 490.66, 491.66; CSIR 942, 943, 945, 946, 1126; PREM 48558 Zea mays: Stolk & Samson (1983) undetermined host: Stolk & Samson (1983) * P. pusillum G. Smith parvum (Raper & Fennell) Stolk & Scott soil: Scott (1968b), CSIR 973, 1054, 1058; MCP. PPRI 3263; PREM 48557, 48881. 48887, 49194 meridianum Scott anatolicum (see E. euglaucum) anatolicum (see E. euglaucum) soil: Scott (1968a, b); Stolk & Samson (1983); CBS 314.67, 217.71, 219.71; CSIR 1052, 1037, 1036, 1103; PREM 48884 baarnense (Van Beyma) Stolk & Scott Acacia mollesjuna: CBS 339.61 soil: Scott (1968b), CSIR 1059, 1070, 1071, 1090, 1106, 1107, 1130; PPRI 3259 brefeldianum (see E. javanicum var. javanicum) catenatum Scott soil: Scott (1968a); Stolk & Samson (1983); CBS 325.67; CSIR 1097; PREM 48556 cinnamopurpureum Scott & Stolk Pinus spp.: CBS 492.66; CSIR 946 soil: Scott (1968b); Stolk & Samson (1983); CBS 490.66, 491.66; CSIR 942, 943, 945, 946, 1126; PREM 48558 Zea mays: Stolk & Samson (1983) undetermined host: Stolk & Samson (1983) * P. pusillum G. Smith baarnense (Van Beyma) Stolk & Scott Acacia mollesjuna: CBS 339.61 soil: Scott (1968b), CSIR 1059, 1070, 1071, 1090, 1106, 1107, 1130; PPRI 3259 * P. meridianum Scott soil: IMI 0 6 209 ochrosalmoneum Scott &. Stolk soil: Scott (1968b), Stolk & Samson (1983); CBS 515.67; CSIR 1094; PREM 48886 Zea mays: Stolk & Samson (1983); CBS 489.66; CSIR 145 * P. ochrosalmoneum Udagawa Zea mays: IMI 116 248 hirayamae (see E. euglaucum) Iris spp.: Doidge et al. (1953) ^ P. inusitatum Scott pp g ( ) Litchi chinensis: Doidge et al. (1953); Roth (1963) soil: IMI 136 214 g Lupinus spp.: Van Warmelo (1967) p Mangifera indica: Wehner et al. (1981) Malus sylvestris: Doidge et al. (1953); Verwoerd (1929) Malus sylvestris: Doidge et al. (1953); Verwoerd (1929) Medicago sativa: Lamprecht (1988); Marasas & Bredell (1973); Van soil: Stolk & Samson (1983); CBS 211.71 undetermined host: Stolk & Samson (1983) = brefeldianum (B. Dodge) Stolk & Scott apple juice: CBS 291.62 soil: Scott (1968b); Stolk & Samson (1984), CBS, CSIR 1002, 1010, 1011, 1012, 1013; 1028, 1029, 1030, 1068, 1069, 1108, 1109; PPRI 3260; PREM 48555 undetermined host: Stolk & Samson (1983) ^ P. brefeldianum B. Dodge apple juice: Van der Spuy et al. (1975) = ehrlichii (Klebahn) Stolk & Scott soil: Scott (1968b), CSIR 1025, 1026, 1027; MCP, ; PPRI 3262, 3695; PREM 49195, 49362 = javanicum (Van Beyma) Stolk & Scott Arachis hypogaea: CSIR 416, 417, 419, 420, 421, 424; PREM 48259 soil: Scott (1968b), CSIR 1004, 1005, 1006, 1007, 1008, 1009, 1015, 1018, 1019, 1025, 1026, 1027, 1110; 48382, 48550; * P. javanicum Van Beyma soil: Eicker (1969, 1970, 1973); Martin (1960); Papendorf (1976); MCP 123 soil: Stolk & Samson (1983); CBS 211.71 undetermined host: Stolk & Samson (1983) = brefeldianum (B. Dodge) Stolk & Scott apple juice: CBS 291.62 soil: Scott (1968b); Stolk & Samson (1984), CBS, CSIR 1002, 1010, 1011, 1012, 1013; 1028, 1029, 1030, 1068, 1069, 1108, 1109; PPRI 3260; PREM 48555 undetermined host: Stolk & Samson (1983) ^ P. brefeldianum B. Dodge apple juice: Van der Spuy et al. (1975) = ehrlichii (Klebahn) Stolk & Scott soil: Scott (1968b), CSIR 1025, 1026, 1027; MCP, ; PPRI 3262, 3695; PREM 49195, 49362 = javanicum (Van Beyma) Stolk & Scott Arachis hypogaea: CSIR 416, 417, 419, 420, 421, 424; PREM 48259 soil: Scott (1968b), CSIR 1004, 1005, 1006, 1007, 1008, 1009, 1015, 1018, 1019, 1025, 1026, 1027, 1110; 48382, 48550; * P. javanicum Van Beyma soil: Eicker (1969, 1970, 1973); Martin (1960); Papendorf (1976); MCP 123 molasses meal: Roth (1968) soil: CSIR 1088, 1089 javanicum Van Beyma soil: Eicker (1969, 1970, 1973); Martin (1960); Papendorf (1976); MCP 123 Zea mays: Van der Westhuizen & Bredell (1972) lapidosum Scott & Stolk soil: Scott (1968b); CBS 318.66, CSIR 1035; PREM 48880 Zea mays: CSIR 1093 unrecorded host: Stolk & Samson (1983) * P. lapidosum Raper & Fennell soil: IMI 113 748; PREM 48880; UCT meridianum Scott soil: Scott (1968a, b); Stolk & Samson (1983); CBS 314.67, 217.71, 219.71; CSIR 1052, 1037, 1036, 1103; PREM 48884 * P. meridianum Scott soil: IMI 0 6 209 ochrosalmoneum Scott & Stolk waksmanii Zaleski Barleria obtusa: PPRI 3704; PREM 49884 Encephalartos spp.: PPRI 4283 Medicago sativa: PREM 44474 mushroom casing: Smit (1984) soil: Eicker (1975); Papendorf (1976); MCP 40 Zea mays: Van der Westhuizen & Bredell (1972); MRC 203 waksmanii Zaleski Barleria obtusa: PPRI 3704; PREM 49884 Encephalartos spp.: PPRI 4283 Medicago sativa: PREM 44474 mushroom casing: Smit (1984) soil: Eicker (1975); Papendorf (1976); MCP 40 Zea mays: Van der Westhuizen & Bredell (1972); MRC 203 Warmelo (1967); PREM 44530. 4 Musa spp.: Roth & Loest (1965) mushroom casing: Smit (1984) Narcissus spp.: Doidge et al. (1953) nuts and dried fruit: Wehner & Rabie (1970) nuts and dried fruit: Wehner & Rabie (1970) Panicum coloratum: Eicker (1976) Pinus spp.: Lundquist (1987) Prunus persica: Doidge et al. (1953) Prunus salicina: Doidge et al. (1953) g ( ) recalcitrant seed: Berjak et al. (1989); Mycock & Berjak (1990) recalcitrant seed: Berjak et al. (1989); Mycock & Berjak (199 Raphanus sativus var. longipinnatus: Holtzhausen (1978) Raphanus sativus var. longipinnatus: Holtzhausen (1978) Saccharum officinarum: Doidge (1950) solitum (see P. aurantiogriseum) soppii (see P. miczynskii) spiculisporum (see T. trachyspermus) viridicatum Westling aerospora. Thom (1930); Doidge (1950) Arachis hypogaea: MRC 292 birdseed: PREM 4221 cereal and legume products: Scott (1965) cheese: MRC 1132 Hordeum wlgare: MRC 1761, 2669, 2830 material: Doidge (1950) natural gum: Roth (1968) soil: CSIR 15. 405. 407; PREM 48906 Zea mays Hutchison et al. (1973); Pitt (1979); Van der Westhuizen & Bredell (1972); CSIR 255. 349. 354. 396. 413. 425. 430. 460. 570. 663. 724; MRC 422 = oli\ino\iride Biourge Allium cepa: PREM 44769 = palitans Westling Allium cepa PREM 44758 flannel: Doidge (1950) Zea mays Van der Westhuizen &. Bredell (1972) vulpinum (Cooke & Massee) Siefert & Samson dung: PPRI 3727; PREM 49880 = cla\iforme Bain spiculisporum (see T. trachyspermus) viridicatum Westling aerospora. Thom (1930); Doidge (1950) Arachis hypogaea: MRC 292 birdseed: PREM 4221 cereal and legume products: Scott (1965) cheese: MRC 1132 Hordeum wlgare: MRC 1761, 2669, 2830 material: Doidge (1950) natural gum: Roth (1968) soil: CSIR 15. 405. 407; PREM 48906 Zea mays Hutchison et al. (1973); Pitt (1979); Van der Westhuizen & Bredell (1972); CSIR 255. 349. 354. 396. 413. 425. 430. 460. 570. 663. 724; MRC 422 = oli\ino\iride Biourge Allium cepa: PREM 44769 = palitans Westling Allium cepa PREM 44758 flannel: Doidge (1950) Zea mays Van der Westhuizen &. Bredell (1972) vulpinum (Cooke & Massee) Siefert & Samson dung: PPRI 3727; PREM 49880 = cla\iforme Bain spinulosum Thom spinulosum Thom Allium cepa Naude & Jooste (1989); PREM 48012. 48561. 48871. 48872, 48873 pp soil: Cohen (1950); Papendorf (1976); MCP 165; UCT undetermined host: Thom (1930) soil: CSIR 15. 405. 407; PREM 48906 stolkiae (see E. stolkiae) stoloniferum (see P. hrevicompactum) vulpinum (Cooke & Massee) Siefert & Samson dung: PPRI 3727; PREM 49880 = cla\iforme Bain 90 Bothalia 22,1 (1992) wortmannii (see T. wortmannii) Penicillium species undetermined aerospora: Ordman (1963, 1970); Ordman & Etter (1956); Radmore (1986); Van der Merwe et al. (1979) Arachis hypogaea: Ferreira & Lutchman (1989); Marasas & Van Rensburg (1986); Van Warmelo (1967) Cenchrus ciliaris: Bezuidenhout (1977) cheese: Liick et al.(1976); Liick & Wehner (1979) Citrus sinensis: Doidge (1950); Doidge & Van der Plank (1936); Roth (1967); Verwoerd (1929) Crucifera spp.: Holtzhausen & Knox-Davies (1974) Cucumis melo: Doidge et al. (1953) Eucalyptus spp.: Lundquist & Baxter (1985) fodder: Dutton & Westlake (1985) foodstuff: Martin & Keen (1978) Hordeum vulgare: Liibben & Rabie (1989) indigenous seed: Isaacs & Benic (1990) Iris spp.: Doidge et al. (1953) Litchi chinensis: Doidge et al. (1953); Roth (1963) Lupinus spp.: Van Warmelo (1967) Mangifera indica: Wehner et al. (1981) Malus sylvestris: Doidge et al. (1953); Verwoerd (1929) Medicago sativa: Lamprecht (1988); Marasas & Bredell (1973); Van erubescens (see E. terrenum) Penicillium species undetermined aerospora: Ordman (1963, 1970); Ordman & Etter (1956); Radmore (1986); Van der Merwe et al. (1979) Arachis hypogaea: Ferreira & Lutchman (1989); Marasas & Van Rensburg (1986); Van Warmelo (1967) Cenchrus ciliaris: Bezuidenhout (1977) cheese: Liick et al.(1976); Liick & Wehner (1979) Citrus sinensis: Doidge (1950); Doidge & Van der Plank (1936); Roth (1967); Verwoerd (1929) Crucifera spp.: Holtzhausen & Knox-Davies (1974) Cucumis melo: Doidge et al. (1953) Eucalyptus spp.: Lundquist & Baxter (1985) fodder: Dutton & Westlake (1985) foodstuff: Martin & Keen (1978) Hordeum vulgare: Liibben & Rabie (1989) indigenous seed: Isaacs & Benic (1990) Iris spp.: Doidge et al. (1953) Litchi chinensis: Doidge et al. (1953); Roth (1963) Lupinus spp.: Van Warmelo (1967) Mangifera indica: Wehner et al. (1981) Malus sylvestris: Doidge et al. (1953); Verwoerd (1929) Zea mays: CBS 238.65; CSIR 445 f pp Cucumis melo: Doidge et al. (1953) * P. hiravamae Scott & Stolk Eucalyptus spp.: Lundquist & Baxter (1985) fodder: Dutton & Westlake (1985) spiculisporus (see T. trachvspermus) thermophilus Stolk Celtis africana litter: Pitt (1979); Stolk & Samson (1972); CBS 116.72 * P dupontii Griffin & Maubl. apple juice: Van der Spuy et al. (1975) compost: IMI 197 483 mushroom compost: Eicker (1977) thermophilus Stolk Celtis africana litter: Pitt (1979); Stolk & Samson (1972); CBS 116.72 trachvspermus (Shear) Stolk & Samson Manihot esculenta: Pitt (1979); MRC 724 contaminant: PPRI 3885 = spiculisporus (Lehman) C.R. Benjamin soil: CSIR 955, 956 ^ P spiculisporum Lehman undetermined host: MCP 1125 # P. terrenum Scott soil: IMI 136 208 soil: CSIR 955, 956 ^ P spiculisporum Lehman undetermined host: MCP 1125 pinetorum Stolk soil: Allsopp etal. (1987); Scott (1968b); CBS 328.71; CSIR 1092, 1125; PPRI 3490; PREM 48883; UCT soil: Allsopp etal. (1987); Scott (1968b); CBS 328.71; CSIR 1092, 1125; PPRI 3490; PREM 48883; UCT Bothalia 22.1 (1992) 91 Bothalia 22.1 (1992) ^ P. pinetorum Stolk soil: CSIR 1092 senticosum Scott soil: Scott (1968a, b); Stolk & Samson (1983); CBS 30.67, 329.71; CSIR 1042, 1104; IMI 216 905; PREM 48882 # P. senticosum Scott soil: IMI 216 905 flavus (Klocker) Stolk & Samson apple juice: Pitt (1979) contaminant: PPRI 37W; PREM 48577 Encephalartos laevifolius: PPRI 320; PREM 49074 wine bottle cork: MCP 27 = flavus var. macrosporus Stolk & Samson fruit: Stolk & Samson (1972); CBS 317.63; IMI 197 487 soil: Stolk & Samson (1972); CBS 226.72; PPRI 3791 = vermiculatus (Dang.) C.R. Benjamin soil: CSIR 960, 962 , 963 * P. dangeardii Pitt apple juice: IMI 197 478 * P. vermiculatum Dang. apple juice: Van der Spuy et al. (1975) soil: Eicker (1969, 1973) luteus (Zukal) C.R. Benjamin ^ P. luteum Zukal aerospora: Roth (1968) Citrus sinensis: Doidge (1950) flannel: Doidge (1950); PREM 33288 molasses meal: Roth (1968) natural gum: Roth (1968) spiculisporus (see T. trachvspermus) thermophilus Stolk Celtis africana litter: Pitt (1979); Stolk & Samson (1972); CBS 116.72 * P dupontii Griffin & Maubl. apple juice: Van der Spuy et al. (1975) compost: IMI 197 483 mushroom compost: Eicker (1977) trachvspermus (Shear) Stolk & Samson Manihot esculenta: Pitt (1979); MRC 724 contaminant: PPRI 3885 = spiculisporus (Lehman) C.R. Benjamin soil: CSIR 955, 956 ^ P spiculisporum Lehman undetermined host: MCP 1125 vermiculatus (see T. flavus) wortmannii (Klocker) C.R. Benjamin Arachis hypogaea: MRC 332 Oryza scuiva: Pitt (1979) soil: Allsopp et al. (1987); MCP 1134; Stolk & Samson (1972); CBS 293.53; CSIR 954, 957, 964, 965 Whtsoma marginaia: PPRI 3675 * P. wortmannii Klocker = flavus var. macrosporus Stolk & Samson fruit: Stolk & Samson (1972); CBS 317.63; IMI 197 487 soil: Stolk & Samson (1972); CBS 226.72; PPRI 3791 shearii Stolk & Scott Medicago spp.: Lamprecht (1988); PPRI 4017; PREM 48322 soil: Scott (1968b); CSIR 1003, 1016, 1017; PREM 48549 Zea mays: CSIR 722 * P. dangeardii Pitt apple juice: IMI 197 478 Zea mays: CSIR 722 stolkiae Scott soil: Scott (1968a, b); Stolk & Samson (1983); CBS 315.67, 330.71, 331.71; CSIR 1003, 1041, 1074; PREM 48552 # P. pinetorum Stolk stolkiae Scott soil: IMI 136 210 terrenum Scott soil: Scott (1968a, b), Stolk & Samson (1983); CBS 30.67, 212.71, 20.71, 220.71, 327.71; CSIR 972, 1020, 1021, 1022, 1023, 1024; PPRI 3266 = aluuiceum Scott soil: Scott (1968a); Stolk & Samson (1983); CBS 317.67; CSIR 1039, 1056, 1091, 1100, 1101; PPRI 3488; PREM 48885 = erubescens Scott soil: Scott (1968a, b); Stolk & Samson (1983); CBS 318.67, 319.67; CSIR 944, 974, 1040, 1032, 1034, 1038, 1040, 1061; PPRI 3261; PREM 48554, 49199 P. alutaceum Scott soil: IMI 136 243 # P. erubescens Scott soil: IMI 136 404 # P. terrenum Scott soil: IMI 136 208 Eupenicillium species undetermined mushroom casing: Smit (1984) soil: CSIR 1127, 1128, 1129; PPRI; UCT GENUS TALAROMYCES avellaneus (Thom & 'I\iresson) C.R. Benjamin (anamorph: Merimbla ingelheimense, Pitt 1979) soil: CSIR 958, 959 bacillosporus (Swift) C.R Benjamin (anamorph: Geosmithia swiftii Pitt 1979) soil: CSIR 961 * P. vermiculatum Dang. apple juice: Van der Spuy et al. (1975) soil: Eicker (1969, 1973) stolkiae Scott soil: Scott (1968a, b); Stolk & Samson (1983); CBS 315.67, 330.71, 331.71; CSIR 1003, 1041, 1074; PREM 48552 # P lki S luteus (Zukal) C.R. Benjamin ^ P. luteum Zukal aerospora: Roth (1968) Citrus sinensis: Doidge (1950) flannel: Doidge (1950); PREM 33288 molasses meal: Roth (1968) natural gum: Roth (1968) soil: IMI 136 210 GENUS TALAROMYCES wortmannii (Klocker) C.R. Benjamin Arachis hypogaea: MRC 332 Oryza scuiva: Pitt (1979) soil: Allsopp et al. (1987); MCP 1134; Stolk & Samson (1972); CBS 293.53; CSIR 954, 957, 964, 965 Whtsoma marginaia: PPRI 3675 * P. wortmannii Klocker Oryza sativa: MRC 243 avellaneus (Thom & 'I\iresson) C.R. Benjamin (anamorph: Merimbla ingelheimense, Pitt 1979) soil: CSIR 958, 959 avellaneus (Thom & 'I\iresson) C.R. Benjamin (anamorph: Merimbla ingelheimense, Pitt 1979) soil: CSIR 958, 959 bacillosporus (Swift) C.R Benjamin (anamorph: Geosmithia swiftii Pitt 1979) soil: CSIR 961 bacillosporus (Swift) C.R Benjamin (anamorph: Geosmithia swiftii Pitt 1979) soil: CSIR 961 bacillosporus (Swift) C.R Benjamin (anamorph: Geosmithia swiftii Pitt 1979) soil: CSIR 961
https://openalex.org/W2800704546	https://discovery.dundee.ac.uk/ws/files/30131874/Final_Published_Version.pdf	English	null	Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases	Nature communications	2,018	cc-by	3,243	Citation for published version (APA): Blue Mountains Eye Study—GWAS group, NEIGHBORHOOD Consortium, Wellcome Trust Case Control Consortium 2, & Souzeau, E. (2018). Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases. Nature Communications, 9(1), 1864. Article 1864. https://doi.org/10.1038/s41467-018-03646-6 University of Dundee Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Blue Mountains Eye Study—GWAS group, NEIGHBORHOOD Consortium, Wellcome Trust Case Control Consortium 2, & Souzeau, E. (2018). Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases. Nature Communications, 9(1), 1864. Article 1864. https://doi.org/10.1038/s41467-018-03646-6 Citation for published version (APA): Blue Mountains Eye Study—GWAS group, NEIGHBORHOOD Consortium, Wellcome Trust Case Control Consortium 2, & Souzeau, E. (2018). Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases. Nature Communications, 9(1), 1864. Article 1864. https://doi.org/10.1038/s41467-018-03646-6 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 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/. Adriana I. Iglesias 1,2,3, Aniket Mishra4, Veronique Vitart5, Yelena Bykhovskaya6,7, René Höhn8,9, Henriët Springelkamp1, Gabriel Cuellar-Partida10, Puya Gharahkhani 10, Jessica N. Cooke Bailey 11,12, Colin E. Willoughby13,14, Xiaohui Li 15,16, Seyhan Yazar5,17, Abhishek Nag18, Anthony P. Khawaja 19,20, Ozren Polašek21, David Siscovick22,23, Paul Mitchell24, Yih Chung Tham25, Jonathan L. Haines 11,12, Lisa S. Kearns26, Caroline Hayward 5, Yuan Shi25, Elisabeth M. van Leeuwen1, Kent D. Taylor15,16, Blue Mountains Eye Study - GWAS group, Pieter Bonnemaijer 1, Jerome I. Rotter15,16, Nicholas G. Martin27, Tanja Zeller28,29, Richard A. Mills30, Emmanuelle Souzeau 30, Sandra E. Stafﬁeri 26, Jost B. Jonas 31, Irene Schmidtmann32, Thibaud Boutin5, Jae H. Kang 33, Sionne E.M. Lucas34, Tien Yin Wong25,35,36, Manfred E. Beutel37, James F. Wilson 5,38, NEIGHBORHOOD consortium, Wellcome Trust Case Control Consortium 2 (WTCCC2), André G. Uitterlinden2,39,40, Eranga N. Vithana25, Paul J. Foster 20, Pirro G. Hysi 18, Alex W. Hewitt 26,41, Chiea Chuen Khor 42, Louis R. Pasquale 33,43, Grant W. Montgomery 27,44, Adriana I. Iglesias 1,2,3, Aniket Mishra4, Veronique Vitart5, Yelena Bykhovskaya6,7, René Höhn8,9, Henriët Springelkamp1, Gabriel Cuellar-Partida10, Puya Gharahkhani 10, Jessica N. Cooke Bailey 11,12, Colin E. Willoughby13,14, Xiaohui Li 15,16, Seyhan Yazar5,17, Abhishek Nag18, Anthony P. Khawaja 19,20, Ozren Polašek21, David Siscovick22,23, Paul Mitchell24, Yih Chung Tham25, Jonathan L. Haines 11,12, Lisa S. Kearns26, Caroline Hayward 5, Yuan Shi25, Elisabeth M. van Leeuwen1, Kent D. Taylor15,16, Blue Mountains Eye Study - GWAS group, Pieter Bonnemaijer 1, Jerome I. Rotter15,16, Nicholas G. Martin27, Tanja Zeller28,29, Richard A. Mills30, Emmanuelle Souzeau 30, Sandra E. Stafﬁeri 26, Jost B. Jonas 31, Irene Schmidtmann32, Thibaud Boutin5, Jae H. Kang 33, Sionne E.M. : Nature Communications https://doi.org/10.1038/s41467-018-03646-6; published online 14 May 2018 Correction to: Nature Communications https://doi.org/10.1038/s41467-018-03646-6; published online Emmanuelle Souzeau, who contributed to analysis of data, was inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article. Author Correction: Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases Adriana I. Iglesias et al.# NATURE COMMUNICATIONS \| (2019) 10:155 \| https://doi.org/10.1038/s41467-018-07819-1 \| www.nature.com/naturecommunications © The Author(s) 2019 Blue Mountains Eye Study - GWAS group Blue Mountains Eye Study GWAS group Jie Jin Wang24, Elena Rochtchina24, John Attia46, Rodney Scott46, Elizabeth G. Holliday46, Tien Yin Wong26, Paul N. Baird26, Jing Xie26, Michael Inouye47, Ananth Viswanathan20 & Xueling Sim36 46University of Newcastle, Newcastle, NSW 2308, Australia. 47Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia 46University of Newcastle, Newcastle, NSW 2308, Australia. 47Walter and Eliza Hall Institute of Medical Research 46University of Newcastle, Newcastle, NSW 2308, Australia. 47Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia Lucas34, Tien Yin Wong25,35,36, Manfred E. Beutel37, James F. Wilson 5,38, NEIGHBORHOOD consortium, Wellcome Trust Case Control Consortium 2 (WTCCC2), André G. Uitterlinden2,39,40, Eranga N. Vithana25, Paul J. Foster 20, Pirro G. Hysi 18, Alex W. Hewitt 26,41, Chiea Chuen Khor 42, Louis R. Pasquale 33,43, Grant W. Montgomery 27,44, Correspondence and requests for materials should be addressed to S.M. (email: Stuart.MacGregor@qimrberghofer.edu.au). #A full list of authors and their afﬁliations appears at the end of the paper. 1 AUTHOR CORRECTION 1Department of Ophthalmology, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. 2Department of Epidemiology, Erasmus Medica Center, 3000 CA Rotterdam, The Netherlands. 3Department of Clinical Genetics, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands 4University of Bordeaux, Bordeaux Population Health Research Center, INSERM UMR 1219, F-33000 Bordeaux, France. 5Institute of Genetics and Molecular Medicine, Medical Research Council Human Genetics Unit, University of Edinburgh, Edinburgh EH42XU, UK. 6Regenerative Medicine Institute and Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. 7Cornea Genetic Eye Institute, Los Angeles, CA 90048, USA. 8Department of Ophthalmology, University Medical Center Mainz, 55131 Mainz, Germany. 9Department of Ophthalmology, Inselspital, University Hospital Bern, University of Bern, CH-3010 Bern, Switzerland. 10Statistical Genetics, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4029, Australia. 11Department of Population and Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH 44106, USA. 12Institute for Computational Biology, Case Western Reserve University, Cleveland, OH 44106, USA. 13Biomedical Sciences Research Institute, Ulster University, Belfast, Northern Ireland BT52 1SA, UK. 14Royal Victoria Hospital, Belfast Health and Social Care Trust, Belfast, Northern Ireland BT12 6BA, UK. 15Institute for Translational Genomics and Population Sciences and Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90509, USA. 16Division of Genomic Outcomes, Departments of Pediatrics and Medicine, Harbor-UCLA Medical Center, Torrance, CA 90502, USA. 17Centre for Ophthalmology and Visual Science, University o Western Australia, Lions Eye Institute, Perth, WA WA 6009, Australia. 18Department of Twin Research and Genetic Epidemiology, King’s College London, London WC2R 2LS, UK. 19Department of Public Health and Primary Care, Institute of Public Health, University of Cambridge School of Clinical Medicine, Cambridge CB2 0SR, UK. 20NIHR Biomedical Research Centre, Moorﬁelds Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London EC1V 9EL, UK. 21Faculty of Medicine, University of Split, HR-21000 Split, Croatia. 22Departments of Medicine and Epidemiology and Cardiovascular Health Research Unit, University of Washington, Washington, WA 98101, USA. 23The New York Academy of Medicine, New York, NY 10029, USA. 24Centre for Vision Research, Department of Ophthalmology and Westmead Institute for Medical Research University of Sydney, Sydney, NSW 2145, Australia. 25Singapore Eye Research Institute, Singapore National Eye Centre, Singapore 168751, Singapore. 26Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC 3002, Australia. 27Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane QLD 4029, Australia. 28Department of General and Interventional Cardiology, University Heart Center Hamburg, 20251 Hamburg, Germany. 29German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany. 30Department of Ophthalmology, Flinders University, Adelaide SA 5042, Australia. 31Department of Ophthalmology, Medical Faculty Mannheim of the Ruprecht-Karls-University of Heidelberg, 68167 Mannheim, Germany. 32Institute for Medical Biostatistics, Epidemiology and Informatics, University Medical Center Mainz, 55131 Mainz, Germany. 33Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA. 34Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS 7005, Australia. 35Ophthalmology & Visual Sciences Academic Clinical Program (Eye ACP), Duke-NUS Medical School, Singapore 169857, Singapore. 36Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117549, Singapore. 37Department of Psychosomatic Medicine and Psychotherapy, University Medical Center Mainz, Mainz 55131, Germany. 38Centre for Global Health Research, Usher Institute for Population Health Sciences and Informatics, University o Edinburgh, Edinburgh EH16 4UX, UK. 39Department of Internal Medicine, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. 40Netherlands Consortium for Healthy Ageing, Netherlands Genomics Initiative, 2593 HW The Hague, The Netherlands. 41School of Medicine, Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS 7005, Australia. 42Genome Institute of Singapore, 60 Biopolis Street Singapore 138672, Singapore. 43Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear Inﬁrmary, Boston, MA 02114, USA. 44Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4067, Australia. 45Department of Ophthalmology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands. These authors contributed equally: Adriana I. Iglesias, Aniket Mishra Veronique Vitart. These authors jointly supervised this work: Jamie E. Craig, Yaron S. Rabinowitz, Janey L. Wiggs, Kathryn P. Burdon, Cornelia M van Duijn, Stuart MacGregor. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-018-07819-1 Caroline C.W. Klaver 1,2,45, Tin Aung25,35,36, Norbert Pfeiffer8, David A. Mackey17, Christopher J. Hammond 18, Ching-Yu Cheng25,35,36, Jamie E. Craig30, Yaron S. Rabinowitz6,7, Janey L. Wiggs 43, Kathryn P. Burdon 34, Cornelia M. van Duijn2 & Stuart MacGregor 10 1Department of Ophthalmology, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. 2Department of Epidemiology, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. 3Department of Clinical Genetics, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. 4University of Bordeaux, Bordeaux Population Health Research Center, INSERM UMR 1219, F-33000 Bordeaux, France. 5Institute of Genetics and Molecular Medicine, Medical Research Council Human Genetics Unit, University of Edinburgh, Edinburgh EH42XU, UK. 6Regenerative Medicine Institute and Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. 7Cornea Genetic Eye Institute, Los Angeles, CA 90048, USA. 8Department of Ophthalmology, University Medical Center Mainz, 55131 Mainz, Germany. 9Department of Ophthalmology, Inselspital, University Hospital Bern, University of Bern, CH-3010 Bern, Switzerland. 10Statistical Genetics, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4029, Australia. 11Department of Population and Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH 44106, USA. 12Institute for Computational Biology, Case Western Reserve University, Cleveland, OH 44106, USA. 13Biomedical Sciences Research Institute, Ulster University, Belfast, Northern Ireland BT52 1SA, UK. 14Royal Victoria Hospital, Belfast Health and Social Care Trust, Belfast, Northern Ireland BT12 6BA, UK. 15Institute for Translational Genomics and Population Sciences and Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90509, USA. 16Division of Genomic Outcomes, Departments of Pediatrics and Medicine, Harbor-UCLA Medical Center, Torrance, CA 90502, USA. 17Centre for Ophthalmology and Visual Science, University of Western Australia, Lions Eye Institute, Perth, WA WA 6009, Australia. 18Department of Twin Research and Genetic Epidemiology, King’s College London, London WC2R 2LS, UK. 19Department of Public Health and Primary Care, Institute of Public Health, University of Cambridge School of Clinical Medicine, Cambridge CB2 0SR, UK. 20NIHR Biomedical Research Centre, Moorﬁelds Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London EC1V 9EL, UK. 21Faculty of Medicine, University of Split, HR-21000 Split, Croatia. 22Departments of Medicine and Epidemiology and Cardiovascular Health Research Unit, University of Washington, Washington, WA 98101, USA. 23The New York Academy of Medicine, New York, NY 10029, USA. 24Centre for Vision Research, Department of Ophthalmology and Westmead Institute for Medical Research, University of Sydney, Sydney, NSW 2145, Australia. 25Singapore Eye Research Institute, Singapore National Eye Centre, Singapore 168751, Singapore. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-018-07819-1 26Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC 3002, Australia. 27Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane QLD 4029, Australia. 28Department of General and Interventional Cardiology, University Heart Center Hamburg, 20251 Hamburg, Germany. 29German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany. 30Department of Ophthalmology, Flinders University, Adelaide SA 5042, Australia. 31Department of Ophthalmology, Medical Faculty Mannheim of the Ruprecht-Karls-University of Heidelberg, 68167 Mannheim, Germany. 32Institute for Medical Biostatistics, Epidemiology and Informatics, University Medical Center Mainz, 55131 Mainz, Germany. 33Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA. 34Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS 7005, Australia. 35Ophthalmology & Visual Sciences Academic Clinical Program (Eye ACP), Duke-NUS Medical School, Singapore 169857, Singapore. 36Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117549, Singapore. 37Department of Psychosomatic Medicine and Psychotherapy, University Medical Center Mainz, Mainz 55131, Germany. 38Centre for Global Health Research, Usher Institute for Population Health Sciences and Informatics, University of Edinburgh, Edinburgh EH16 4UX, UK. 39Department of Internal Medicine, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. 40Netherlands Consortium for Healthy Ageing, Netherlands Genomics Initiative, 2593 HW The Hague, The Netherlands. 41School of Medicine, Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS 7005, Australia. 42Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore. 43Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear Inﬁrmary, Boston, MA 02114, USA. 44Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4067, Australia. 45Department of Ophthalmology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands. These authors contributed equally: Adriana I. Iglesias, Aniket Mishra, Veronique Vitart. These authors jointly supervised this work: Jamie E. Craig, Yaron S. Rabinowitz, Janey L. Wiggs, Kathryn P. Burdon, Cornelia M. van Duijn, Stuart MacGregor. NATURE COMMUNICATIONS \| (2019) 10:155 \| https://doi.org/10.1038/s41467-018-07819-1 \| www.nature.com/naturecommunication 68Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX37BN, UK. 69Dept Statistics, University of Oxford, Oxford OX1 3TG, UK. 70Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. 71Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, 100 Roberts Road, Subiaco, WA 6008, Australia. 72Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Cambridge CB2 0XY, UK. 73Department of Psychosis Studies, NIHR Biomedical Research Centre for Mental Health at the Institute of Psychiatry, King’s College London and The South London and Maudsley NHS Foundation Trust, Denmark Hill, London SE5 8AF, UK. 74University of Queensland Diamantina Institute, Brisbane, QLD 4102, Australia. 75Dept Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK. 76Dept Epidemiology and Public Health, University College London, London WC1E 6BT, UK. 77Neuropsychiatric Genetics Research Group, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Eire, Ireland. 78Molecular and Physiological Sciences, The Wellcome Trust, London NW1 2BE, UK. 79Department of Oncology, Old Road Campus, University of Oxford, Oxford OX3 7DQ, UK. 80Digestive Diseases Centre, Leicester Royal Inﬁrmary, Leicester LE7 7HH, UK. 81Centre for Digestive Diseases, Queen Mary University of London, London E1 2AD, UK. 82Clinical Neurosciences, St George’s University of London, London SW17 0RE, UK. 83King’s College London Dept Medical and Molecular Genetics, King’s Health Partners, Guy’s Hospital, London SE1 9RT, UK. 84Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. 85King’s College London Social, Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, Denmark Hill, London SE5 8AF, UK. 86University of Cambridge Dept Clinical Neurosciences, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK. 87Dept Molecular Neuroscience, Institute of Neurology, Queen Square, London WC1N 3BG, UK NEIGHBORHOOD consortium R. Rand Allingham48, Murray H. Brilliant49, Donald L. Budenz50, William G. Christen51, John Fingert52,53, David S. Friedman54, Douglas Gaasterland55, Terry Gaasterland56, Michael A. Hauser48,57, Peter Kraft58, Richard K. Lee59, Paul R. Lichter60, Yutao Liu48,57, Stephanie J. Loomis43, Sayoko E. Moroi60, Margaret A. Pericak-Vance61, Anthony Realini62, Julia E. Richards60, Joel S. Schuman63, William K. Scott61, Kuldev Singh64, Arthur J. Sit65, Douglas Vollrath66, Robert N. Weinreb67, Gadi Wollstein63, Donald J. Zack54 & Kang Zhang67 R. Rand Allingham48, Murray H. Brilliant49, Donald L. Budenz50, William G. Christen51, John Fingert52,53, David S. Friedman54, Douglas Gaasterland55, Terry Gaasterland56, Michael A. Hauser48,57, Peter Kraft58, Richard K. Lee59, Paul R. Lichter60, Yutao Liu48,57, Stephanie J. Loomis43, Sayoko E. Moroi60, Margaret A. Pericak-Vance61, Anthony Realini62, Julia E. Richards60, Joel S. Schuman63, William K. Scott61, Kuldev Singh64, Arthur J. Sit65, Douglas Vollrath66, Robert N. Weinreb67, Gadi Wollstein63, Donald J. Zack54 & Kang Zhang67 2 2 p y y y y ept Clinical Neurosciences, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK. 87Dept Molecular Neuroscience, S L d WC1N 3BG UK AUTHOR CORRECTION NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-018-07819-1 48Department of Ophthalmology, Duke University Medical Center, Durham, NC 27705, USA. 49Center for Human Genetics, Marshﬁeld Clinic Research Foundation, Marshﬁeld, WI, USA. 50Department of Ophthalmology, University of North Carolina, Chapel Hill, NC 27517, USA. 51Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02114, USA. 52Department of Ophthalmology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA. 53Department of Anatomy/Cell Biology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA. 54Wilmer Eye Institute, John Hopkins University, Baltimore, MD 21287, USA. 55Eye Doctors of Washington, Chevy Chase, MD 20815, USA. 56Scripps Genome Center, University of California at San Diego, San Diego, CA 92037, USA. 57Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA. 58Department of Biostatistics, Harvard School of Public Health, Boston, MA 02115, USA. 59Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA. 60Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105, USA. 61Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, USA. 62Department of Ophthalmology, WVU Eye Institute, Morgantown, WV 26506, USA. 63Department of Ophthalmology, UPMC Eye Center, University of Pittsburgh, Pittsburgh, PA 15213, USA. 64Department of Ophthalmology, Stanford University, Palo Alto, CA 94303, USA. 65Department of Ophthalmology, Mayo Clinic, Rochester, MN 55905, USA. 66Department of Genetics, Stanford University, Palo Alto, CA 94305, USA. 67Department of Ophthalmology, Hamilton Eye Center, University of California, San Diego, CA 92093, USA Wellcome Trust Case Control Consortium 2 (WTCCC2) Wellcome Trust Case Control Consortium 2 (WTCCC2) Peter Donnelly68,69, Ines Barroso70, Jenefer M. Blackwell71,72, Elvira Bramon73, Matthew A. Brown74, Juan P. Casas75,76, Aiden Corvin77, Panos Deloukas70, Audrey Duncanson78, Janusz Jankowski79,80,81, Hugh S. Markus82, Christopher G. Mathew83, Colin N.A. Palmer84, Robert Plomin85, Anna Rautanen68, Stephen J. Sawcer86, Richard C. Trembath83, Nicholas W. Wood87, Chris C.A. Spencer68, Gavin Band68, Céline Bellenguez68, Colin Freeman68, Garrett Hellenthal68, Eleni Giannoulatou68, Matti Pirinen68, Richard Pearson68, Amy Strange68, Zhan Su68, Damjan Vukcevic68, Cordelia Langford70, Sarah E. Hunt70, Sarah Edkins70, Rhian Gwilliam70, Hannah Blackburn70, Suzannah J. Bumpstead70, Serge Dronov70, Matthew Gillman70, Emma Gray70, Naomi Hammond70, Alagurevathi Jayakumar70, Owen T. McCann70, Jennifer Liddle70, Simon C. Potter70, Radhi Ravindrarajah70, Michelle Ricketts70, Matthew Waller70, Paul Weston70, Sara Widaa70 & Pamela Whittaker70 Wellcome Trust Case Control Consortium 2 (WTCCC2) Peter Donnelly68,69, Ines Barroso70, Jenefer M. Blackwell71,72, Elvira Bramon73, Matthew A. Brown74, Juan P. Casas75,76, Aiden Corvin77, Panos Deloukas70, Audrey Duncanson78, Janusz Jankowski79,80,81, Hugh S. Markus82, Christopher G. Mathew83, Colin N.A. Palmer84, Robert Plomin85, Anna Rautanen68, Stephen J. Sawcer86, Richard C. Trembath83, Nicholas W. Wood87, Chris C.A. Spencer68, Gavin Band68, Céline Bellenguez68, Colin Freeman68, Garrett Hellenthal68, Eleni Giannoulatou68, Matti Pirinen68, Richard Pearson68, Amy Strange68, Zhan Su68, Damjan Vukcevic68, Cordelia Langford70, Sarah E. Hunt70, Sarah Edkins70, Rhian Gwilliam70, Hannah Blackburn70, Suzannah J. Bumpstead70, Serge Dronov70, Matthew Gillman70, Emma Gray70, Naomi Hammond70, Alagurevathi Jayakumar70, Owen T. McCann70, Jennifer Liddle70, Simon C. Potter70, Radhi Ravindrarajah70, Michelle Ricketts70, Matthew Waller70, Paul Weston70, Sara Widaa70 & Pamela Whittaker70 3 NATURE COMMUNICATIONS \| (2019) 10:155 \| https://doi.org/10.1038/s41467-018-07819-1 \| www.nature.com/naturecommunication
https://openalex.org/W2967242776	https://seer.ufrgs.br/index.php/EmQuestao/article/download/86324/53336	Portuguese	null	Educação para a competência em informação e as ações realizadas por bibliotecários	Em Questão	2,019	cc-by	12,155	E-ISSN 1808-5245 E-ISSN 1808-5245 Adriana Rosecler Alcará Doutora; Universidade Estadual de Londrina, Londrina, PR, Brasil; adrianaalcara@gmail.com Resumo: O objetivo da pesquisa foi verificar as ações que os bibliotecários desenvolvem para a formação de habilidades para a competência em informação dos usuários, bem como quais são as suas necessidades em relação a essas ações. Para tanto, foi realizada uma pesquisa de campo, descritiva, com abordagem qualitativa, tendo como participantes sete bibliotecárias de diferentes tipos de bibliotecas. Os dados foram coletados por meio de um grupo de foco para identificar suas experiências, ações e necessidades para as práticas educativas voltadas à competência em informação. Para análise dos dados, apoiou-se na técnica de análise de conteúdo. Entre os resultados foi possível perceber que os bibliotecários têm realizado diversas atividades para a formação de seus usuários que podem contribuir para a competência em informação. Conclui-se, no entanto, que ainda é necessário estreitar os laços entre os campos de atuação e instituições de ensino, de forma a atender os bibliotecários quanto às suas necessidades relativas à educação para competência em informação. Da mesma forma, evidencia-se a relevância de tornar a competência em informação integrada ao processo de formação dos bibliotecários, abordando-a como um conteúdo transversal presente em diferentes disciplinas, tendo em vista a formação de profissionais preparados para buscar e utilizar informações de forma mais crítica, ética e reflexiva. Palavras-chave: Competência em informação. Formação de habilidades informacionais. Formação para competência em informação. Educação para a competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda Mestranda; Universidade Estadual de Londrina, Londrina, PR, Brasil; anamirandamm@gmail.com Ana Maria Mendes Miranda Mestranda; Universidade Estadual de Londrina, Londrina, PR, Brasil; anamirandamm@gmail.com Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 1 Introdução As formas de aprender têm se modificado ao longo dos tempos, passando de um processo passivo de recepção de conteúdo para uma aprendizagem e construção de conhecimento mais dinâmicas. Nessa perspectiva, a escola tem se pautado em \| 13 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 E-ISSN 1808-5245 um discurso para educação ativa, que visa desenvolver, nos sujeitos, habilidades metacognitivas e reflexivas. Pretende, nesta perspectiva, mais do que transmitir conteúdos, formar os estudantes para a avaliação de problemas em contextos reais que auxiliem em seu processo de desenvolvimento, não apenas acadêmico, mas também social (GASQUE; CASARIN, 2016; LIBÂNEO, 2004). um discurso para educação ativa, que visa desenvolver, nos sujeitos, habilidades metacognitivas e reflexivas. Pretende, nesta perspectiva, mais do que transmitir conteúdos, formar os estudantes para a avaliação de problemas em contextos reais que auxiliem em seu processo de desenvolvimento, não apenas acadêmico, mas também social (GASQUE; CASARIN, 2016; LIBÂNEO, 2004). Colaborando com isso, as Diretrizes Curriculares Nacionais do Ministério da Educação (BRASIL, 2013) apontam que, para a sobrevivência no atual ambiente informacional e tecnológico, é necessário que as pessoas possuam condições de aprendizagem contínua ao longo da vida. Para isso, é requerido que os estudantes, para além de adquirirem informações, desenvolvam habilidades no intuito de realizar com autonomia as atividades acadêmicas e de aprendizagem, de tal forma que continuem aprendendo durante toda a vida. Não basta, para a aprendizagem, que o estudante memorize o conteúdo ministrado em sala de aula, mas que seja “[...] flexível, criativo, capaz de encontrar soluções inovadoras para os problemas de amanhã [...]” (LIBÂNEO, 2004, p. 5), ou seja, que ele saiba como agir e interagir com o mundo ao seu redor. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 1 Introdução Para Belluzzo, Santos e Almeida Junior (2014), essas ações críticas e reflexivas frente ao mundo podem ser consideradas competência em informação, que pode ser definida como o resultado da internalização, construção ou potencialização de habilidades atitudinais, operacionais, cognitivas e comunicacionais que tornam o indivíduo capaz de suprir suas necessidades informacionais de forma rápida e consciente. O conjunto dessas habilidades permeia todo o processo de aprendizagem, desde a percepção de suas necessidades, as estratégias para localizar a informação necessária, a análise crítica destas informações, assim como permeia todo o processo consciente de tomada de decisão (DUDZIAK, 2008). Sendo as habilidades informacionais essenciais à busca e uso da informação, considera-se que tanto as habilidades quanto a informação são essenciais para que o ser humano desenvolva suas atividades e o seu fazer social. Nesse sentido, é possível refletir que o processo de formação de habilidades para a competência em informação não é necessário apenas para o contexto acadêmico. É importante que o estudante, independente das atividades que realizará, tenha suas habilidades desenvolvidas, uma vez que é por meio \| 14 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 p Ana Maria Mendes Miranda e Adriana Rosecler Alcará destas que ele se posiciona como cidadão diante do mundo; busca, analisa e utiliza a informação de forma crítica e consciente; consolidando, assim, seu papel social. É fato que as habilidades informacionais para a competência em informação devem ser desenvolvidas em todos os ambientes de inserção e atuação do sujeito, mas é na educação e por meio dela que estas possuem maior potencial de ampliação. Para Hatschbach (2002), é no trabalho educativo que se constata uma necessidade de aprendizado contínuo, é através dele que se desperta a capacidade de análise e que se auxilia no resgate da cidadania, assim como incentiva um protagonismo na construção de uma sociedade mais igualitária e emancipadora. Dessa forma, ter as habilidades informacionais desenvolvidas é, segundo a autora, um elemento essencial na educação moderna. Tanto na educação básica quanto no ensino superior, as bibliotecas são instituições apropriadas para auxiliar no desenvolvimento de habilidades nos estudantes. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx doi org/10 19132/1808 5245253 13 39 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 1 Introdução E, conforme apontam estudos realizados no âmbito das bibliotecas universitárias (MATA; ALCARÁ, 2016; MIRANDA; ALCARÁ, 2017), os bibliotecários têm sido os grandes responsáveis no desenvolvimento de ações para a promoção de habilidades para a competência em informação. Entretanto, esses estudos também evidenciaram que, apesar dos esforços de bibliotecas e bibliotecários, as ações ainda se realizam de forma isolada e sem uma periodicidade, uma vez que a maioria delas não está integrada a projetos ou programas institucionais, dificultando, assim, a manutenção e alcance destas práticas educativas. O documento da Association of College and Research Libraries (ACRL, 2016) sinaliza que o bibliotecário, em parceria com outros profissionais, pode redesenhar as atividades de formação dos usuários, cursos ou até mesmo currículos. Assim, para que o bibliotecário esteja preparado para isso, é imprescindível que sua formação promova o desenvolvimento de habilidades informacionais e o instrumentalize para tornar-se multiplicador destas habilidades, desenvolvendo-as no âmbito das bibliotecas por meio da formação dos usuários. Nesse contexto, o objetivo desta pesquisa foi verificar as ações que os bibliotecários desenvolvem para a formação de habilidades para a \| 15 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 competência em informação dos usuários e quais são as suas necessidades em relação a essas ações. Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará competência em informação dos usuários e quais são as suas necessidades em relação a essas ações. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 2 Habilidades para a competência em informação A competência em informação vem da tradução do termo em inglês Information Literacy, que surgiu em 1974, no relatório The information service environment relationships and priorities, elaborado pelo bibliotecário americano Paul Zurkowski. De acordo com Dudziak (2003, p. 24), esse termo também foi traduzido como alfabetização informacional, letramento, literacia e fluência informacional, porém, “[...] a utilização da expressão competência em informação parece ser a mais adequada em função de sua definição [...]”. Isso porque, segundo a autora, a competência em informação se refere “[...] a um saber agir responsável e reconhecido, que implica mobilizar, integrar, transferir conhecimentos, recursos, habilidades, que agreguem valor, direcionados à informação e seu vasto universo.” No Brasil, os estudos sobre a competência em informação tiveram, entre os seus precursores, Caregnato (2000), Belluzzo (2001) e Hatschbach (2002) (DUDZIAK, 2003). Na atualidade, o tema possui uma vasta produção de pesquisas e tem sido amplamente debatido em eventos técnicos e científicos da Biblioteconomia, Arquivologia e Ciência da Informação. Durante algum tempo, as habilidades no uso das Tecnologias de Informação e Comunicação foram priorizadas para o desenvolvimento dessas competências. No entanto, com o passar dos anos, a tecnologia tornou-se mais acessível, a informação passou a estar disponível em meio digital, e o contato com aparelhos e dispositivos digitais iniciou-se cada vez mais cedo. Dessa forma, o enfoque, que antes era na utilização da tecnologia, passou a ser na percepção do sujeito quanto à sua necessidade de informação, assim como no reconhecimento de fontes para buscá-la e na sua utilização de forma crítica e mais reflexiva. As autoras Vitorino e Piantola (2009, p. 135) seguem nesta direção apontando que: \| 16 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará [...] a ideia inicialmente aceita de que a competência informacional consiste essencialmente em conjunto de habilidades individuais ligadas à manipulação da informação em um suporte digital constitui apenas uma das muitas dimensões sugeridas pelo termo, que vem crescendo em complexidade à medida que as pesquisas sobre o tema evoluem. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 2 Habilidades para a competência em informação Sendo a competência essencial no processo de busca e uso da informação, esta se faz \| 17 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 E-ISSN 1808-5245 necessária a todas as pessoas que utilizam informação para desenvolver suas atividades, sejam elas acadêmicas, cotidianas ou profissionais. Assim, todos devem ter suas habilidades informacionais desenvolvidas, no intuito de melhor realizar suas ações frente à sociedade na qual se inserem. necessária a todas as pessoas que utilizam informação para desenvolver suas atividades, sejam elas acadêmicas, cotidianas ou profissionais. Assim, todos devem ter suas habilidades informacionais desenvolvidas, no intuito de melhor realizar suas ações frente à sociedade na qual se inserem. As habilidades mencionadas são consideradas habilidades informacionais e são definidas por Gasque (2013) como ações específicas que o sujeito necessita para alcançar a competência em informação. Dessa forma, é indispensável que o sujeito domine uma série de habilidades informacionais para que possa ser considerado competente. As habilidades mencionadas são consideradas habilidades informacionais e são definidas por Gasque (2013) como ações específicas que o sujeito necessita para alcançar a competência em informação. Dessa forma, é indispensável que o sujeito domine uma série de habilidades informacionais para que possa ser considerado competente. Ainda quanto às habilidades informacionais, é importante que a pessoa saiba reconhecer suas necessidades, formule questões sobre as informações que precisa buscar, explore as fontes de informação para entender melhor sobre o assunto, determine o foco da busca, identifique quais termos melhor representam o conteúdo, saiba utilizar estratégias para buscar e selecionar informações pertinentes e avalie as fontes de informação considerando sua confiabilidade, atualidade, entre outros fatores. Além de ler e compreender os conteúdos apresentados, assim como assimilá-los e comunicá-los de forma que os demais possam compreendê-los. Borges e colaboradores (2012) salientam, também, que essas habilidades não são importantes apenas para a ascensão profissional ou acadêmica, mas para participação social, já que diversas ações sociais foram transferidas do meio físico para o meio digital. 2 Habilidades para a competência em informação A competência em informação é um conceito dinâmico que tem se modificado, buscando contemplar as mais diversas habilidades que se fazem necessárias para a pessoa lidar de forma adequada com a informação, tais como, planejamento, ações reflexivas no processo de busca e uso da informação, avaliação dos resultados, entre outras. Gasque (2013, p. 5) afirma que a competência em informação é a “[...] capacidade do aprendiz de mobilizar o próprio conhecimento que o ajuda a agir em determinada situação.” A autora reforça que, durante o processo para desenvolver as habilidades, os indivíduos vão aperfeiçoando suas capacidades para identificar sua necessidade de informação, buscá-la e usá-la de maneira eficaz e eficiente, considerando seus mais diversos aspectos. A American Library Association (ALA) publicou, em 2000, um documento que definiu competência em informação como [...] um conjunto de habilidades que exigem que os indivíduos reconheçam quando as informações são necessárias e tenham a capacidade de localizar, avaliar e usar efetivamente as informações necessárias. No ano de 2016, a ACRL publicou um novo e atualizado documento sobre a competência em informação, reformulando os conceitos do antigo modelo e os moldes nos quais este era estruturado. Nesse sentido, define a competência em informação como um grupo integrado de habilidades que perpassa a descoberta da informação, a percepção de sua produção e do seu valor e o seu uso “[...] na criação de novos conhecimentos e a participação ética nas comunidades de aprendizagem.” (ACRL, 2016, p. 03, tradução nossa). Em síntese, a competência em informação pode ser vista como o resultado do processo de apropriação, desenvolvimento e potencialização de habilidades que tornam alguém capaz de realizar atividades voltadas para localizar e utilizar a informação de forma eficaz e consciente. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 Q , g , , , p , / doi: http://dx.doi.org/10.19132/1808-5245253.13-39 2 Habilidades para a competência em informação Muitas ações de caráter público são propostas em meios digitais, tais como as petições eletrônicas, as enquetes públicas e os grupos de debates online. Para que o cidadão possa participar ativamente do desenvolvimento dessas ações, é importante que o mesmo tenha domínio das habilidades tanto digitais como cognitivas, sociais e comunicativas. É importante ressaltar que, para que essas habilidades sejam consideradas bem desenvolvidas, o indivíduo precisa aplicar estratégias de busca, seleção, uso e avaliação da informação, utilizando-se do senso crítico e da ética, tanto para produzir como para receber informações. Considerando todos os aspectos relacionados ao desenvolvimento de habilidades informacionais e, consequentemente, a busca pela competência em informação, é importante que o bibliotecário tenha sua formação pautada nesse processo, não \| 18 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 apenas se tornando competente em informação, mas preparado para sua atuação enquanto formador dessas habilidades informacionais. Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 apenas se tornando competente em informação, mas preparado para sua atuação enquanto formador dessas habilidades informacionais. apenas se tornando competente em informação, mas preparado para sua atuação enquanto formador dessas habilidades informacionais. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 3 Ações para a formação da competência em informação Como já mencionado, o bibliotecário é um dos grandes responsáveis pela promoção de habilidades informacionais. Seu papel pode ser desenvolvido nas mais diversas bibliotecas, entretanto, é importante que o ensino dessas habilidades se inicie ainda na educação básica e seja aprimorado no ensino superior, de forma que os usuários aprendam a aprender. Na sequência, será apresentado um breve relato de ações que vêm sendo promovidas por bibliotecários, tendo em vista a formação das habilidades informacionais dos usuários. O objetivo desta seção é evidenciar a necessidade de o bibliotecário ter, ao longo de sua formação, seja na graduação ou na pós-graduação, conteúdos pedagógicos que o instrumentalize para suas futuras práticas educativas voltadas à competência em informação. Guedes e Farias (2007) entrevistaram bibliotecários de bibliotecas escolares da rede particular de Natal (RN) e seus resultados apontaram que mais da metade deles auxiliavam os estudantes no momento da busca, mas não possuíam ações de formação coletiva. As autoras também questionaram de que forma os bibliotecários procuravam auxiliar os estudantes no processo de compreensão da informação, e metade dos entrevistados indicaram realizar atividades com toda a comunidade acadêmica, com intuito de abordar a pesquisa bibliográfica, orientação no uso das fontes, organização de feiras científicas e culturais, ações para incentivo à leitura, entre outras. Entretanto, poucos afirmaram desenvolver ações para auxiliar no desenvolvimento dos trabalhos acadêmicos, como orientações à normalização e apresentação destes trabalhos. Sobre esse assunto, os bibliotecários afirmaram que esta responsabilidade é, em grande parte, dos professores e que fica a cargo da biblioteca apenas a orientação individual para os estudantes que procuram ajuda. Silva, Neves e Gomes (2013) aplicaram um questionário com os estudantes de uma escola do Instituto Federal da Bahia (IFBA), com o objetivo \| 19 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 de investigar como percebiam a biblioteca escolar. Os estudantes mencionaram uma série de ações que poderiam ser desenvolvidas pela biblioteca, buscando melhorar suas experiências de aprendizagem, assim como fomentar o desenvolvimento de habilidades. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 Q , g , , , p , / doi: http://dx.doi.org/10.19132/1808-5245253.13-39 3 Ações para a formação da competência em informação A autora constatou que os bibliotecários têm desenvolvido ações, mas reforça a necessidade de formação de bibliotecários com um perfil de pesquisador, de \| 20 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 maneira que os mesmos possam contribuir para a formação de estudantes pesquisadores por meio da sua atuação em bibliotecas escolares. Quanto às ações realizadas em bibliotecas universitárias, Miranda e Alcará (2017) compilaram algumas iniciativas de formação em Instituições de Ensino Superior (IES) no Brasil, entre as citadas, é relevante destacar Freitas e Vieira (2015) e Peres, Miranda e Simeão (2016), em que os autores relatam um programa de competência desenvolvido na Universidade de Brasília (UnB), com o apoio de bibliotecários, docentes e administradores, visando o desenvolvimento de habilidades em estudantes de graduação e a realização de oficinas para estudantes do ensino médio. Vale, também, relatar a iniciativa de um programa de formação continuada de usuários na Universidade Federal do Paraná (UFPR) (UEZU; ARAÚJO; ESTEVÃO, 2014). Essas iniciativas são programas de formação institucionais, envolvendo a equipe da biblioteca e o corpo docente e administrativo da instituição. Além dos programas mencionados, Miranda e Alcará (2017) descrevem diversas iniciativas de formação de usuários, por meio de oficinas de busca da informação, treinamentos, guias de fontes de informação, tutoriais para uso de bases de dados, minicursos sobre as normas da Associação Brasileira de Normas Técnicas (ABNT), entre outros. Ainda em relação às ações promovidas pelas IES ou pelas bibliotecas universitárias, pode-se citar a experiência da Universidade Federal de Santa Maria (UFSM) na oferta do Curso de Pesquisa Bibliográfica: fontes de informações da Área de Saúde na Internet, realizada por meio do Departamento de Enfermagem e pelo Programa de Pós-Graduação em Enfermagem desde 2009. O curso em questão foi ofertado durante três anos, inicialmente com o objetivo de atualizar profissionais e estudantes em relação à pesquisa bibliográfica, e, posteriormente, abrangendo a pesquisa avançada, tornando-se parte das atividades promovidas anualmente por demanda da comunidade (FONSECA; PAULA; PADOIN, 2013). Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 3 Ações para a formação da competência em informação As ações mencionadas foram: clube do livro, jogos interativos, sorteio de livro, atividades dinâmicas de incentivo à leitura, debates, post de poesias, café científico, gincana, sarau/oficina de poesias, leitura em grupo, oficina de pesquisa, divulgação de documentários nacionais e espaço de descanso. Durante o tempo disponibilizado para a aplicação do questionário, a biblioteca promoveu a primeira ação voltada para a promoção de habilidades, sendo esta um concurso de desenho, realizado em parceria com a professora de Língua Portuguesa, onde o tema a ser desenhado seria em consonância com os conteúdos de literatura ministrados em sala de aula. Bedin, Chagas e Sena (2015), ao realizarem uma pesquisa com bibliotecários da Rede de Bibliotecas Escolares de Florianópolis, notaram algumas melhoras na participação destes na realização de atividades nas escolas. Quanto a essas atividades, os profissionais relataram que promovem a formação dos estudantes para a pesquisa autônoma na biblioteca, e as ações são feitas de forma coletiva, com o intuito de compartilhar os conhecimentos gerados pelos próprios estudantes. No entanto, quando questionados sobre a realização de atividades para reconhecimento de fontes de informação digitais e seu manuseio, assim como análise consciente de suas informações, os profissionais não mostraram realizar atividades nesse sentido. Na mesma perspectiva, Bedin (2017), ao levantar as ações promovidas por bibliotecários escolares em Florianópolis, encontrou algumas iniciativas de atuação na biblioteca escolar, entre elas pode-se destacar: a participação nas reuniões da escola; trabalho em cooperação com o professor; planejamento no uso da biblioteca e na pesquisa escolar; participação no processo de pesquisa escolar; uso de fontes de pesquisa no ambiente digital; uso de métodos de pesquisa; uso das redes sociais; jornal do colégio; exposições de projetos; indicação de leitura; falando com o autor; feira de livros; sacola literária; filmes; concursos; piquenique literário; uma noite na biblioteca; e debates. 3 Ações para a formação da competência em informação Buscando complementar as ações promovidas por bibliotecários, ressalta-se também o estudo de Almeida (2015), que investigou a biblioteca do Instituto Federal de Educação da Paraíba (IFPB) – Campus Sousa e propôs um modelo de Projeto Educativo para desenvolvimento de competência em \| 21 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 E-ISSN 1808-5245 informação. O autor aponta a importância da realização de projetos que visem a competência para além das atividades de rotina que a promovem, salientando que projetos tendem a oferecer uma especificidade e complexidade maior e, desta forma, apresentam resultados mais satisfatórios. Entre as atividades incluídas no projeto estão as seguintes: oficinas sobre organização de dados das nuvens; elaboração e atualização do currículo Lattes; uso dos diretórios de grupo de pesquisa; minicursos sobre acesso e uso do Portal de Periódicos da Capes; acesso às bibliotecas digitais de teses e dissertações; uso dos buscadores; e uso das normas da ABNT. O autor também propõe uma série de atividades culturais como: café com o autor; sarau poético; concurso literário; exposição fotográfica; e festival de cinema. Na perspectiva dessas atividades, Almeida (2015) utiliza-se do termo biblioteca aprendente, que reflete a visão das autoras da presente pesquisa quanto à necessidade de bibliotecas que sejam parte da aprendizagem dos sujeitos, auxiliando no seu desenvolvimento enquanto ser social. No intuito de concretizar esta visão, aponta-se a necessidade de formação de bibliotecários capacitados para seu papel educativo enquanto formadores de habilidades para a competência em informação de seus usuários. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 4 Procedimentos metodológicos O estudo caracteriza-se como uma pesquisa de campo, descritiva, com abordagem qualitativa. A coleta de dados ocorreu por meio de um grupo de foco com a finalidade de investigar as ações que os bibliotecários desenvolvem para a formação de habilidades para a competência em informação dos usuários, bem como quais são as suas necessidades em relação a essas ações. O grupo de foco, segundo Di Chiara (2005), é uma técnica para a coleta de dados que requer a formação de um grupo pequeno e homogêneo, que represente o universo a ser pesquisado. Para a realização da coleta de dados cria- se uma situação informal para a discussão dos tópicos de interesse da pesquisa. Nesse sentido, foi selecionada para participar da pesquisa uma amostragem de bibliotecários de diferentes tipos de bibliotecas. Vale ressaltar que foi utilizada uma seleção por conveniência, que de acordo com Gil (2012, \| 22 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 E-ISSN 1808-5245 p Ana Maria Mendes Miranda e Adriana Rosecler Alcará p.94), “[...] é destituída de qualquer rigor estatístico”. Nesse caso, “[...] o pesquisador seleciona os elementos a que tem acesso, admitindo que estes possam, de alguma forma, representar o universo. Aplica-se esse tipo de amostragem em estudos exploratórios ou qualitativos, onde não é requerido elevado nível de precisão”. O convite para os bibliotecários foi enviado por e-mail e o encontro com esses profissionais foi organizado pelo grupo de pesquisa (cujas autoras são integrantes), com o objetivo de aproximar os estudos realizados pelo grupo com as práticas profissionais. Para tanto, os membros do grupo e as autoras desta pesquisa realizaram algumas reuniões antes do grupo de foco, buscando definir melhores estratégias para esta atividade. Assim, o grupo de foco foi realizado com a presença de sete bibliotecárias (sendo três de biblioteca escolar, uma de biblioteca pública, duas de biblioteca universitária e uma de biblioteca universitária especializada) e os membros do grupo de pesquisa (cinco integrantes). 4 Procedimentos metodológicos Entres os integrantes do grupo, dois membros atuaram como mediadores das discussões e os demais tiveram o papel de observadores. Antes de iniciar a discussão, as bibliotecárias foram convidadas a assinar o Termo de Consentimento Livre e Esclarecido (TCLE) e, em seguida, com base no roteiro pré-definido a partir dos objetivos da pesquisa, iniciou-se o grupo de foco. Os dados obtidos a partir da discussão do grupo de foco foram registrados em áudio e, em seguida, transcritos. Posteriormente, apoiando-se na técnica de análise de conteúdo de Bardin (1977), os dados foram organizados em categorias, analisados e interpretados, tendo a literatura como apoio. Algumas falas das bibliotecárias participantes do estudo foram inseridas durante a apresentação e discussão dos resultados e, visando preservar sua identidade, foram identificadas com numerais de 1 a 7. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 5 Apresentação e discussão dos dados Os resultados foram agrupados e serão apresentados segundo duas categorias de análise, a saber: Ações dos bibliotecários que podem contribuir para a formação de habilidades para a competência em informação; Processo e necessidade de formação do bibliotecário em relação à competência em informação. \| 23 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 5.1 Ações realizadas pelos bibliotecários que podem contribuir para a formação da competência em informação Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 5.1 Ações realizadas pelos bibliotecários que podem contribuir para a formação da competência em informação A intenção desta parte da pesquisa consistia em identificar ações para formação de usuários em relação à busca e seleção de informações no acervo e na web; avaliação de fontes de informação; compartilhamento da informação; orientações à pesquisa em âmbito escolar e acadêmico; atividades lúdicas; e uso ético e crítico da informação; que podem contribuir para a formação de habilidades para a competência em informação. Quanto ao processo de busca e seleção da informação por parte de seus usuários, algumas das participantes se referiram às estratégias que utilizam para facilitar a localização no acervo. Nesse sentido, duas das participantes que atuam em biblioteca escolar mencionaram o uso das cores, assim como a separação por tipo de materiais (mais finos, com mais ilustrações), além da organização tradicional do acervo, buscando facilitar para que os estudantes menores possam se localizar na biblioteca. Aguiar (2012, p. 41) salienta que a organização da informação na biblioteca escolar é muito significativa, pois [...] favorece a recuperação da informação de forma independente, e por isso auxilia o processo de aprendizagem das crianças, de um modo geral e em particular àquelas dos anos iniciais do ensino fundamental, não só para as pesquisas escolares, mas para toda vida acadêmica. [...] favorece a recuperação da informação de forma independente, e por isso auxilia o processo de aprendizagem das crianças, de um modo geral e em particular àquelas dos anos iniciais do ensino fundamental, não só para as pesquisas escolares, mas para toda vida acadêmica. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 Q , g , , , p , / doi: http://dx.doi.org/10.19132/1808-5245253.13-39 [...] favorece a recuperação da informação de forma independente, e por isso auxilia o processo de aprendizagem das crianças, de um modo geral e em particular àquelas dos anos iniciais do ensino fundamental, não só para as pesquisas escolares, mas para toda vida acadêmica. 5 Apresentação e discussão dos dados Guedes e Farias (2007) se referem ao ambiente da biblioteca e a importância deste espaço ser bem sinalizado, organizado, autoexplicativo, de forma que o estudante possa aprender a se localizar dentro do mesmo, desenvolvendo sua autonomia. Assim como no estudo realizado pelas autoras, em que a maioria dos bibliotecários afirmou que o espaço é bem organizado e apresenta estratégias que facilitam seu entendimento pelo público, na presente pesquisa isso também ficou evidente. A Participante 2 expôs que na instituição em que trabalha “[...] foi dividido o acervo também, em I e J, infantil e juvenil, para o pessoal saber, e os alunos começarem a conhecer onde está [o material e ter independência para acessá-lo]”. Esta participante também mencionou que colocou faixas coloridas nas estantes, indicando a faixa etária de cada parte do \| 24 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 acervo, possibilitando, assim, que os estudantes reconheçam os materiais adequados para sua idade. Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 p Ana Maria Mendes Miranda e Adriana Rosecler Alcará acervo, possibilitando, assim, que os estudantes reconheçam os materiais adequados para sua idade. A participante 1 também citou uma atividade realizada com a professora de Literatura, em que duas vezes por ano fazem uma gincana na qual as crianças precisam localizar materiais no acervo, possibilitando, de maneira lúdica, a localização em relação aos materiais e a independência no uso da biblioteca. As bibliotecárias que atuam nas bibliotecas escolares e na biblioteca pública expuseram sobre diversas ações promovidas de forma lúdica, buscando auxiliar as crianças em seu processo de aprendizagem e autonomia na busca e uso da informação. Nesse sentido, a Participante 3 relatou que fazem workshops, oficinas e cursos. Destacou uma ação específica, desenvolvida pela biblioteca, denominada friendship (amizade), em que os usuários escrevem cartas para pessoas de outros países. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 5 Apresentação e discussão dos dados Essa bibliotecária atua em uma biblioteca de escola de idiomas, espaço em que desenvolve várias ações para a aprendizagem de outro idioma. Já a Participante 4 reportou que durante as férias escolares a biblioteca oferece entre “[...] as programações um dia de produção de história, no outro, jogos, no outro, dobradura, palestras [...]”. Outra ação mencionada pelas Participantes 3 e 4 é o encontro às cegas, em que a biblioteca organiza uma mesa com livros embrulhados e os estudantes escolhem sem saber qual o título ou tema do livro. Nos relatos dessas participantes, percebe-se o envolvimento dos usuários nessas atividades e a relevância para sua interação com a biblioteca. Assim como, visualizou-se a importância das ações realizadas pelas bibliotecárias, que podem contribuir tanto para a autonomia dos usuários na busca da informação como para o uso e produção da informação, que são essenciais para a formação de habilidades para a competência em informação Vale ainda mencionar que as atividades citadas pelas bibliotecárias participantes desta pesquisa se aproximam das ações já relatadas por bibliotecários escolares nos estudos de Guedes e Farias (2007), Silva, Neves e Gomes (2013) e Bedin (2017), as quais visam promover a aprendizagem das crianças por meio de ações lúdicas. A Participante 2 expôs um projeto realizado pela biblioteca escolar onde atua, e como as crianças reagiram a ele. Ela comentou que iniciou um projeto de \| 25 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 E-ISSN 1808-5245 leitura com estudantes do 1º ao 5º ano, no qual eles contavam histórias aos mais pequenos e uma das regras do projeto era que as histórias precisavam ser de algum livro. Umas das alunas inventou uma história e escreveu um livro para realizar a contação aos colegas. O que se pode ressaltar na fala dessa bibliotecária é que, além de participar das atividades, os estudantes também colaboram na sua elaboração. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx doi org/10 19132/1808 5245253 13 39 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 5 Apresentação e discussão dos dados Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 p Ana Maria Mendes Miranda e Adriana Rosecler Alcará atividades que convidem a comunidade a participar ativamente do espaço destas unidades informacionais. atividades que convidem a comunidade a participar ativamente do espaço destas unidades informacionais. As participantes deste estudo mencionaram, também, as ações realizadas para capacitação e formação de usuários no uso de bases de dados ou demais fontes de informação. No âmbito das bibliotecas universitárias, a Participante 5 relatou sobre as capacitações no uso de base de dados na semana de recepção aos novos estudantes, ainda complementou que, após estas atividades, eles sempre retornam à biblioteca para tirar dúvidas durante seu processo de formação. Semelhante a isso, as Participantes 6 e 7 citaram que, no início de cada ano letivo, oferecem treinamentos básicos que auxiliam o usuário na busca do material pelo nome do autor e título. Assim como orientam os usuários na pesquisa para elaboração de artigos, especialmente no uso do Portal de Periódicos da Capes e recursos disponíveis no Instituto Brasileiro de Informação em Ciência e Tecnologia (IBICT). Em relação ao uso de fontes de informação para pesquisa, é imprescindível a participação dos bibliotecários nesse processo. Conforme argumenta Soares (2014), a pesquisa é um momento importante para o desenvolvimento de habilidades informacionais, já que depende da busca, seleção, avaliação e uso das informações. Embora a competência em informação não se restrinja apenas às habilidades de busca e uso da informação, vale salientar que esses são os primeiros passos para desenvolvê-la, já que a autonomia para buscar, selecionar e usar a informação precede o processo de uso reflexivo e as ações de produção e compartilhamento da informação, inerentes à competência em informação. No âmbito das bibliotecas escolares, apesar das diferenças no processo de busca da informação quando comparadas às universitárias, é possível perceber, através das ações mencionadas, os esforços das bibliotecárias em auxiliar seus estudantes no uso das fontes. Nesse sentido, a Participante 1 apontou que auxilia os estudantes do ensino médio na seleção de fontes confiáveis por meio de uma lista de sites, que fica disponível ao lado dos computadores utilizados por eles durante as pesquisas na internet. Bedin, Chagas e Sena (2015, p. 5 Apresentação e discussão dos dados E isso é relevante para a formação da competência em informação, já que, de acordo com a ACRL (2016), a competência em informação inclui diversas habilidades, dentre elas, a produção da informação, que, nesse caso, pôde ser evidenciada quando a estudante criou e escreveu uma história para ser contada aos seus colegas. A Participante 3 também relatou atividades desenvolvidas pelos próprios usuários, denominadas “[...] movimento fazer (Maker Space), que é onde você volta a fazer as coisas que você delegava para as indústrias, descobre como é feito [...]”. Ela menciona que planejam realizar uma atividade de programação de jogos, onde os estudantes poderão desenvolver seus próprios jogos eletrônicos. Tal atividade auxilia na promoção da criatividade, pois o estudante não vai apenas participar de jogos ou brincadeiras, mas auxiliar no seu processo de criação e desenvolvimento. Em relação ao movimento citado pela participante, Santos Neto e Zaninelli (2017, p. 02) dissertam que [...] o maker space seja um dispositivo para inovar o contexto biblioteconômico, vislumbra-se que ele possa ser inserido em diversos tipos de equipamentos informacionais, isto é, universitários, públicos, especializados, escolares etc. Os autores reiteram que, nas bibliotecas, este movimento tem como objetivo não apenas o acesso às tecnologias, mas a formação em relação à competência em informação e competência digital, para que estes usuários possam, de maneira autônoma, tornar tangível seus projetos e ideias. Esse conceito necessita de uma aproximação entre a comunidade de usuários e as tecnologias e, por meio disso, é possível que as bibliotecas incentivem a criatividade de seus usuários. O movimento Maker pode, ainda, ser citado como caminho para a aproximação entre as bibliotecas e suas comunidades, criando \| 26 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 atividades que convidem a comunidade a participar ativamente do espaço destas Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 atividades que convidem a comunidade a participar ativamente do espaço destas unidades informacionais. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 5 Apresentação e discussão dos dados 370), em sua pesquisa com bibliotecários escolares, observaram que, em relação à formação para utilização de fontes de \| 27 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 informação na web, “[...] os dados mostraram um índice negativo, pouco explorado no ambiente escolar.” Percebe-se necessidade de mais atividades referentes à avaliação de fontes de informação da web. Apesar dos esforços relatados pelas bibliotecárias participantes da presente pesquisa, pode-se notar que ainda não há uma percepção dos administradores das instituições e professores em relação à importância dos recursos informacionais para a pesquisa escolar e formação dos estudantes. No sentido da avaliação das fontes de informação, Tomaél, Alcará e Silva (2016) ressaltam que esse procedimento sempre foi relevante e tem se intensificado com a proliferação de fontes de informação digitais, tornando-se cada vez mais necessário. Assim sendo, as autoras propõem alguns parâmetros para avaliação das fontes de informação digitais, entre eles estão a confiabilidade, a credibilidade, a precisão, a integridade e a relevância da informação. Em alguns relatos, foi possível perceber que os bibliotecários têm procurado auxiliar seus usuários na seleção das fontes de informação, principalmente na web. A participante 5 diz que nem sempre os estudantes sabem a confiabilidade de fontes, principalmente na internet, então ela auxilia no estabelecimento de critérios, tais como a confiabilidade e a atualidade das informações recuperadas. Nesse caso, a bibliotecária orienta os usuários a identificarem a procedência e o autor responsável e suas credenciais, assim como a data de criação ou atualização da fonte de informação. Retomando as atividades mencionadas pelas participantes bibliotecárias de bibliotecas escolares, é relevante citar as atividades realizadas em parceria com os professores. Nesse caso, vale destacar que algumas enfatizaram que os professores solicitam sua ajuda no momento da seleção do material para as atividades das disciplinas. Porém, relataram que não há uma sistemática na realização dessas parcerias, e que, nem sempre, os professores avisam com antecedência sobre as pesquisas que serão realizadas pelos estudantes, inviabilizando assim o planejamento e a organização do material bibliográfico para subsidiar as pesquisas escolares. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 5 Apresentação e discussão dos dados Conforme aponta Bedin (2017), diversos profissionais citaram os professores como parte essencial para o bom desenvolvimento de seu trabalho, sendo que as parcerias se dão por meio da “[...] escolha do acervo, o \| 28 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 p Ana Maria Mendes Miranda e Adriana Rosecler Alcará acompanhamento do planejamento de sala de aula e o desenvolvimento de projetos [...]”. Para bibliotecários que atuam em instituições de ensino, o desenvolvimento de atividades alinhadas com os docentes é de grande ajuda, não apenas na interação da biblioteca com a instituição e na sua valorização enquanto parte do processo de aprendizagem, mas também é essencial para o desenvolvimento de habilidades transversais necessárias aos estudantes, com as quais a biblioteca tem muito a colaborar. Já em relação às parcerias nas bibliotecas universitárias, a Participante 5 comentou que existe um trabalho sendo realizado com os professores, por meio de um projeto denominado “Caminhos Jurídicos”, no qual ela está inserida e realiza orientações e atividades com vistas à busca e uso da informação. A ação mencionada pela bibliotecária vai ao encontro da proposta realizada pelo documento da ACRL (2016, p. 21) de oferecer “[...] recursos para aperfeiçoar a competência do corpo docente [...]”, assim como incluir ações para a formação da competência em informação no currículo dos cursos. Dessa forma, é possível que não só os professores se tornem mais capacitados para formar seus estudantes, como também entendam as necessidades de ações voltadas para a competência em informação no currículo de forma integrada. Vale salientar que as bibliotecárias também auxiliam seus usuários na apresentação dos conteúdos, seja em relação à normalização bibliográfica, por meio da orientação e disponibilização das normas, ou demais necessidades, como o uso de power point. A Participante 1, responsável por uma biblioteca escolar, informou que os estudantes já constroem projetos de pesquisa científica, e que estes procuram auxílio da biblioteca para sanar necessidades ligadas a este projeto. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 5 Apresentação e discussão dos dados Ela menciona uma atividade denominada “Projetão na Iniciação Científica”, em que os estudantes são orientados não apenas na busca da informação, mas também na elaboração e apresentação de trabalhos. Segundo ela, os estudantes apresentam dificuldades para inserir anexos em e-mails, organizar slides e até mesmo para criar seus e-mails. Ainda relacionado à apresentação e elaboração de trabalhos científico, a Participante 5, que é responsável por uma biblioteca universitária especializada, relatou sobre uma semana de atividade da qual a biblioteca é parte, e que ministra palestras sobre \| 29 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 [...] como fazer projeto, como fazer o TCC, normalizar, fazer pesquisa em bases de dados, então a gente leva isso na semana jurídica, e a gente tem notado que além dos nossos alunos, os alunos das [demais] universidades da região têm ido participar também. (PARTICIPANTE 5) Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 [...] como fazer projeto, como fazer o TCC, normalizar, fazer pesquisa em bases de dados, então a gente leva isso na semana jurídica, e a gente tem notado que além dos nossos alunos, os alunos das [demais] universidades da região têm ido participar também. (PARTICIPANTE 5) Semelhante à ação citada pela bibliotecária, Lima e colaboradores (2017) mencionam a Semana de Metodologia e Produção Científica, promovida pela Universidade Federal do Ceará (UFC), na qual diversas atividades são ministradas com o objetivo de auxiliar os estudantes na realização de suas pesquisas acadêmicas. Para os autores, essas ações contribuem de forma positiva para minimizar as lacunas informacionais dos indivíduos, mostrando, ainda, que a biblioteca é adequada para auxiliar nos processos de pesquisa e fomentar discussões em relação à construção do conhecimento. Em relação ao uso de recursos informacionais digitais, algumas participantes da pesquisa evidenciaram sua importância na formação da competência em informação, como pode ser identificado no relato da Participante 3: “Quando eu entendo essa competência informacional, eu vejo essa parte de você ensinar a fazer uma pesquisa, ele aprender a usar os recursos, [que] hoje você necessita para a aprendizagem [...]”. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 5 Apresentação e discussão dos dados As Participantes 3 e 5 relatam o uso de mídias sociais para alcançar a comunidade de usuários, a participante 5 citou o uso de recursos, em especial o Facebook. Apesar destas duas menções, pode-se perceber que ainda são poucas as ações que fazem uso de mídias sociais como ferramenta para auxiliar no compartilhamento da informação e na formação de seus usuários. A participante 3 também salientou sobre os conteúdos que são trabalhados nas atividades promovidas pela biblioteca: “[...] a gente traz ciência, tecnologia, engenharia, artes, matemática, liderança, empreendedorismo, esses são os temas”. De acordo com ela, essa atividade possibilita ao estudante o conhecimento e o aprendizado sobre as diferentes áreas e permite a visão do todo, para que o próprio estudante possa descobrir a área que mais se identifica. Assim como permite respeitar e aceitar as diferenças em relação às opções dos demais estudantes. Essa integração entre os conteúdos também é considerada por Dudziak (2005), ao apontar que conteúdos pré-determinados podem estreitar os \| 30 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 E-ISSN 1808-5245 horizontes e diminuir as oportunidades de descoberta ativa dos estudantes. Para a autora, a transdisciplinaridade beneficia o ensino, que vai além do projeto pedagógico voltado para o contexto disciplinar, e questiona fragmentações da realidade, sendo também esse o sentido da competência em informação. Ela ainda complementa que a biblioteca pode ser o ponto de mudança, agregando novos valores não condicionados à educação tradicional e à separação de conteúdos por disciplinas, mas centralizando aspectos multidisciplinares e convergindo-os em atividades que considerem as “[...] experiências pessoais dos aprendizes, reconhecendo suas diferenças e complexidade.” (DUDZIAK, 2005, p. 9). [...] a gente tem o grupo no WhatsApp, e-mail, essas coisas, e tem um encontro também, no mínimo uma vez por ano, em que todas as bibliotecárias se encontram, fora as outras atividades que são por Skype. E então, a gente acaba conversando bastante, tanto esses colegas do próprio trabalho quanto com os outros das outras instituições. (PARTICIPANTE 5) Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 5.2 Processo e necessidades de formação do bibliotecário em relação à competência em informação Na categoria relacionada à formação dos bibliotecários, foi questionado sobre que formas utilizam para obter formação voltada para a competência em informação. Nesse sentido, além da graduação em Biblioteconomia, as bibliotecárias ressaltaram os recursos e fontes utilizadas para complementação de sua formação. A Participante 5 mencionou que buscou ajuda de outros profissionais da mesma área para ampliar a sua formação: “[...] para me capacitar, para poder capacitá-los, eu entrei nos grupos jurídicos de São Paulo, já que é uma biblioteca especializada em Direito.” A Participante 7 destacou que, para tirar dúvidas, trocar ideias e se atualizar em algumas situações, também faz contato com outros bibliotecários: A Participante 3 também enfatizou que desenvolveu parcerias com outras bibliotecas, com as quais realizava encontros mensais e citou que precisou buscar formação em outras áreas, além da Biblioteconomia, para poder atuar na sua unidade de informação. Para tal, ela buscou formação \| 31 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 complementar em cursos de extensão e pós-graduação, mencionou também a participação em cursos de empreendedorismo e o uso do YouTube para assistir cursos e palestras relevantes à formação de suas habilidades Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 E-ISSN 1808-5245 p Ana Maria Mendes Miranda e Adriana Rosecler Alcará complementar em cursos de extensão e pós-graduação, mencionou também a participação em cursos de empreendedorismo e o uso do YouTube para assistir cursos e palestras relevantes à formação de suas habilidades. É possível notar que a formação exigida destes bibliotecários requer conhecimentos multidisciplinares, que vão muito além dos aspectos ligados apenas à Biblioteconomia ou à Ciência da Informação. Isso reforça a importância do aprender a aprender ao longo da vida, endossando que, para a competência em informação, são requeridas habilidades diversificadas, as quais se constituem em pilares importantes que auxiliam o bibliotecário a reconhecer suas necessidades e buscar informações que o ajudem a supri-las. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 5.2 Processo e necessidades de formação do bibliotecário em relação à competência em informação Algumas participantes reportaram-se ao processo de formação ocorrido durante a graduação. A participante 5 ressaltou que seu interesse para desenvolver ações de capacitação dos usuários começou durante a frequência em algumas disciplinas, e também apontou um evento de Biblioteconomia como parte importante da sua formação enquanto multiplicadora de habilidades. Soares (2014) também considera, em sua pesquisa, que os eventos da área auxiliam muito na formação dos profissionais e no desenvolvimento de ações educativas no âmbito das bibliotecas. Evidencia-se, assim, que os eventos técnicos e científicos podem ser considerados uma importante fonte de informação, aprendizagem e compartilhamento de experiências para profissionais e podem contribuir para a formação continuada dos mesmos. Já a Participante 6 ressaltou que, durante a graduação, aprendeu a auxiliar no desenvolvimento da autonomia do usuário. A partir dos relatos das bibliotecárias, foi possível perceber que a graduação não é suficiente para a educação para a competência em informação. Isso se evidenciou especialmente porque, na ocasião da formação, as bibliotecárias participantes desta pesquisa não tiveram a oportunidade de refletir sobre as questões relativas à competência em informação nas disciplinas que frequentaram. Assim como não tiveram disciplinas específicas sobre a temática, com exceção de uma das participantes que teve a oportunidade de frequentar uma disciplina de competência em informação no mestrado. Ainda se referindo ao curso de graduação, as participantes da pesquisa consideram que a formação em Biblioteconomia forneceu a base para a atuação \| 32 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 p Ana Maria Mendes Miranda e Adriana Rosecler Alcará profissional, porém, assim que ingressaram no mundo do trabalho, precisaram buscar a formação continuada. Também foi possível perceber que estas profissionais se mostram flexíveis ao desenvolver ações nas bibliotecas, buscando contextualizar os conteúdos aprendidos na academia às necessidades apresentadas pela unidade de informação. De forma geral, os resultados indicam que as participantes deste estudo têm buscado diferentes formações e recursos para suprir as necessidades de novos conhecimentos e desenvolver ações que visem à formação de seus usuários. 5.2 Processo e necessidades de formação do bibliotecário em relação à competência em informação Em relação a essa formação complementar, ficou evidente que algumas das participantes são bastante proativas quanto à busca de novas ideias e aprendizagem para a sua atuação. Na continuidade da discussão sobre as questões relacionadas à formação das bibliotecárias, indagou-se, também, sobre a necessidade de alguma formação que gostariam de ter para sua atuação profissional. Para tal, as bibliotecárias referiram-se a algumas atividades que poderiam ser oferecidas para contribuir com a formação das habilidades informacionais e que auxiliariam no desenvolvimento de ações para a formação da competência em informação. Em relação à biblioteca escolar, destacaram a falta dos conteúdos que abordassem atividades lúdicas e pedagógicas para apoiar nas ações de formação para competência em informação. Já no que tange às formações voltadas para bibliotecas universitárias, foram mencionadas as constantes mudanças nas fontes de informação e bases de dados. Nesse sentido, uma das participantes externou seu interesse, e talvez de outros profissionais da área, em participar de cursos e oficinas sobre estas ferramentas, para aprimorar seus conhecimentos sobre o tema e, consequentemente, obter melhores condições para capacitar seus usuários. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 6 Considerações finais As ações relatadas pelas bibliotecárias são bastante variadas, de acordo com o contexto de cada unidade de informação. Também foi possível notar que, apesar das dificuldades que algumas bibliotecárias apresentaram para desenvolver estas \| 33 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 ações, elas têm envidado esforços para que estas aconteçam da melhor forma. E, mesmo não sendo diretamente relacionadas à competência em informação, as ações que desenvolvem podem contribuir tanto para a formação dos usuários quanto às habilidades para a competência em informação, seja para maior autonomia no uso da biblioteca e na busca da informação, ou para o uso mais reflexivo da informação. Quanto às necessidades de formação das bibliotecárias, é visível a demanda por formação complementar, além da graduação, especialmente no que se refere à falta de formação pedagógica dessas bibliotecárias. Isso porque, nos relatos sobre sua formação na graduação, explicitaram que não tiveram disciplinas voltadas à competência em informação e às práticas pedagógicas para desenvolver ações de formação para os usuários. Ficou evidente que, em relação a esse aspecto, elas buscam conhecimentos em cursos de educação continuada e por meio do compartilhamento de experiências de outros profissionais. Nesse sentido, é importante uma maior aproximação entre os campos de atuação do bibliotecário e as instituições de ensino, de modo a atender às necessidades de educação para a competência em informação. Assim como é imprescindível integrar a competência em informação ao longo da formação dos bibliotecários, seja por meio de disciplinas específicas para tal ou de modo transversal nos currículos dos cursos de Biblioteconomia. Durante o grupo de foco, o compartilhamento e a reflexão sobre as experiências e habilidades já desenvolvidas por alguns profissionais chamou a atenção de outras participantes, que demonstraram interesse em receber formação das bibliotecárias que possuíam mais domínio em determinada área de atuação. Esse aspecto nos permite constatar que muitos bibliotecários têm desenvolvido ações no intuito de formar seus usuários, revelando ser preciso disponibilizar espaços nas instituições de ensino superior para que estas ações sejam reconhecidas e compartilhadas com estudantes e demais profissionais. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 6 Considerações finais Assim como se evidenciou a relevância do bibliotecário multiplicador da competência em informação. Outro resultado que merece destaque foi a possibilidade de vislumbrar a competência em informação como algo que vai além das habilidades para \| 34 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará buscar e usar a informação, como relatado na fala de uma das participantes do grupo de foco ao mencionar a sua percepção em relação ao papel da biblioteca: [...] é esse apresentar, esse compartilhar, esse momento de aprendizagem junto [com o usuário]; você passa a atividade em que um está ensinando o outro; eles ensinam a gente, nem tudo a gente sabe, muitas vezes a gente aprende com eles, muita coisa, e ensina também. A fala da bibliotecária contempla uma dimensão da competência que é ressaltada nesta pesquisa, que está na possibilidade de compartilhar as experiências de aprendizagem de maneira a colaborar com a formação do outro. Para finalizar, vale enfatizar que a competência em informação não perpassa apenas aspectos profissionais ou acadêmicos da vida de um indivíduo, ela deve ser adotada como uma das maneiras de formar cidadãos mais reflexivos frente ao ambiente informacional, mais conscientes de seu papel social, mais autônomos no processo de busca de informações e mais éticos na produção e compartilhamento de conteúdo. Assim, a visão de educação para a competência em informação colabora para uma formação profissional adequada às necessidades atuais da sociedade, permitindo às instituições de ensino formarem pessoas mais dispostas e capazes de utilizar a informação em prol de uma sociedade mais justa e igualitária. AGUIAR, N. C. 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In: SIMPÓSIO DE ENGENHARIA DE PRODUÇÃO DA UNESP, 7., 2001, Bauru. Anais [...] Bauru: UNESP, 2001. p. 32-42. BELLUZZO, R. C. B.; SANTOS, C. A.; ALMEIDA JÚNIOR, O. F. A competência em informação e sua avaliação sob a ótica da mediação da informação: reflexões e aproximações teóricas. Informação e Informação, Londrina, v. 19, n. 2, p. 60-77, maio/ago., 2014. BORGES, J. et al. Competências infocomunicacionais: um conceito em desenvolvimento. Tendências da Pesquisa Brasileira em Ciência da Informação, João Pessoa, v. 5, n. 1, p. 1-23, 2012. BRASIL. Ministério da Educação. Secretaria de Educação Básica. Diretrizes Curriculares Nacionais Gerais da Educação Básica. Brasília: MEC, 2013. CAREGNATO, S. E. O desenvolvimento de habilidades informacionais: o papel das bibliotecas universitárias no contexto da informação digital em rede. Revista de Biblioteconomia & Comunicação, Porto Alegre, v. 8, p. 47-55, 2000. DI CHIARA, I. G. Grupo de foco. In: VALENTIM, M. L. P. (org.). Métodos qualitativos de pesquisa em Ciência da Informação. São Paulo: Pólis, 2005. DUDZIAK, E. A. Competência em Informação: melhores práticas educacionais voltadas para a Information Literacy. In: CONGRESSO BRASILEIRO DE BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO, 21., 2005, Curitiba. Anais [...] Curitiba: FEBAB, 2005. DUDZIAK, E. A. Competência em Informação: melhores práticas educacionais voltadas para a Information Literacy. In: CONGRESSO BRASILEIRO DE Ê BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO, 21., 2005, Curitiba. Anais [...] Curitiba: FEBAB, 2005. DUDZIAK, E. A. Information literacy: princípios, filosofia e prática. Ciência da Informação, Brasília, v. 32, n. 1, p. 23-35, 2003. DUDZIAK, E. A. Os faróis da sociedade de informação: uma análise crítica sobre a situação da competência em informação no Brasil. Informação e Sociedade, João Pessoa, v. 18, n. 2, p. 41-53, maio/ago. 2008. DUDZIAK, E. A. Os faróis da sociedade de informação: uma análise crítica sobre a situação da competência em informação no Brasil. Referências AGUIAR, N. C. Organização da informação em bibliotecas escolares: contribuições para a competência informacional infantil. Biblioteca Escolar em Revista, São Paulo, v. 1, n. 2, p. 31-44, 2012. AGUIAR, N. C. Organização da informação em bibliotecas escolares: contribuições para a competência informacional infantil. Biblioteca Escolar em Revista, São Paulo, v. 1, n. 2, p. 31-44, 2012. ALMEIDA, J. L. S. A biblioteca como organização aprendente: o desenvolvimento de competências em informação no Instituto Federal de Educação, Ciência e Tecnologia da Paraíba. 2015. Dissertação (Mestrado Profissional em Gestão nas Organizações Aprendentes) - Universidade Federal da Paraíba, 2015. AMERICAN LIBRARY ASSOCIATION. Information literacy competency standards for higher education. Chicago: ACRL/ALA, 2000. ASSOCIATION OF COLLEGE AND RESEARCH LIBRARIES. Framework for information literacy for higher education. Chicago: ACRL, 2016. ASSOCIATION OF COLLEGE AND RESEARCH LIBRARIES. Framework for information literacy for higher education. Chicago: ACRL, 2016. \| 35 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 E-ISSN 1808-5245 BARDIN, L. Análise de conteúdo. Lisboa: Edições 70, 1977. Informação e Sociedade, João Pessoa, v. 18, n. 2, p. 41-53, maio/ago. 2008. \| 36 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 FONSECA, E. R.; PAULA, C. C.; PADOIN, S. M. de M. Curso de Pesquisa Bibliográfica (CPB): uma experiência da UFSM/RS para competência em informação na área de saúde. In: CONGRESSO BRASILEIRO DE BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO (CBBD), 25., 2013, Florianópolis. Anais [...] Florianópolis: FEBAB, 2013. FREITAS, F.; VIEIRA, J. S. A competência informacional e a semana universitária na Universidade Brasília. In: CONGRESSO BRASILEIRO DE FREITAS, F.; VIEIRA, J. S. A competência informacional e a semana universitária na Universidade Brasília. In: CONGRESSO BRASILEIRO DE BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO, 26., 2015, São Paulo. Anais [...] São Paulo: FEBAB, 2015. BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO, 26., 2015, São Paulo. Anais [...] São Paulo: FEBAB, 2015. GASQUE, K. C. G. D. Competência em Informação: conceitos, características e desafios. Atoz, Curitiba, v. 2, n. 1, p. 5-9, jan./jun. 2013. GASQUE, K. C. G. D.; CASARIN, Helen de C. S. Bibliotecas escolares: tendências globais. Em Questão, Porto Alegre, v. 22, n. 3, p. 36-55, 2016. GIL, A. C. Métodos e técnicas de pesquisa social. 6. ed. São Paulo: Atlas, 2012. GUEDES, C. A.; FARIAS, G. Information literacy: uma análise nas bibliotecas escolares da rede privada em Natal/RN. Revista Digital de Biblioteconomia e Ciência da Informação, Campinas, v. 4, n. 2, p. 110-133, 2007. HATSCHBACH, M. H. L. Information literacy: aspectos conceituais e iniciativas em ambiente digital para o estudante de nível superior. 2002. Dissertação (Mestrado em Ciência da Informação) – Escola de Comunicação, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2002. LIBÂNEO, J. C. A aprendizagem escolar e a formação de professores na perspectiva da psicologia histórico-cultural e da teoria da atividade. Educar, Curitiba, n. 24, p. 113-147, 2004. LIMA, J. S. et al. Semana de metodologia & produção científica: contribuições da biblioteca universitária para a formação acadêmica. In: CONGRESSO Ê LIMA, J. S. et al. Semana de metodologia & produção científica: contribuições da biblioteca universitária para a formação acadêmica. In: CONGRESSO Ê LIMA, J. S. et al. Semana de metodologia & produção científica: contribuições da biblioteca universitária para a formação acadêmica. BARDIN, L. Análise de conteúdo. Lisboa: Edições 70, 1977. In: CONGRESSO BRASILEIRO DE BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO, 27., 2017, Fortaleza. Anais [...] Fortaleza: FEBAB, 2017. MATA, M. L.; ALCARÁ, A. R. Análise das práticas educacionais dos bibliotecários em bibliotecas universitárias com enfoque na educação de usuários e na competência em informação. In: ENCONTRO NACIONAL DE PESQUISA EM CIÊNCIA DA INFORMAÇÃO 17 2016 B hi A i [ ] BRASILEIRO DE BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO, 27., 2017, Fortaleza. Anais [...] Fortaleza: FEBAB, 2017. MATA, M. L.; ALCARÁ, A. R. Análise das práticas educacionais dos bibliotecários em bibliotecas universitárias com enfoque na educação de usuários e na competência em informação. In: ENCONTRO NACIONAL DE PESQUISA EM CIÊNCIA DA INFORMAÇÃO, 17., 2016, Bahia. Anais [...] Bahia: UFBA, 2016. MIRANDA, A. M. M.; ALCARÁ, A. R. Ações para o desenvolvimento e formação das habilidades informacionais. In: SEMINÁRIO EM CIÊNCIA DA INFORMAÇÃO, 7., 2017, Londrina. Anais [...] Londrina: Departamento de Ciência da Informação, 2017. p. 171-186. \| 37 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 E-ISSN 1808-5245 PERES, M.; MIRANDA, A.; SIMEÃO, E. Promoção de competências em informação: formação para iniciação científica no ensino superior e comunidades. Revista Ibero-americana de Ciêencia da Informação, Brasília, v. 9, n. 1, p. 213-220, 2016. SANTOS NETO, J. A.; ZANINELLI, T. B. Bibliotecas com MakerSpaces: tendência ou necessidade de inovação? In: CONGRESSO BRASILEIRO DE Ã Ê SANTOS NETO, J. A.; ZANINELLI, T. B. Bibliotecas com MakerSpaces: tendência ou necessidade de inovação? In: CONGRESSO BRASILEIRO DE BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO 27 2017 F t l A i [ ] F t l FEBAB 2017 SANTOS NETO, J. A.; ZANINELLI, T. B. Bibliotecas com MakerSpaces: tendência ou necessidade de inovação? In: CONGRESSO BRASILEIRO DE BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO, 27., 2017, Fortaleza. Anais [...] Fortaleza: FEBAB, 2017. BIBLIOTECONOMIA, DOCUMENTAÇÃO E CIÊNCIA DA INFORMAÇÃO, 27., 2017, Fortaleza. Anais [...] Fortaleza: FEBAB, 2017. SILVA, A. S. R.; NEVES, D. A. B.; GOMES, M. Y. F. S. Avaliação da biblioteca escolar para o desenvolvimento de competências informacionais: a experiência da biblioteca do Instituto Federal da Bahia – Campus Camaçari. Biblioteca Escolar em Revista, São Paulo, v. 2, n. 1, p. 20-40, 2013. SOARES, L. V. O. A formação como aliada no exercício do papel educativo do bibliotecário na escola. 2014. BARDIN, L. Análise de conteúdo. Lisboa: Edições 70, 1977. Dissertação (Mestrado em Ciência da Informação) - Escola de Ciência da Informação, Universidade Federal de Minas Gerais, Belo Horizonte, 2014. TOMAÉL, M. I.; ALCARÁ, A. R.; SILVA, T. E. Fontes de informação digital: critérios de qualidade. In: TOMAÉL, M. I.; ALCARÁ, A. R. (org.). Fontes de informação digital. Londrina: Eduel, 2016. UEZU, D.; ARAÚJO, P. C.; ESTEVÃO, J. S. B. A educação de usuários sistematizada e institucionalizada: o caso do programa de educação continuada de usuário do sistema de bibliotecas (SIBI) da Universidade Federal do Paraná UEZU, D.; ARAÚJO, P. C.; ESTEVÃO, J. S. B. A educação de usuários sistematizada e institucionalizada: o caso do programa de educação continuada de usuário do sistema de bibliotecas (SIBI) da Universidade Federal do Paraná (UFPR). In: SEMINÁRIO NACIONAL DE BIBLIOTECAS UNIVERSITÁRIAS, 18., 2014, Belo Horizonte. Anais [...] Belo Horizonte, UFMG, 2014. ( ) (UFPR). In: SEMINÁRIO NACIONAL DE BIBLIOTECAS UNIVERSITÁRIAS, 18., 2014, Belo Horizonte. Anais [...] Belo Horizonte, UFMG, 2014. VITORINO, E. V.; PIANTOLA, D. Competência informacional: bases históricas e conceituais: construindo significados. Ciência da Informaçã, Brasília, v. 38, n. 3, p. 130-141, set./dez., 2009. Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Education for information literacy and actions performed by librarians Abstract: The objective of the research was to verify the actions that the librarians develop for the formation of skills for the users information literacy and their needs in relation to these actions. For that, it was carried out a field, descriptive, and qualitative research , with seven librarians from different types of libraries as participants. The data were collected through a focus group with some librarians to identify their experiences, actions and needs for educational practices focused on information literacy. Data analysis was based on the \| 38 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Em Questão, Porto Alegre, v. 25, n. 3, p. 13-39, set./dez. 2019 doi: http://dx.doi.org/10.19132/1808-5245253.13-39 Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará Educação para competência em informação e as ações realizadas por bibliotecários Ana Maria Mendes Miranda e Adriana Rosecler Alcará E-ISSN 1808-5245 content analysis technique. Among the results it was possible to notice that the librarians have carried out several activities to train their users, that can contribute to the information literacy. It is concluded, however, that it is still necessary to strengthen the links between the fields of activity and educational institutions, in order to assist librarians with their needs regarding education for information literacy. Likewise, it is evident the relevance of making information literacy integrated into the process of training librarians, approaching it as a transversal content present in different disciplines, in view of training professionals prepared to seek and use information in a more critical, ethical and reflective way. content analysis technique. Among the results it was possible to notice that the librarians have carried out several activities to train their users, that can contribute to the information literacy. It is concluded, however, that it is still necessary to strengthen the links between the fields of activity and educational institutions, in order to assist librarians with their needs regarding education for information literacy. Likewise, it is evident the relevance of making information literacy integrated into the process of training librarians, approaching it as a transversal content present in different disciplines, in view of training professionals prepared to seek and use information in a more critical, ethical and reflective way. Keywords: Information literacy. Formation of informational skills. Training for information literacy. Recebido: 30/08/2018 Aceito: 03/02/2019 \| 39
https://openalex.org/W2016074962	http://ainfo.cnptia.embrapa.br/digital/bitstream/item/92751/1/Temperamento-Acta-Animal-Science.pdf	English	null	Breed and selection line differences in the temperament of beef cattle	Acta Scientiarum. Animal Sciences	2,013	cc-by	5,286	renças de temperamento entre raças e linhas de seleção para gado de corte Diferenças de temperamento entre raças e linhas de seleção para gado de corte RESUMO. O temperamento de quatro raças bovinas foi avaliado utilizando-se o teste de velocidade de fuga (FT) e o escore de comportamento (BST). FT foi definida como o tempo necessário para animais percorrerem uma distância de 2 m após a pesagem. BST foi baseada no comportamento dos animais na balança, amostrando-se quatro categorias de comportamento: movimentos, intensidade de respiração, vocalizações e coices. Os coeficientes de herdabilidade de FT e BST foram estimados com uso de um modelo de máxima verossimilhança restrita, considerando meio irmãos paternos. Caracu apresentou menores médias para BST do que as demais raças. Nelore apresentou resultados intermediários, seguida por Guzerat e Gyr com médias mais elevadas (p < 0,05). Resultados similares foram observados para FT, mas as médias de Caracu e Nelore não diferiram entre si. Observou-se baixa associação entre FT e BST (rp= -0,36; p < 0,01). A correlação entre rank de touros ordenados pelos seus valores preditos (p) para FT e BST foi moderada e negativa (rs = -0,63; p < 0,001). A herdabilidade de FT e BST foi de 0,35 e 0,34, respectivamente. A comparação de rebanhos Nelore com diferentes critérios de seleção para peso corporal mostrou que linhas de seleção podem modular positivamente o temperamento de Bos indicus. Palavras-chave: vacas de corte, comportamento, medo, manejo. Ubiratan Piovezan1, Joslaine Noely dos Santos Gonçalves Cyrillo2 and Mateus José Rodrigues Paranhos da Costa3 1Empresa Brasileira de Pesquisa Agropecuária, Cx. Postal 109, 79320-900, Corumbá, Mato Grosso do Sul, Brazil. 2Estação Experimental de Sertãozinho, Instituto de Zootecnia, Sertãozinho, São Paulo, Brazil. 3Departamento de Zootecnia, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual de São Paulo, Jaboticabal, São Paulo, Brazil. Author for correspondence. E-mail: piovezan@cpap.embrapa.br ABSTRACT. The temperament of four beef cattle breeds were measured using a flight time test (FT) and a behavior score test (BST). FT was defined as the time taken by animals to cross a distance of 2 m after weight scale. The BST used a visual assessment of cattle behavior in which the results of four categories defined the score: movements, breathing intensity, vocalization and kicking. FT and BST coefficients of heritability were estimated using the restricted maximum likelihood, considering half siblings. Caracu presented a lower BST value than the other breeds. Nellore presented intermediate results, followed by Guzerat and Gyr with similar and higher means (p < 0.05). Similar results were observed with FT, but Caracu and Nellore did not differ from each other. A low association between FT and BST was found (rp= -0.36; p < 0.01). The correlation between a sire’s ranking, according to the predicted breeding values (p) estimated for FT and BST, was moderate and negative (rs = -0.63; p < 0.001). Heritability estimates for FT and BST were 0.35 and 0.34, respectively. Inside Nellore breed, herds with different selection criteria for weight were compared. Our results show that selection line based on weight might positively modulate temperament of Bos indicus cattle. Keywords: beef cows, behavior, fear, husbandry. Keywords: beef cows, behavior, fear, husbandry. Diferenças de temperamento entre raças e linhas de seleção para gado de corte Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013 Acta Scientiarum. Animal Sciences http://www.uem.br/acta ISSN printed: 1806-2636 ISSN on-line: 1807-8672 Acta Scientiarum Doi: 10.4025/actascianimsci.v35i2.16426 http://www.uem.br/acta ISSN printed: 1806-2636 ISSN on-line: 1807-8672 Acta Scientiarum Doi: 10.4025/actascianimsci.v35i2.16426 http://www.uem.br/acta ISSN printed: 1806-2636 ISSN on-line: 1807-8672 Doi: 10 4025/actascianims Palavras-chave: vacas de corte, comportamento, medo, manejo. Material and methods The temperament assessments were conducted during weighing handling procedures, which started in the calving season (April of 1996, when cows and calves were weighed just after calving), and included assessments at the beginning of the breeding season (around 2 months later), at the end of breeding season (3 months after the previous assessment) and during weaning procedures (approximately 3 months after the end of breeding season). Two methods were used to assess cattle temperament: the BST for the reactions of cows during weighing, adapted from Fordyce et al. (1982), and the FT as described by Burrow (1997). All measurements were conducted during and immediately after weighing. BST was scored first, followed by FT. This study was conducted on a research farm (Estação Experimental de Zootecnia de Sertãozinho, EEZS; 21º09’ S, 48º05’W) in Sertãozinho city, State of São Paulo, Brazil. Four breeds of beef cattle were raised in the EEZS, under similar handling conditions: three Bos indicus breeds (Nellore, Guzerat and Gyr) and one native breed, Caracu, a Bos taurus that originated in Brazil through the crossing-breeding of Iberian cattle. The herds had been selected based on live weight selection criteria since 1981. Reproductive failure and old age were the criteria normally used to define which cows would be culled. However, in some cases, health, udder problems and bad temperament were also considered. More details of the EEZS cattle selective breeding program have been described in Souza et al. (2010). y The BST included four variables. The first of these variables was cow movement intensity (CM), which was scored from 1 to 5. CM = 1 when the cow remained quiet most of the time inside the weigh-scale, presenting only occasional head and tail movements. CM = 2 when the cow changed its position inside the weigh-scale only a few times but did not keep its hooves in the same position for more than few seconds; occasional movements of head and tail were present. CM = 3 when the cow presented frequent movements inside the weigh- scale, with frequent and vigorous head and tail movements. CM = 4, when the cow rarely remained still inside the weigh-scale, presenting frequent, vigorous and abrupt movements of the head and tail. CM = 5, when the cow was in continuous movement, presenting strong head and tail movements that included jumping and forcing the weigh-scale walls with its head. y Temperament measurements Temperament measurements Acta Scientiarum. Animal Sciences Introduction clear that knowledge about cattle temperament has practical and economic implications (BIRO; STAMPS, 2008; RÉALE et al., 2007). It is accepted that animals with excitable temperaments are more difficult and dangerous to handle than those with calm temperaments (BURROW, 2001; MARTIN; RÉALE, 2008; RÉALE et al., 2007). It is also accepted that animals with poor temperaments are generally less productive (KING et al., 2006). Animal reactions toward humans have been assessed in numerous ways and studied in different scenarios, which has aimed to solve practical problems during handling or to define which animals should not be selected to reproduce in a breeding program. It is The practical definition of temperament is based on the intensity of a bovine’s reactions to specific situations or handling procedures (MAFFEI et al., 2006). Nevertheless, other approaches to animal temperament adopt a wider concept and define the variable as a pool of individual psycho physiological traits that determine emotional reactions (RÉALE et al., 2007). Indeed, such approaches make the practical assessment of temperament much more complex. Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013 Acta Scientiarum. Animal Sciences 208 Piovezan et al. In the present study, cattle temperament was assessed using two measures, flight time (FT) and behavior score test (BST), to test the following hypotheses: 1) temperament varies between and within cattle breeds; 2) differences in temperament within breeds can be explained by additive genetic inheritance; and 3) there is a strong relationship between the FT and BST methods of assessing cattle temperament. of grass. Approximately 30 days before calving, the cows were driven into the paddocks where they remained in groups until 24h after calving. All paddocks were situated next to the farmyard in places with intense movements of people and animals around them. In the calving paddocks, cows were fed once a day with silage of corn or sorghum with hay and cotton seeds as concentrate. y Temperament measurements Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013 where: N = number of offspring, R =0.25 (half sibling relationships), h2 = heritability, t = 0.25h2 (for paternal half sibling), Ps = average of sibling, and Pc = average of the contemporary group. The waiting time in the corral (TW), defined as the length of time from arrival in the corral until release from the weigh-scales was also measured. During weighing, the farm employees scored the cows according to their body condition (BC). Because BC did not vary, it was represented by two classes of condition: poor and good. The Spearman coefficient of correlation was used to compare the rankings of 30 sires sorted by their predicted breeding values (p) estimated by FT and BST. The Pearson correlation coefficient between FT and BST was also estimated. Tukey test were used to compare BST and FT means between breeds and between herds within the Nellore breed. Data analyses The adherence of BST and FT distribution of probability to the normal distribution was tested by the procedure PROC Univariate, and the components of variance were estimated using the restricted maximum likelihood methodology (REML) by the procedure, Proc Varcomp, using the SAS software (LITTEL et al., 1991), according the following model: Reactivity of the cattle in the weight scale 209 collection. BST was recorded for 1550 cows, including 364 Nellore, 536 Guzerat, 257 Gyr and 393 Caracu. Yijklm = dependent variable BST or FT, μ = general average, ri = fixed effect of breed, cj = fixed effect of body condition, pik = random effect of sire within breeds, aijl = random effect of animal within sires and breeds, and eijklm = random error. collection. BST was recorded for 1550 cows, including 364 Nellore, 536 Guzerat, 257 Gyr and 393 Caracu. Each behavioral variable was recorded independently during weighing, and the final BST was defined by the combination of these measurements, according to the following criteria: Each behavioral variable was recorded independently during weighing, and the final BST was defined by the combination of these measurements, according to the following criteria: j Three other variables (TW = waiting time in the corral, EO = entrance order, and AGE = cow age) were included in the model as covariables for adjustment purposes. The heritability of BST and FT considered the information of paternal half-sisters (FALCONER; MACKAY, 1996), and the heritabilities were calculated according to the following equation: g g BST = 1 when: CM = 1, BI = 0 or 1, VO = 0 or 1 and KIC = 0; BST = 2 when: CM = 1 and BI ≥ 2; or CM = 2 and BI = 0, 1 or 2 (if VO or KIC = 0); or CM = 2 and BI = 3, independent of VO and KIC results. p BST = 3 when: CM = 2 (if BI ≥ 2); or CM = 3 BST = 3 when: CM = 2 (if BI ≥ 2); or CM = 3 and BI = 0, 1 or 2 (if VO or KIC = 0); or CM = 3 and BI = 3, independent of VO and KIC results. h2 = 4σ2p / (σ2pe + σ2 error) ( ); and BI = 0, 1 or 2 (if VO or KIC = 0); or CM = 3 and BI = 3, independent of VO and KIC results. ( ) and BI = 0, 1 or 2 (if VO or KIC = 0); or CM = 3 and BI = 3, independent of VO and KIC results. where: 2 BST = 4 when: CM = 3 (if BI ≥ 2); or CM = 4 and BI = 0, 1, 2 (if VO or KIC = 0); or CM = 4 and BI = 3, independent of VO and KIC results. BST = 4 when: CM = 3 (if BI ≥ 2); or CM = 4 and BI = 0, 1, 2 (if VO or KIC = 0); or CM = 4 and BI = 3, independent of VO and KIC results. h2 = heritability coefficient, σ2p = sire effect, and σ2 pe + σ2 error = total variance. p BST = 5 when: CM = 4 (if BI ≥ 2); or CM = 5, independent of the other results. Sires with fewer than four progeny in the population were not considered in the statistical analysis. The standard error of h2 was estimated according to Falconer (1989). p FT was measured as the time taken by a cow to cross a 2 m distance immediately after leaving the weigh-scale. Times were recorded by a digital chronometer connected to photoelectric cells fitted just after the scale exit and 2 m ahead. The chronometer was switched on when the cow crossed the first pair of photoelectric cells and switched off when it reached the second pair of cells. The reasoning behind this methodology is that cattle with an undesirable temperament (more reactive) will run when leaving the scale, presenting lower times than those with good temperaments. We recorded FT for 345 cows (88 Nellore, 113 Guzerat, 50 Gyr and 94 Caracu) during the calving season. The data analyses performed only for the Nellore breed considered the fixed effect of herd instead of the breed fixed effect in the model and did not consider the component of variance of animal within sire and breed. The predicted breeding values (p) of sires for FT and AS were calculated by: P = Pc +Rh2 [n / 1+(n-1)t] (Ps – Pc) Acta Scientiarum. Animal Sciences Material and methods The second BST variable was breathing intensity (BI), which was scored from 0 to 3, where BI = 0 for no audible breath, BI = 1 for audible breath, BI = 2 for very deep breath, or BI = 3 for snorting or snoring. The third variable was vocalization (VO), where VO = 0 for no vocalization or VO = 1 for one or more vocalizations. The final BST variable was kicking (KIC), where KIC = 0 for no kicking or KIC = 1 for one or more kicks. The repeatability within and between observers had been tested previously for all measurements, and high correlations (from 90 to 95%) were achieved before initiating BST data There were three herds of the Nellore breed in the EEZS, which were defined according to different selection criteria for live weight. In the Selection herd (NeS), selective breeding was applied using only sires and cows with live weight performance above the herd average. In the control herd (NeC), no selective breeding was applied. In the traditional herd (NeT), selective criteria were similar to those used in the selection herd (MERCADANTE et al., 2003). Prior to this work, carried out during the years of 1996 and 1997, no temperament trait had been considered in the EEZS selection program. The percentage of maiden female heifers was approximately 20%, and the stocking rate was approximately two Animal Unit (cow) per ha. Cows observed in this study were at least 3 years old; pasture grazed and had received mineral supplementation. The herds were kept in different paddocks and went to the corral six times per year. The cows were handled separately during temperament assessment. All farm cows were submitted to similar conditions and experienced the same handling routines. The calving season coincides with the end of the dry season, which is characterized by high air temperature, low air humidity, high intensity of solar radiation, and low availability and poor quality Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013 Acta Scientiarum. Animal Sciences Reactivity of the cattle in the weight scale Results and discussion The BST statistical analysis showed significant effects of breed, sire within breed and animal within sire and breed (p < 0.01), but no significant effects of body condition (R2 = 0.72, CV = 37.17%). There was a significant effect of breed (p < 0.01, R2 = 0.36, CV = 58.86%) on FT. Acta Scientiarum. Animal Sciences Yijklm =μ + ri + cj + pik + aijl + eijklm where: Yijklm =μ + ri + cj + pik + aijl + eijklm Yijklm =μ + ri + cj + pik + aijl + eijklm The Guzerat and Gyr breeds presented the highest scores for BST, which did not differ Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013 Acta Scientiarum. Animal Sciences 210 Piovezan et al. between each other. These breeds were followed by Nellore and Caracu (Table 1).The Gyr and Guzerat breeds had the lowest flight times, and their means did not differ from each other. Nellore and Caracu had higher FT means and did not differ from each other (Table 1). possibilities were considered to explain the significant effect of waiting time in the corral on the temperament of the Nellore breed: cattle with more excitable temperaments are the last to leave the corral because they try to avoid going through the facilities, or the cattle get more excitable as time passes due to a negative environmental effect on them. Table 1. Behavior score test (BST) and flight time (FT) means and standard errors, according to cattle breed. BST BST FT FT Breeds Mean ± SE N Mean±SE N Caracu 1.52c ± 0.83 393 2.52a ± 1.21 94 Gyr 2.48a ± 1.22 257 1.51b ± 0.97 50 Guzerat 2.59a ± 1.33 536 1.64 b ± 1.18 113 Nellore 2.20b ± 1.21 364 2.14a ± 1.18 88 BST = Behavioral score test, FT = Flight time (seconds). Means followed by the same letter do not differ from each other (Tukey, p < 0.05). Table 1. Behavior score test (BST) and flight time (FT) means and standard errors, according to cattle breed. g The difference in temperament between the selection herd (NeS) and control herd (NeC) of the Nelore breed suggests that breeding selection based on live weight could affect cattle temperament in a desirable way. Although this was not planned, it may be that a genetic correlation exists. Results and discussion As a result, differences among line selection herds could be a side effect of the breeding program to increase live weight at 378 days of age in the EEZS. The Nellore Selection herd has been submitted to the highest selection pressure for live weight for decades. The observed status of temperament is, in part, a consequence of the selection criteria adopted in the past. Favorable correlated responses in temperament following selection for live weight at 378 days in Nellore cattle was conferred from the comparison between herds. This result is in agreement with studies in which poor temperaments were associated with low performances (BURROW; PRAYAGA, 2004; PRAYAGA et al., 2009). Hence, the observed response may be specific only to these herds (e.g., it may be due to the use of one or two sires that happened to have a high expected breeding value for live weight and also good temperament). BST = Behavioral score test, FT = Flight time (seconds). Means followed by the same letter do not differ from each other (Tukey, p < 0.05). BST = Behavioral score test, FT = Flight time (seconds). Means followed by the same letter do not differ from each other (Tukey, p < 0.05). For the Nellore breed, we found significant effects of AGE and TW (p < 0.05 and p < 0.01, respectively) only for BST. The mean age (± SD) of the studied cows was 6.28 ± 2.76 years. The average time waiting in the corral was of 2.34 ± 1.54h. Significant effects (p < 0.05) of herd and sire within herd were found in Nellore (Table 2). Table 2. Behavior score test (BST) means and standard errors according to herds within the Nellore breed. Table 2. Behavior score test (BST) means and standard errors according to herds within the Nellore breed. Table 2. Behavior score test (BST) means and standard errors according to herds within the Nellore breed. Herds N Mean ±SE Control 68 2.69a ± 1.437 Selection 147 2.13bc ± 1.185 Traditional 189 1.97c ± 1.038 Means followed by the same letter do not differ from each other (Tukey, p < 0.05). The BST and FT heritability estimates (± SE) were 0.34 ± 0.000244 and 0.35 ± 0.000764, respectively. The Pearson’s correlation coefficient between BST and FT was negative and significant (rp = -0.36; p < 0.01). Acta Scientiarum. Animal Sciences References FIGUEIREDO, L. G. G.; ELER, J. P.; MOURÃO, G. B.; FERRAZ, J. B. S.; BALIEIRO, J. C. C.; MATTOS, E. C. Análise genética do temperamento em uma população da FIGUEIREDO, L. G. G.; ELER, J. P.; MOURÃO, G. B.; FERRAZ, J. B. S.; BALIEIRO, J. C. C.; MATTOS, E. C. Análise genética do temperamento em uma população da raça Nelore. Livestock Research for Rural Development, v. 17, n. 17, p. 1-7, 2005. FORDYCE, G.; GODDARD, M.; SEIFERT, G. W. The measurement of temperament in cattle and effect of experience and genotype. Animal Production in Australia, v. 14, p. 329-332, 1982. Another point to consider about cattle reactivity is the consequence of decreasing the cattle response in Brazilian field conditions. It may be that less responsive cows are not desirable in circumstances in which the herd faces predator attack. Such situations are still common in certain parts of Brazil, where young calves can be attacked by pumas (Puma concolor), jaguars (Panthera onca) and vultures (Cathartes spp). raça Nelore. Livestock Research for Rural Development, v. 17, n. 17, p. 1-7, 2005. FORDYCE, G.; GODDARD, M.; SEIFERT, G. W. The measurement of temperament in cattle and effect of experience and genotype. Animal Production in Australia, v. 14, p. 329-332, 1982. FORDYCE, G.; HOWITT, C. J.; HOLROYD, R. G.; O'ROURKE, P. K.; ENTWISTLE, K. W. The performance of Brahman-Shorthorn and Sahiwal- Shorthorn beef cattle in the dry tropics of northern Queensland - Scrotal circumference, temperament, ectoparasite resistance, and the genetics of growth and other traits in bulls. Australian Journal of Experimental Agriculture, v. 36, n. 1, p. 9-17, 1996. FORDYCE, G.; HOWITT, C. J.; HOLROYD, R. G.; O'ROURKE, P. K.; ENTWISTLE, K. W. The performance of Brahman-Shorthorn and Sahiwal- Shorthorn beef cattle in the dry tropics of northern Queensland - Scrotal circumference, temperament, ectoparasite resistance, and the genetics of growth and other traits in bulls. Australian Journal of Experimental Agriculture, v. 36, n. 1, p. 9-17, 1996. GAULY, M.; MATIAK, H.; HOFFMAN, K.; KRAUS, M.; ERHARDT, G. Estimating genetic variability in temperamental traits in German Angus and Simental Cattle. Applied Animal Behaviour Science, v. 74, n. 2, p. 109-119, 2001. In resume, the temperament varied between and within cattle breeds; differences within breeds can be explained by additive genetic inheritance. Results and discussion The sire rank according to predicted breeding values estimated for FT and BST was negatively correlated and significant (rs = -0.63; p < 0.001; N = 30). g g p ) Even thought feel animals have been evaluated in this study (345), the FT heritability coefficient found was similar to those estimated by Burrow (2001) and Nkrumah et al. (2007), who reported h2 from 0.40 to 0.44 and from 0.31 to 0.67, respectively. The same occurred with BST, which is also in agreement with the heritability estimates reported in the literature. In the literature, values of heritability range from 0.16 (FIGUEIREDO et al., 2005) to 0.67 (FORDYCE et al., 1982), when subjective scores were adopted. The maximum value of heritability determined was 0.70 (FORDYCE et al., 1996) when using the flight distance test. Independent of the method to measure temperament, previous studies found moderate to high heritability coefficients when estimated by mixed models of analysis (BURROW, 2001; GAULY et al., 2001). The heritability values presented here corroborates evidence of additive genetic factors controlling cattle responses to human handling. These results assure that temperament can be controlled by selection and are useful knowledge for Brazilian farmers, who usually raise Zebu or Zebu cross-breed cattle. Both temperament measures indicated significant differences among the breeds. The Bos taurus breed (Caracu) scores were better than the Bos indicus breeds (Nellore, Guzerat and Gyr). These results are in agreement with the opinion of EEZS livestock workers, who indicated, prior to data collection, that the Caracu herd was easier to handle than the other breeds and that the Guzerat and Gyr breeds were more difficult to handle. These conclusions indicate that BST and FT assessments have practical utility as indicators of cattle temperament, and they can be used for selection of cattle with better temperament. The data analysis of the Nellore breed confirmed that age and waiting time in the corral were also important in the expression of cattle temperament. Similar results were reported by Burrow (1997).Two Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013 Acta Scientiarum. Animal Sciences 211 References Phenotypic association between the flight time and behavior score tests was low, but the association between the predicted breeding values of sires according those traits was moderate to high. Selection focused on live weight did not adversely affect the temperament of Bos indicus. Reactivity of the cattle in the weight scale The analyses determined a low association between BST and FT. If we consider that the behavior score is a measurement that restricts bovine movements and that flight time does not involve restriction, our results are comparable with the reported by Curley Jr. et al. (2006), which found low association between measurements with and without cattle movement restriction. References References BIRO, P. A.; STAMPS, J. A. Are animal personality traits linked to life-history productivity? Trends in Ecology and Evolution, v. 23, n. 7, p. 361-368, 2008. BURROW, H. M. Measurements of temperament and their relationships with performance traits of beef cattle. Animal Breeding Abstracts, v. 65, n. 7, p. 477-495, 1997. BURROW, H. M. Variances and covariances between productive and adaptive traits and temperament in a composite breed of tropical beef cattle. Livestock Production Science, v. 70, n. 3, p. 213-233, 2001. BURROW, H. M. Variances and covariances between productive and adaptive traits and temperament in a composite breed of tropical beef cattle. Livestock The correlation between sire ranking according to predicted breeding values estimated for FT and BST was moderate and negative. Sire selection based on these measures might not achieve similar results. p p p composite breed of tropical beef cattle. Livestock Production Science, v. 70, n. 3, p. 213-233, 2001. BURROW, H. M.; PRAYAGA, K. C. Correlated responses in productive and adaptive traits and temperament following selection for growth and heat resistance in tropical beef cattle. Livestock Production Science, v. 86, n. 1, p. 143-161, 2004. g Although it is possible and useful to include cattle temperament as a criterion for selective breeding, it is still not common in the management of Brazilian herds. This fact is probably because there is no clear definition of which measurement system should be used. Petherick et al. (2009) suggested that FT would be more related to innate components of temperament, while BST is supposed to be more related to the learned components of that trait. If this is the case, then age effects will be significant for BST, whereas they are likely to be less significant with FT. Otherwise, our study corroborates that idea. Consequently, the quality of handling routines and the farm facilities help define which temperament measurement is more suitable. CURLEY JR., K. O.; PASCHAL, J. C.; WELSH, J. T. H.; RANDEL, R. D. Technical note: Exit velocity as a measure of cattle temperament is repeatable and associated with serum concentration of cortisol in Brahman bulls. Journal of Animal Science, v. 84, n. 11, p. 3100-3103, 2006 Journal of Animal Science, v. 84, n. 11, p. 3100-3103, 2006. FALCONER, D. S. Introduction to quantitative genetics. Harlow: Longman Sci and Tech, 1989. FALCONER, D. S.; MACKAY, T. F. C. Introduction to quantitative genetics. 4th ed. New York: Longman, 1996. Acta Scientiarum. Animal Sciences License information: 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 work is properly cited. Acta Scientiarum. Animal Sciences Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013 Conclusion KING, D. A.; SCHUEHLE PFEIFFER, C. E.; RANDEL, R. D.; WELSH JR., T. H.; OLIPHINT, R. A.; BAIRD, B. E.; CURLEY JR., K. O.; VANN, R. C.; HALE, D. S.; SAVELL, J. W. Influence of animal temperament and stress responsiveness on the carcass quality and beef tenderness of feedlot cattle. Meat Science, v. 74, n. 3, p. 546-556, 2006. We conclude that breed influences the temperament of beef cattle. On average, Bos indicus breeds had poorer temperament evaluations. Our study found those flight time and behavior score test are both indicative of temperament and are moderately heritable. Therefore, these measures could be useful in cattle selection programs to improve both innate temperaments and responsiveness to training. LITTEL, R. C.; FREUND, R. J.; SPECTOR, P. C. SAS System for linear models. 3rd ed. Cary: SAS Institute Inc., 1991. Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013 Acta Scientiarum. Animal Sciences 212 Piovezan et al. Piovezan et al. MAFFEI, W. E.; BERGMANN, J. A. G.; PINOTTI, M.; OLIVEIRA, M. E. C.; SILVA, C. Q. Animal reactivity in a mobile cage: a new methodology to access bovine temperament. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v. 58, n. 6, p. 1123-1131, 2006. MARTIN, J. G. A.; RÉALE, D. Temperament, risk assessment and habituation to novelty in eastern chipmunks, Tamiasstriatus. Animal Behaviour, v. 75, n. 1, p. 309-318, 2008. PRAYAGA, K. C.; CORBET, N. J.; JOHNSTON, D. J.; WOLCOTT, M. L.; FORDYCE, G.; BURROW, H. M. Genetics of adaptive traits in heifers and their relationship to growth, pubertal and carcass traits in two tropical beef cattle genotypes. Animal Production Science, v. 49, n. 6, p. 413-425, 2009. RÉALE, D.; READER, S. M.; SOL, D.; MCDOUGALL, P. T.; DINGEMANSE, N. J. Integrating animal temperament within ecology and evolution. Biological Reviews, v. 82, n. 2, p. 291-318, 2007. MERCADANTE, M. E. Z.; PACKER, I. U.; RAZOOK, A. G.; CYRILLO, J. N. S. G.; FIGUEIREDO, L. A. Direct and correlated responses to selection for yearling weight on reproductive performance of Nelore cows. Journal of Animal Science, v. 81, n. 2, p. 376-384, 2003. SOUZA, F. R. P.; MERCADANTE, M. E. Z.; FONSECA, L. F. S.; FERREIRA, L. M. S.; REGATIERI, SOUZA, F. R. P.; MERCADANTE, M. E. Z.; FONSECA, L. F. S.; FERREIRA, L. M. S.; REGATIERI, I. C.; AYRES, D. R.; TONHATI, H.; SILVA, S. L.; RAZOOK, A. G.; ALBUQUERQUE, L. G. Assessment of DGAT1 and LEP gene polymorphisms in three Nelore (Bos indicus) lines selected for growth and their relationship with growth and carcass traits. Journal of Animal Science, v. 88, n. 2, p. 435-441, 2010. NKRUMAH, J. D.; CREWS JR., D. H.; BASARAB, J. A.; PRICE, M. A.; OKINE, E. K.; WANG, Z.; LI, C.; MOORE, S. S. Genetic and phenotypic relationships of feeding behavior and temperament with performance, feed efficiency, ultrasound, and carcass merit of beef cattle. Journal of Animal Science, v. 85, n. 10, p. 2382-2390, 2007. Received on March 30, 2012. Accepted on May 28, 2012. Received on March 30, 2012. PETHERICK, J. C.; DOOGAN, V. J.; HOLROYD, R. G.; OLSSON, P.; VENUS, B. K. Quality of handling and holding yard environment, and beef cattle temperament: 1. Relationships with flight speed and fear of humans Applied PETHERICK, J. C.; DOOGAN, V. J.; HOLROYD, R. G.; OLSSON, P.; VENUS, B. K. Piovezan et al. Quality of handling and holding yard environment, and beef cattle temperament: 1. Relationships with flight speed and fear of humans. Applied Animal Behaviour Science, v. 120, n. 1, p. 18-27, 2009. Accepted on May 28, 2012. Relationships with flight speed and fear of humans. Applied Animal Behaviour Science, v. 120, n. 1, p. 18-27, 2009. p g p pp Animal Behaviour Science, v. 120, n. 1, p. 18-27, 2009. Maringá, v. 35, n. 2, p. 207-212, Apr.-June, 2013
https://openalex.org/W4313513167	https://papiro.unizar.es/ojs/index.php/tropelias/article/download/5494/4475	es		Víctor Said Armesto. Contexto histórico-cultural y testimonios epistolares de su quehacer filológico	Tropelías/Tropelías	2,009	cc-by	0
https://openalex.org/W2124330529	https://europepmc.org/articles/pmc3854453?pdf=render	English	null	Physical activity patterns among South-Asian adults: a systematic review	The international journal of behavioural nutrition and physical activity	2,013	cc-by	8,525	REVIEW Open Access Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 6 Keywords: Physical activity, Inactivity, South Asia, Adults Studies in the United Kingdom have shown that the risk of diabetes is 3 to 5 times higher for immigrants from Bangladesh, India and Pakistan compared with the native white Caucasian population, with an associated increased risk of complications, morbidity and mortality [3]. South Asia has the highest number of patients with diabetes and the prevalence of diabetes among adults is over 10% in many parts of the region [4]. This increased metabolic risk among South Asians appears to be multi-factorial, where unhealthy dietary habits and physical inactivity are coupled with genetic predisposition [5]. Physical inactivity increases the risk of developing abdominal adiposity, diabetes and cardiovascular disease [6]. Furthermore, physical inactivity is considered the fourth leading risk factor for global Physical activity patterns among South-Asian adults: a systematic review Chathuranga D Ranasinghe1, Priyanga Ranasinghe2, Ranil Jayawardena3 and Anoop Misra4 Chathuranga D Ranasinghe1, Priyanga Ranasinghe2, Ranil Jayawardena3 and Anoop Misra4 © 2013 Ranasinghe 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. * Correspondence: chath_r@yahoo.com.au 1Allied Health Sciences Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka Full list of author information is available at the end of the article Abstract Physical activity (PA) has many beneficial physical and mental health effects. Physical inactivity is considered the fourth leading risk factor for global mortality. At present there are no systematic reviews on PA patterns among South Asian adults residing in the region. The present study aims to systematically evaluate studies on PA patterns in South Asian countries. A five-staged comprehensive search of the literature was conducted in Medline, Web of Science and SciVerse Scopus using keywords ‘Exercise’, ‘Walking’, ‘Physical activity’, ‘Inactivity’, ‘Physical Activity Questionnaire’, ‘International Physical Activity Questionnaire’, ‘IPAQ’, ‘Global Physical Activity Questionnaire’ and ‘GPAQ’, combined with individual country names. The search was restricted to English language articles conducted in humans and published before 31st December 2012. To obtain additional data a manual search of the reference lists of articles was performed. Data were also retrieved from the search of relevant web sites and online resources. The total number of hits obtained from the initial search was 1,771. The total number of research articles included in the present review is eleven (India-8, Sri Lanka-2, Pakistan-1). In addition, eleven country reports (Nepal-3, Bangladesh-2, India-2, Sri Lanka-2, Bhutan-1, Maldives-1) of World Health Organization STEPS survey from the South-Asian countries were retrieved online. In the research articles the overall prevalence of inactivity was as follows; India (18.5%-88.4%), Pakistan (60.1%) and Sri Lanka (11.0%-31.8%). STEPS survey reports were available from all countries except Pakistan. Overall in majority of STEPS surveys females were more inactive compared to males. Furthermore, leisure related inactivity was >75% in studies reporting inactivity in this domain and people were more active in transport domain when compared with the other domains. In conclusion, our results show that there is a wide variation in the prevalence of physical inactivity among South-Asian adults within and between countries. Furthermore, physical inactivity in South Asian adults was associated with several socio-demographic characteristics. Majority of South Asian adults were inactive during their leisure time. These Factors need to be considered when planning future interventions and research aimed at improving PA in the region. Keywords: Physical activity, Inactivity, South Asia, Adults Search strategy A fi d A five staged comprehensive search of the literature was conducted in the following databases; PubMed® (U.S. National Library of Medicine, USA), Web of Science® [v.5.4] (Thomson Reuters, USA) and SciVerse Scopus® (Elsevier Properties S.A, USA) for studies published before 31st December 2012. During the first stage the above databases were searched using the following search criteria. The Medline database was searched using the MeSH (Medical Subject Headings) terms ‘Exercise’ and ‘Walking’, with keywords ‘Physical activity’, ‘Inactivity’, ‘Physical Activity Questionnaire’, ‘International Physical Activity Questionnaire’, ‘IPAQ’, ‘Global Physical Activity Questionnaire’ and ‘GPAQ’. The search terms were com- bined with the names of the individual South-Asian countries; ‘Sri Lanka’, ‘Lanka’, ‘Ceylon’, ‘India’, ‘Bangladesh’, ‘Pakistan’, ‘Nepal’, ‘Bhutan’, ‘Maldives’. The Web of Science database was searched using all of the above search terms in article topic. In the SciVerse Scopus database the above terms were searched in the article title, abstract or keywords. In the United Kingdom the levels of physical activity have long been known to be lower in the South Asian people than in the general white Caucasian population [14]. The desire to walk, cycle and participate in sports and recreational activity is low among South Asians in comparison with the general population in United Kingdom [14]. Data from the Health Survey for England found that South Asians were 60% less likely than native Caucasians to meet government recommendations for physical activity [15]. Likewise, a systematic review by Fischbacher et al. (2004) examined physical activity levels among South Asians and compared it to the general United Kingdom population and noted that rates were 50–75% less in South Asians [16]. Lack of understanding about benefits, communication gap with health care professionals, cultural beliefs and lack of culturally sensi- tive facilities are some of the potential barriers for physical activity in South Asians [17]. Swaminathan and Vaz, systematically reviewed the physical activity level of children in India [18]. Although there are several stud- ies from individual South Asian countries on the level of physical activity among adults in each country, at present there are no comprehensive systematic reviews on physical activity patterns among South Asian adults residing in the region. The aim of this exercise is to systematically evaluate published work on physical activ- ity from individual South Asian countries and summarize as for a common region enabling comparison with other regions. Introduction South Asia, commonly known as the Indian sub-continent, is home to almost one-fifth of the world’s population and is comprised of many diverse ethnic, linguistic and religious groups [1]. Altogether there are 7 countries in the region namely; Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan and Sri Lanka. Although there are signifi- cant cultural differences between regional countries, South Asians are an inherently high-risk group for developing abdominal adiposity, diabetes, cardiovascular diseases [2]. * Correspondence: chath_r@yahoo.com.au 1Allied Health Sciences Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka Full list of author information is available at the end of the article Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Page 2 of 11 Page 2 of 11 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Search strategy A fi d In the second stage the total hits obtained from searching these three databases were pooled together and duplicates were removed. This was followed by screening of the retrieved articles by reading the article title in the third stage and abstracts in stage four. In the fifth stage individual manuscripts were screened, and those not satisfying inclusion criteria (given below) were excluded. To obtain additional data a manual search of the reference lists of articles selected in stage five was performed. Wherever possible forward citations of the studies retrieved during the literature search was traced and screened for possible inclusion. Furthermore, data were also retrieved from the search of relevant web sites and online resources. This search process was conducted independently by two reviewers (PR and RJ) and the final group of articles to be included in the review was deter- mined after an iterative consensus process. The initial literature search using the above search criteria identified the following number of articles in the respective databases; Medline® (n = 121), Web of Science® (n = 424) and SciVerse Scopus® (n = 1226). The search strategy is summarized in Figure 1. Methods mortality causing an estimated 3.2 million annual deaths (6% of global deaths) [7]. A systematic review of published studies reporting physical activity among South Asian adults residing in South Asia was undertaken in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement (Additional file 1) [19]. Physical activity is defined as any bodily movement produced by skeletal muscles that substantially elevates energy expenditure [8]. It is an established fact that physical activity has many beneficial health effects. Regular (≥3 times per week) moderate intensity physical activity such as brisk walking, dancing and gardening decreases the risk of non-communicable disease [9]. Adequate phys- ical activity is also known to increase mental well being [10]. The Harvard alumni study has shown that the alumni mortality rates were significantly lower among those who were physically active, even after adjusting for other lifestyle risk factors [10]. Regular physical activity has been reported to lower blood pressure in adults with hypertension [11]. Physical activity assists in weight loss or a reduction in visceral fat, which could ultimately help in reducing blood pressure [12]. Participation in regular physical activity improves blood glucose control by increasing insulin sensitivity and can prevent or delay onset of type 2 diabetes [13]. Hence it is evident that regular physical activity has numerous cardio-metabolic beneficial effects and the ongoing worldwide epidemic of cardiovascular and metabolic disease is evidence to the fact that the general population is physically inactive. Results The total number of research studies included in the present review is eleven (India-8, Sri Lanka-2, Pakistan-1). In addition, country reports (n = 11, Nepal-3, Bangladesh-2, India-2, Sri Lanka-2, Bhutan-1, Maldives-1) of World Health Organization (WHO) STEPS survey for non-communicable disease risk factors for the South-Asian countries were re- trieved by searching the WHO website (http://www.who. int/chp/steps/reports/en/index.html). The sample size in research studies varied from 416–6,940 and the study setting for research studies were urban (n = 2), rural (n = 3) or both (n = 6). In the STEPS surveys sample sizes were between 2,026 and 44,491. Most research studies used self designed interviewer administered questionnaires Quality assessment adults (>15 years of age), b) Cross‐sectional study design or being the first phase of a longitudinal study, c) Geographic- ally and temporally defined population from any of the South Asian countries mentioned above, d) Being an original study presenting data on physical activity, e) Evaluating physical activity using questionnaires, f) Published in English, or with detailed summaries in English and g) Peer-reviewed fully published research papers. Studies limited to adults engaged in a particular profession, based in hospitals/institutions and confined to those with diagnosed illnesses were excluded. In addition conference proceedings, editorials, commentaries and book chapters/book reviews were excluded. All included research studies were assessed for quality and assigned a rating of either “good” (6–7 points), “fair” (4–5 points) or “poor” (1–3 points) by two reviewers (DCR and RJ). The following seven criteria were used and each one was given a point; 1) appropriateness of research design, 2) appropriate recruitment strategy, 3) adequate response rate (>50%) 4) representativeness of sample, 5) usage of objective and reliable measures to assess physical activity 6) power calculation/justification of numbers and 7) appropriateness of statistical analysis. Inclusion and exclusion criteria The following inclusion criteria were used; a) Population- based studies among healthy non-institutionalized human Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 3 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Page 3 of 11 Figure 1 Summarized search strategy. Figure 1 Summarized search strategy. Findings from research studies d h ll l In India the overall prevalence of inactivity was 18.5%- 88.4% (Table 1). In Indian males the prevalence of inactivity was 12.7%-66.2%. Majority of the studies (n = 5, 62.5%) reported a prevalence of inactivity < 23% in Indian males. In Indian females the inactivity prevalence was 17.0%-79.6%, while majority of the studies (n = 5, 62.5%) reported it to be > 39.5%. The prevalence of inactivity in urban areas of India was reported as 20.7%-88.7%, while in rural areas it was 6.6%-88.1%. One study conducted in rural India reported that physical inactivity was more at leisure time (males - 85.2%, females - 97.3%), and at work (male - 57.2%, female - 59.9%) and less during transport (male - 18.8%, female - 45.7%) [23]. A similar study in both urban and rural areas of India reported inactivity more at leisure time (74.0%) and less at work (31.0%) [24]. There were two research studies on physical activity from Sri Lanka [30,31]. One study was conducted on a nationally representative sample and the other in the most populous western province of Sri Lanka. Both studies looked at urban and rural settings and Inactivity was defined using a uniformed method (IPAQ). The prevalence of Inactivity was 11% in the national sample and 31.8% in In India the overall prevalence of inactivity was 18.5%- 88.4% (Table 1). In Indian males the prevalence of inactivity was 12.7%-66.2%. Majority of the studies (n = 5, 62.5%) reported a prevalence of inactivity < 23% in Indian males. In Indian females the inactivity prevalence was 17.0%-79.6%, while majority of the studies (n = 5, 62.5%) reported it to be > 39.5%. The prevalence of inactivity in urban areas of India was reported as 20.7%-88.7%, while in rural areas it was 6.6%-88.1%. One study conducted in rural India reported that physical inactivity was more at leisure time (males - 85.2%, females - 97.3%), and at work (male - 57.2%, female - 59.9%) and less during transport (male - 18.8%, female - 45.7%) [23]. A similar study in both urban and rural areas of India reported inactivity more at leisure time (74.0%) and less at work (31.0%) [24]. Only one STEPS survey has been carried out in Bhutan in the capital city Thimphu (2007, n = 2,484). The overall inactivity prevalence was 58.6% and females (69.6%) were more inactive than males (49.8%). Findings from STEPS surveys In the STEPS survey reports there was uniformity in the sampling and the definition of inactivity. Overall in majority of studies females were more inactive compared to males (Table 2). Furthermore, leisure related inactivity was >75% in studies reporting inactivity in this domain and people were more active in the transport domain when compared with other domains (Table 2). Two surveys were done in 2002 (n = 11,409) and 2009 (n = 9,275) in Bangladesh. In 2002, overall prevalence of physical inactivity was 52.3%. However, in this survey physical activity was categorised as light, moderate, heavy based on a likert scale; always, usually, sometimes and never. Inactivity was defined as people who have never done light/moderate activities, which was different to the criteria used in all other WHO STEPS surveys. In the national sample (2009) the overall inactivity prevalence was 27%, with more females (41.3%) being inactive than males (10.5%) (Table 2). Data extraction A separate assessment is not included for the WHO STEPS surveys conducted according to the uniform STEPS study protocol which satisfies all of the above seven criterion used for quality assessment. Definition of inactivity How sedentary lifestyle or inactivity was defined varied significantly in the different studies that used self designed questionnaires to evaluate physical activity (Table 1). Phys- ical Activity Level (PAL) (Estimated total energy expend- iture per 24 hrs / basal metabolic rate) was calculated in 3 studies and a PAL for inactivity was defined (<1.5/<1.69) [20-22]. However, there was a wide variation in the def- inition of physical inactivity in the other studies (Table 1). In the WHO STEPS surveys using the GPAQ, the total of the categorical score in the 3 domains (work, leisure time and transport) classifies an individual into three categor- ies; ‘Inactive’, ‘Moderately active’ and ‘Highly active’. The ‘Inactive’ category includes those who do not perform any physical activity or those reporting some activity, but not enough to meet other categories. The same definition was used in the IPAQ for the ‘Inactive’ category. Data extraction Data were extracted from the included studies by one reviewer using a standardised form and checked for accuracy by a second reviewer. The data extracted from each study were: a) study details (lead author, country/ city, year published/year of survey), b) methods (sample size, sampling method, age of subjects in years and type of questionnaire used, definitions), and c) physical activity data (all adults, males, females and domain specific data). Discrepancies in the extracted data were resolved by discussion, with involvement of a third reviewer when necessary. Page 4 of 11 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 the other study conducted in western province (Table 1). Males (14.6% and 38.5%) were more inactive than females (8.7% and 24.7%). Inactivity of urban adults was 35.2% and higher than rural adults (27.6%) in the study from Western province (Table 1). In the National sample; female gender (OR:2.1), age > 70 (OR:3.8), urban-living (OR:2.5), Muslim ethnicity (Sri Lankan Moor) (OR:2.7), tertiary education (OR:3.6), obesity (OR:1.8), diabetes (OR:1.6), hypertension (OR:1.2) and metabolic syndrome (OR:1.3) all significantly increased odds of being ‘inactive’ [31]. The only study on physical activity conducted in urban Pakistan using IPAQ showed that prevalence of inactivity was 60.1% (males-52.1%, females-69.8%) [29]. for evaluation of physical activity (n = 7). International Physical Activity Questionnaire (IPAQ) was used by 3 research studies and 1 study used WHO STEPS survey tool. All WHO STEPS surveys used Global Physical Activity Questionnaire (GPAQ) for the evaluation of physical activity. Majority of the research studies received a “good” (n = 5) or “fair” (n = 3) ranking on the quality assessment, while only 3 were ranked “poor” (Additional file 2). A separate assessment is not included for the WHO STEPS surveys conducted according to the uniform STEPS study protocol which satisfies all of the above seven criterion used for quality assessment. for evaluation of physical activity (n = 7). International Physical Activity Questionnaire (IPAQ) was used by 3 research studies and 1 study used WHO STEPS survey tool. All WHO STEPS surveys used Global Physical Activity Questionnaire (GPAQ) for the evaluation of physical activity. Majority of the research studies received a “good” (n = 5) or “fair” (n = 3) ranking on the quality assessment, while only 3 were ranked “poor” (Additional file 2). Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 , Rural, M 579, (Ma s, Urban uster sa 416 (M es racterist 782 (mal s Urban minister ys, carryi activities y calcula dex inclu y onnaire, zed, 1.40–1.69 onnaire, physical nactive: hour per week exercise Findings from research studies d h ll l Two large STEPS surveys were done in India in 2003–2005 (6 states, n = 44,491) and 2007–2008 (7 states, n = 39,064). In 2003 the overall prevalence of physical inactivity was 15.8% and in 2007 it was 72.3%. This variation could be attributed to different states used in the two surveys. However Kerala and Maharashtra states were evaluated in both surveys, and the overall prevalence of physical inactivity in Kerala/ Maharashtra has increased from 6.7%/6.8% in 2003 to 75.8%/81.2% in 2007. Male and Female physical inactivity in 2003 was 12.6% and 18.9% respectively. In 2007 preva- lence of Male and Female physical inactivity was 58.5% and 75.7% respectively. Overall females were more inactive than males and urban residents were more inactive than rural (Table 2). The only STEPS survey conducted in Maldives (2004) has been conducted in its’ capital city Malē (n = 2026). p g g at work, home or tran discretionary time es 3,768, Females rban, rural and ng Interviewer administe PAL was calculated an PAL <1.40 extremely in sedentary/lightly active s 260, Females 260), nd rural, Random Interviewer administe Inactive: Sedentary job exercise or cycling, M Sedentary job and som physical exercise and/o OR Standing job and n or cycling ural po at work, discretio Interview PAL was PAL <1.4 sedentary Interview Inactive: exercise Sedentar physical OR Stand or cycling Findings from research studies d h ll l More than 90% (Males 91.2%, Females 94.6%) at work, home or transport and discretionary time ●In males inactivity was 19.7%, while in females it was 17.0% 6,447, (Males 3,768, Females 7-76 yrs, Urban, rural and xed sampling Interviewer administered questionnaire, PAL was calculated and categorized, PAL <1.40 extremely inactive, PAL 1.40–1.69 sedentary/lightly active ●Extreme inactivity prevalence 9.7% (Males 7.4%, Females 12.9%), ●Sedentary/lightly active prevalence 62.1% (Males 58.8%, Females 66.7%) 520, (Males 260, Females 260), rs, Urban and rural, Random Interviewer administered questionnaire, Inactive: Sedentary job and no physical exercise or cycling, Moderately inactive: Sedentary job and some but <1 hour physical exercise and/or cycling per week OR Standing job and no physical exercise or cycling ●Prevalence of inactivity 29.4%* (Males 12.7%, Females 46.1%, Urban 29.6%, Rural 29.2%), ●Prevalence of moderate inactivity 21.5%,* (Males 25.7%, Females 17.3%, Urban 30.0%, Rural 13.1%), ●Inactivity was more in Urban and in females, ●Urban females waist circumference reduced (p < 0.05) with increased physical activity, ●BMI showed a steady decline from inactivity to activity ,825, (Males 650, Females 1175), Rural population Interviewer administered questionnaire, Sedentary lifestyle: those who had never ●40.0% had a sedentary lifestyle (Males 40.8%, Females 39.7%) males it was ales 7.4%, e prevalence .7%, Females valence of %, Females ctivity was females with ed a steady e in all s 85.2%, 8.8%, 57.2% ghest in roup 2.9%, ore than 0%), more 8.9%, n 1.64 in an n the r old not d and cised vity lev 30-1.56 > 58 yr w ove tionary ●Wo ercise les, ● n/day ross ag 6 yrs ag 5%) and w nactivity m %), and le %), ●Ina - 59.9%; ● 4 yrs) and prevalen 1.9% ), ● poration 2 ●Inactivit (31.0%), ● %, RR = 1 killed wo (32.0%, R 3%, RR = prevalenc nder diffe activity w nactivity p %), ●Sed 58.8%, F of inactiv n 29.6%, 5%) and w nactivity m %), and le %), ●Ina - 59.9%; ● 64 yrs) and prevalen 1.9% ), ● poration 2 ●Inactivit (31.0%), ● 7%, RR = 1 killed wo (32.0%, R 3%, RR = prevalenc ender diffe activity w nactivity p %), ●Sed 58.8%, F of inactiv n 29 6%, Females 39.7%) Res ●P and olde fem mal you disc cho exe 40.9 high (rec R a o f m y d c e 4 ( e, e + ure e y onnaire, zed, 1.40–1.69 onnaire, hysical nactive: hour per week exercise ose who had never minister ys, carry activities y calcula dex inclu g home or tran nary time er administer calculated an 0 extremely in /lightly active er administer Sedentary job or cycling, Mo y job and som exercise and/o ng job and n ools an ministe y level diture/E ), Seden ools an ministe y level diture/E ), Seden y y y essm rview sical a rgy ex tabolic L) <1.5 Interview Inactive: light/sede travel, Ina Composit Interview Inactive: light/sede travel, Ina Composit Sedentar at work, discretio Interview PAL was PAL <1.4 sedentar Interview Inactive: exercise Sedentar physical OR Stand or cycling 441), ent pling les 3689), atified 188) pling les 3689), atified 188) pulation s 3,768, Females rban, rural and ng 260, Females 260), d rural, Random ural po 447, (M -76 yrs ed sam 20, (Ma Urban http://www.ijbnpa.org/content/10/1/116 http://www.ijbnpa.org/content/10/1/116 (Males 52.1%, Females 69.8%) ●Females were significantly more inactive than males (OR: 2.1, 95% CI 1.5–3.1, p < 0.001) ●Prevalence of inactivity 31.8%* (Males 38.5%, Females 24.7%), ●Inactivity in urban adults 35.2% (Males 41.0%, Females 29.0), ●Inactivity in rural adults (Males 35.0%, Females 19.0%), ●Physical inactivity had a significant association with high BMI among women irrespective of their urban or rural living ●Prevalence of inactivity 11.0% (Males 14.6%, Females 8.7%) th 2% th 2% ral with % an %, 35.2% l rban or %, rban or %, an 88.7% on incre activity 6 e inactiv < 0.001) * (Males rban ad activity irrespective of thei ivity 11.0% (Males 1 n 88.7% on incre ctivity e inactiv 0.001) (Males ban ad activity ve of thei % (Males 1 4% (U perte ce of %) tly m –3.1, y 31.8 vity in 9.0), ● Exercise .1%), ●P ck of exer Males 52.1 Females ales (OR: Prevalen males 24 Males 41.0 ults (Ma BMI amon rural livin ●Prevalen Females 8 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 7 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Page 7 of 11 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Table 2 Summary of findings from STEPS surveys Country Year of study Sample characteristics Results Inactive (%) Inactivity in each domain (%) Male Female Urban Rural Total Work Transport Recreation Bangladesh 2002 Sample size 11,409 NR NR 50.1# 56.7# 52# (Male 5,625, Female 5,784) Age 25–64 yrs Urban and rural In capital city - Dhaka Bangladesh 2009-2010 Sample size 9,275 10.5 41.3 NR NR 27.0 45.7 44.5 81.9 (Male 4,312, Female 4,963) Age > 25 yrs Urban and rural Bhutan 2007 Sample size 2,484 49.8 69.6 58.6 NA 58.6 69.0 63.2 78.7 (Male 1,138, Female 1,346) Age 25–74 yrs Urban In capital city - Thimpu India 2003-2005 Sample size 44,491 (Male 21,871, Female 22,620) Age 15–64 yrs Urban and rural 6 States in India 1. Assam 8.3 10.7 26.1* 1.6* 9.5* 28.3* 24.6* NR 2. Delhi 25.5 15.5 26.3* NR 20.4* 62.9* 39.3* NR 3. Haryana 16.9 47.6 38.3* 24.6* 32.7* 78.4* 41.9* NR 4. Kerala 4.8 4.3 7.6* 5.9* 6.7* 18.4* 31.8* NR 5. Maharashtra 7.7 6.1 14.9* 1.3* 6.8* 24.5* 21.1* NR 6. http://www.ijbnpa.org/content/10/1/116 Tamil Nadu 16.9 27.2 29.1* 19.9* 22.0* 90.8* 25.1* NR India 2007-2008 Sample size 38,064 (Male 16,891, Female 21,173) Age 24–64 yrs Urban and rural 7 states in India 1. Andhra Pradesh 55.9 79.7 77.5 63.8 67.7 NR NR NR 2.Kerala 64.7 86.2 70.8 74.5 75.8 NR NR NR 3. Madhya Pradesh 33.5 52.0 68.3 31.8 42.3 NR NR NR 4. Maharashtra 75.4 87.7 86.1 77.2 81.2 NR NR NR 5. Mizoram 60.9 82.4 79.1 62.5 71.1 NR NR NR 6. Tamil Nadu 57.3 74.2 79.4 61.6 65.8 NR NR NR 7. Uttarakhand 64.6 69.7 91.6 57.6 67.1 NR NR NR Maldives 2004 Sample size 2,026 NR NR NR NA NR 93.2* NR NR (Male 934, Female 1,092) Age 25–64 yrs Urban (In Male) Table 2 Summary of findings from STEPS surveys Country Year of study Sample characteristics Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 8 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 8 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 Page 8 of 11 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Page 8 of 11 Table 2 Summary of findings from STEPS surveys (Continued) Nepal 2003 Sample size 2,030 73.6 90.9 NR NA 82.3* 82.3* 27.6* 94.5* (Male 1,010, Female 1,020) Age 25–64 yrs Urban Nepal 2004-2005 Sample size 7,792 NR NR NR NR NR 51.5 19.1 86.0 (Male 3,674, Female 4,118) Age 15–64 yrs Urban and rural Nepal 2007 Sample size 4,328 5.2 5.9 NR NR 5.5 10.6 19.0 83.2 (Male 1,907, Female 2,421) Age 15–64 yrs Urban and rural 15 of 75 districts Sri Lanka 2003 Sample size 3,000 12.1 19.1 NR NR 15.6 58.3* NR 94.8* (Male 1,500, Female 1,500) Age 15–74 yrs Urban One of nine provinces - Western Sri Lanka 2006 Sample size 11,680 17.9 31.9 NR NR 25.0 NR NR NR (Male 5,765, Female 5,915) Age 24–64 yrs Urban and rural 5 random districts out of all 25 districts * - calculated from available data; NR – Not reported; NA – Not applicable. http://www.ijbnpa.org/content/10/1/116 Table 2 Summary of findings from STEPS surveys (Continued) - calculated from available data; NR – Not reported; NA – Not applic of the respondents reported that they are physically inactive at work. In contrast STEPS survey data on physical activity were available from all countries except Pakistan. All research studies from the region collectively evaluated physical activity of 26,360 adults, while STEPS surveys contained data on 136,579 adults. We observed a marked variation between research studies in the definition of physical inactivity and in the questionnaires used. Hence, comparisons between studies were undertaken with caution. This barrier was overcome in the STEPS surveys as they used the GPAQ to evaluate physical activity. It is important that future researchers try and adhere to this uniformity in order to derive intra- and inter-regional comparable data and observe secular trends in physical activity. The present study mainly focuses on the preva- lence of physical inactivity and its variations with gender and area of residence. We also tried to identify inactivity levels in different domains (work, transport, leisure) as occupations, transport methods, social and cultural values differ in this region compared to other regions and the developed world. Three STEPS surveys have been conducted in Nepal. In 2003 only an urban sample was taken from the capital city (n = 2,030). In later STEPS surveys both urban and rural samples were studied (2004 n = 7,792 and 2007 n = 4,328). The 2003 survey reported an overall inactivity of 82.9% (Males 74.6%, Females 91.2%) in the urban residents. However in contrast the 2007 national sample (conducted in 15 out of 75 districts) reported that overall inactivity was only 5.5% (Males 5.2%, Females 5.9%) (Table 2). In Sri Lanka two STEPS surveys were done in 2003 (single province, n = 3,000) and 2006 (five districts, n = 11,680). In 2003 overall prevalence of physical inactivity was 15.6% and in 2006 it has increased to 25%. Females (2003–19.1%, 2006–31.9%) were more inactive than males (2003–12.1%, 2006–17.9%) in both studies. Discussion To our knowledge this is the first systematic review evaluating physical inactivity and physical activity patterns among adults from South Asian countries. Published research articles evaluating physical activity prevalence were only available from India, Sri Lanka and Pakistan. We observed several socio-economical factors associated with physical inactivity. Skilled workers and professionals were more inactive than unskilled workers in the region [24]. Similarly, higher education was a significant factor Page 9 of 11 associated with physical inactivity [31]. Among South Indians, Vaz et al. reported that people engaging in rec- reational exercise were inactive in other domains [20,21]. Gender is also an important factor to determining physical activity levels of a population. Several studies reported higher physical inactivity among South Asian women [23,25,29,31,32]. Cultural expectations may restrict the participation of women in certain forms of physical activity in some religious and ethnic groups in the region. The traditional role of South Asian women in taking care of household work and supporting extended family members may limit the time available for them to engage in physical activity, in particularly leisure time physical activities. Low physical activity is one of the contributing risk factors for the higher obesity levels seen among Asian Indians [33]. A qualitative study conducted among South Asians living in United Kingdom found that understanding external motivators and social context of their lives is very crucial for developing successful physical activity interventions [34]. Hence, although exercise is promoted in public health campaigns to increase the overall physical activity level of a population, it is important to under- stand socio-economical factors associated with different populations in order to deliver effective physical activity interventions. which were studied in both surveys also showed marked variation. This could be due to differences in the sampling frames used. However such dramatic increases in inactiv- ity over a short period warrants further investigation. The only surveys conducted in urban capitals of Maldives and Bhutan had significantly higher prevalence of inactivity. In Bhutan inactivity was lower compared to Maldives due to it being mostly an agrarian society with nearly 50% of the labour force engaged in agriculture as compared to Maldives where most are involved in the tourism industry [37]. The most active regional country in the STEPS surveys was Nepal, possibly due to 80% of the Nepalese population being involved in agriculture [38]. Discussion In all regional countries work and transport related activity was higher compared to leisure time activity. Hence it is important to consider individual country profiles, proportions of urbanity and cultures when delivering public health mes- sages focused on leisure time activity. Comparable nationally representative country data are available from the WHO World Health Survey (IPAQ questionnaire) conducted in 2002–2003, where the male and female inactivity prevalence was as follows; India 9.3% & 15.2%, Nepal 6.7% & 9.7% and Sri Lanka 7.3% & 13.8% [39]. The data are fairly comparable to those from WHO STEPS surveys in India (2003) and Nepal (2007). In contrast in Sri Lanka inactivity has markedly increased from the 2002 World Health Survey to the 2006 STEPS survey. Several studies show that IPAQ overestimates inactivity prevalence and under estimates prevalence in rural areas of developing countries [32,40,41]. The STEPS surveys have also been conducted in 80 developing countries around the world allowing for a meaningful comparison across regions [35]. In the Western Pacific region (19 countries) inactivity prevalence ranged from 7.5% in Mongolia to 75.3% in Cook Islands with majority of countries falling between 40%-66%. Eastern Mediterranean (14 countries) prevalence of inactivity ranged from 30%-60%. In Africa (31 countries) lowest prevalence of inactivity was observed in Mozambique (6.4%) and highest in Zimbabwe (79.3%). In nearly half of the African countries inactivity was <29%. East Asian countries like Myanmar (12.7%) and Indonesia (22.6%) had prevalence’s of inactivity comparable with the South Asia and Africans. With the available data we observe that the persons residing in South Asian region are more active compared with the Western Pacific and Mediterranean countries, lying in parallel with East Asia and Africa. In developing countries with agrarian economies inactivity was low irrespective of the region (Nepal, Mongolia, Malawi, Mozambique and Cambodia). However the inactivity prevalence seems to be on the rise in most Western Pacific countries due to recent economic transitions. Hence, countries in the South Asian region in economic transition also need public health planning to enable the people to maintain their already existing active The WHO STEPwise approach was introduced for chronic disease risk factor surveillance in order to close the gap in the availability of data from developing countries and to strengthen their capacity to conduct such surveillances [35]. The GPAQ used in these surveys not only gave uniformity to the gathered data, it also allowed meaningful comparisons. Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Discussion The GPAQ is known to be reliable for surveys in developing countries, adaptable to incorporate cultural differences [36]. The initial STEPS surveys from the region were mostly in sub-populations and confined to logistically favourable areas like capitals. Sri Lanka (2006), Nepal (2007) and Bangladesh (2009) (Inactivity: 25%, 5.5% and 27% respectively) had nationally representative samples in subsequent surveys. Prevalence of overall inactivity reduced in these subsequent surveys as the sample dispersed from populations in urban capitals to the rural parts of the countries. This highlights the effect of urbanity on physical activity. However, in the Sri Lankan STEPS surveys inactivity prevalence has increased in the second survey (2006), which used a national sample. This is probably due to the initial survey (2003) being conducted in more rural areas of the Western province of Sri Lanka, in contrast to Nepal and Bangladesh where the initial surveys were from more urbanised capitals. The two large STPES surveys (2003, 2007) from India contrasted in the prevalence of inactivity, possibly attribut- able to subjective variations in data collection, inter-state variations and differences in time and season of data col- lection. However, data from Kerala and Maharashtra states Page 10 of 11 life styles. We also observe that females were more inactive in the South Asian region when compared to males, a finding which is seen in most other regions and even in developed countries. leisure time. These factors need to be considered when planning future interventions and research aimed at improving PA in the region. However, future researchers should try and adhere to uniformity in definitions and assessment tools in order to derive intra- and inter-regional comparable data and observe secular trends. The International Prevalence Study conducted in 2002– 2004 using IPAQ categorized inactivity to be least in China (6.9%), New Zealand (12.2%), Canada (13.7%), USA (15.9%) and Australia (17.2%) [42]. We observe that South Asian activity levels were in parallel or sometimes better than these developed countries which have longer physical activity promotion strategies, mainly targeted at improving leisure time physical activity. However with increased urbanisation and busy work schedules the leisure time is also reduced. In the South Asian region we observed that activity during work and transport is higher than leisure time activity. Promoting leisure time activity has also become a challenge in South Asia due to cultural and attitudinal barriers. Discussion Hence public health messages in the region should be directed at improving all domains of activity with work and transport policies of the countries supporting them. Competing interests The authors declare that they have no competing interests. Received: 7 April 2013 Accepted: 9 October 2013 Published: 12 October 2013 Received: 7 April 2013 Accepted: 9 October 2013 Published: 12 October 2013 References 1. Gupta M, Singh N, Verma S: South Asians and cardiovascular risk: What clinicians should know. Circulation 2006, 113(25):e924–e929. 1. Gupta M, Singh N, Verma S: South Asians and cardiovascular risk: What clinicians should know. Circulation 2006, 113(25):e924–e929. 2. Eapen D, Kalra GL, Merchant N, Arora A, Khan BV: Metabolic syndrome and cardiovascular disease in South Asians. Vasc Health Risk Manag 2009, 5:731–743. 2. Eapen D, Kalra GL, Merchant N, Arora A, Khan BV: Metabolic syndrome and cardiovascular disease in South Asians. Vasc Health Risk Manag 2009, 5:731–743. 3. Erens B, Primatesta P, Prior G: The health of minority ethnic groups ′99 methodology & documentation. London: The health survey for england; 2001. 4 Jayawardena R Ranasinghe P Byrne NM Soares MJ Katulanda P Hills AP: 3. Erens B, Primatesta P, Prior G: The health of minority ethnic groups ′99 methodology & documentation. London: The health survey for england; 2001. 4. Jayawardena R, Ranasinghe P, Byrne NM, Soares MJ, Katulanda P, Hills AP: Prevalence and trends of the diabetes epidemic in South Asia: a systematic review and meta-analysis. BMC Public Health 2012, 12:380. 5. Gupta M, Brister S: Is South Asian ethnicity an independent cardiovascular risk factor? Can J Cardiol 2006 22(3):193 197 4. Jayawardena R, Ranasinghe P, Byrne NM, Soares MJ, Katulanda P, Hills AP: Prevalence and trends of the diabetes epidemic in South Asia: a systematic review and meta-analysis. BMC Public Health 2012, 12:380. 5. Gupta M, Brister S: Is South Asian ethnicity an independent cardiovascular risk factor? Can J Cardiol 2006, 22(3):193–197. 6. Qin L, Corpeleijn E, Jiang C, Thomas GN, Schooling CM, Zhang W, Cheng KK, Leung GM, Stolk RP, Lam TH: Physical activity, adiposity, and diabetes risk in middle-aged and older Chinese population: The Guangzhou Biobank Cohort Study. Diab Care 2010, 33(11):2342–2348. Authors’ contributions One of the strengths of the current systematic review is the comprehensive and easily replicable search strategy applied to 3 major medical databases. In addition we systematically selected the studies through the application of well-defined inclusion/exclusion criteria. We would also like to highlight several limitations in the present review. Firstly, most of studies were limited to regional populations. As South Asian countries have considerably diverse ethnic groups, the regional findings may not be generalized to whole country or for the entire South Asian region. Secondly, there is no uniformity on physical activity assessment tools. Although several of studies have used IPAQ, differences in the formats used limit comparability. For instance, Arambepola et al. has used longer version of IPAQ whereas Katulanda et al. has used a short version [30,31]. Thirdly, there is clear heterogeneity on the defin- ition of physical inactivity/sedentary life style and physical activity levels. Furthermore, only limited number of articles reported data on the sub-domains of physical activity (work, transport and leisure). In addition, although many studies have shown that resistance exercise improves over- all health and that it is beneficial for South Asians with Diabetes at present there is only very limited data available for South Asians [43,44]. DC, PR and RJ made substantial contribution to conception and study design. DC, PR and RJ were involved in data collection. DC, PR, RJ and AM were involved in refining the study design, statistical analysis and drafting the manuscript. DC, PR, RJ and AM critically revised the manuscript. All authors read and approved the final manuscript. Additional files Additional file 1: PRISMA 2009 checklist. Additional file 2: Assessment of Quality of the included Research Studies. Additional file 1: PRISMA 2009 checklist. Additional file 2: Assessment of Quality of the included Research Studies. Abbreviations Abbreviations GPAQ: Global physical activity questionnaire; IPAQ: International physical activity questionnaire; MeSH: Medical subject heading; MET: Metabolic equivalents; OR: Odds ratio; PAL: Physical activity level; PRISMA: Preferred reporting in systematic reviews & Meta-Analysis; WHO: World Health Organization. Author details 1 1Allied Health Sciences Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka. 2Department of Pharmacology, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka. 3Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia. 4Fortis-C-DOC Centre of Excellence for Diabetes, Metabolic Diseases and Endocrinology, Chirag Enclave, New Delhi, India. Received: 7 April 2013 Accepted: 9 October 2013 Published: 12 October 2013 Conclusions Jepson R, Harris FM, Bowes A, Robertson R, Avan G, Sheikh A: Physical activity in South Asians: An in-depth qualitative study to explore motivations and facilitators. PLoS One 2012, 7(10):e45333. 12. Reaven PD, Barrett-Connor E, Edelstein S: Relation between leisure-time physical activity and blood pressure in older women. Circulation 1991, 83(2):559–565. 35. World Health Organization: WHO Chronic diseases and health promotion, STEPS country reports. http://www.who.int/chp/steps/reports/en/index.html. 13. Hamman RF, Wing RR, Edelstein SL, Lachin JM, Bray GA, Delahanty L, Hoskin M, Kriska AM, Mayer-Davis EJ, Pi-Sunyer X, et al: Effect of weight loss with lifestyle intervention on risk of diabetes. Diabetes Care 2006, 29(9):2102–2107. 36. Armstrong TA, Bull FC: Development of the Global Physical Activity Questionnaire (GPAQ). J Public Health 2006, 14:66–70. Questionnaire (GPAQ). J Public Health 2006, 14:66–70. 37. Central Intelligence Agency: The world factbook; 2013. https://www.cia.gov/ library/publications/the-world-factbook/index.html. 14. Zaman MJ, Jemni M: South Asians, physical exercise and heart disease. Heart 2011, 97(8):607–609. 38. Ertur O: The need for a national urbanization policy in Nepal. Asia Pac Popul J 1994, 9(3):19–36. 15. Williams ED, Stamatakis E, Chandola T, Hamer M: Assessment of physical activity levels in South Asians in the UK: Findings from the Health Survey for England. J Epidemiol Community Health 2011, 65(6):517–521. 39. Guthold R, Ono T, Strong KL, Chatterji S, Morabia A: Worldwide variability in physical inactivity a 51-country survey. Am J Prev Med 2008, 34(6):486–494. 16. Fischbacher CM, Hunt S, Alexander L: How physically active are South Asians in the United Kingdom? A literature review. J Public Health (Oxf) 2004, 26(3):250–258. 40. Ainsworth BE, Macera CA, Jones DA: Comparison of the 2001 BRFSS and the IPAQ physical activity questionnaires. Med Sci Sports Exerc 2006, 38:1584–1592. 17. Horne M, Tierney S: What are the barriers and facilitators to exercise and physical activity uptake and adherence among South Asian older adults: A systematic review of qualitative studies. Prev Med 2012, 55(4):276–284. 41. Craig CL, Marshall AL, Sjöström M: International physical activity questionnaire: 12-country reliability and validity. Med Sci Sports Exerc 2003, 35:1381–1395. 18. Swaminathan S, Vaz M: Childhood physical activity, sports and exercise and noncommunicable disease: A special focus on India. Indian J Pediatr 2013, 80(1):63–70. 42. Bauman A, Bull F, Chey T, Craig CL, Ainsworth BE, Sallis JF, Bowles HR, Hagstromer M, Sjostrom M, Pratt M, et al: The international prevalence study on physical activity: results from 20 countries. Int J Behav Nutr Phys Act 2009, 6:21. Conclusions y , ( ) 7. World Health Organization: Global health risks: Mortality and burden of disease attributable to selected major risks. Geneva: World Health Organization; 2009. There is a wide variation in the prevalence of physical inactivity among South-Asian adults within and between countries. Hence it is difficult to comment about the overall prevalence of physical inactivity in the region. In the South Asian regions females, skilled workers, professionals and those with higher education were more inactive. Majority of South Asian adults were inactive during their 8. Aust NZJ, MedCaspersen CJ, Powell KE, Christenson GM: Physical activity, exercise, and physical fitness: Definitions and distinctions for health-related research. Public Health Rep 1985, 100(2):126–131. 9. Willett WC, Koplan JP, Nugent R, Dusenbury C, Puska P, Gaziano TA: 8. Aust NZJ, MedCaspersen CJ, Powell KE, Christenson GM: Physical activity, exercise, and physical fitness: Definitions and distinctions for health-related research. Public Health Rep 1985, 100(2):126–131. 9. Willett WC, Koplan JP, Nugent R, Dusenbury C, Puska P, Gaziano TA: Prevention of chronic disease by means of diet and lifestyle changes. In Disease control priorities in developing countries. 2nd edition. Edited by 9. Willett WC, Koplan JP, Nugent R, Dusenbury C, Puska P, Gaziano TA: Prevention of chronic disease by means of diet and lifestyle changes. In Disease control priorities in developing countries. 2nd edition. Edited by Page 11 of 11 Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, Jha P, Mills A. Washington (DC): Musgrove P; 2006. Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, Jha P, Mills A. Washington (DC): Musgrove P; 2006. 32. Ekelund U, Sepp H, Barge S: Criterion-related validity of the last 7-day, short form of the International Physical Activity questionnaire in Swedish adults. Public Health Nutr 2006, 9:258–265. 10. Fox KR: The influence of physical activity on mental well-being. Public Health Nutr 1999, 2(3A):411–418. 33. Chopra SM, Misra A, Gulati S, Gupta R: Overweight, obesity and related non-communicable diseases in Asian Indian girls and women. Eur J Clin Nutr 2013, 67(7):688–696. 11. Ishikawa K, Ohta T, Zhang J, Hashimoto S, Tanaka H: Influence of age and gender on exercise training-induced blood pressure reduction in systemic hypertension. Am J Cardiol 1999, 84(2):192–196. 34. Conclusions 19. Moher D, Liberati A, Tetzlaff J, Altman DG, Group P: Preferred reporting items for systematic reviews and meta-analyses: The PRISMA statement. BMJ 2009, 339:b2535. 43. Hills AP, Shultz SP, Soares MJ, Byrne NM, Hunter GR, King NA, Misra A: Resistance training for obese, type 2 diabetic adults: a review of the evidence. Obes Rev 2010, 11(10):740–749. 20. Vaz M, Bharathi AV, Kurpad AV: Exercising’ but not active: Implications for physical activity counselling. Natl Med J India 2006, 19(6):345. 44. Misra A, Alappan NK, Vikram NK, Goel K, Gupta N, Mittal K, Bhatt S, Luthra K: Effect of supervised progressive resistance-exercise training protocol on insulin sensitivity, glycemia, lipids, and body composition in Asian Indians with type 2 diabetes. Diabetes Care 2008, 31(7):1282–1287. 21. Vaz M, Bharathi AV: Perceptions of the intensity of specific physical activities in Bangalore, South India: Implications for exercise prescription. J Assoc Physicians India 2004, 52:541–544. 22. Sullivan R, Kinra S, Ekelund U, Bharathi AV, Vaz M, Kurpad A, Collier T, Reddy KS, Prabhakaran D, Ben-Shlomo Y, et al: Socio-demographic patterning of physical activity across migrant groups in India: results from the Indian Migration Study. PLoS One 2011, 6(10):e24898. doi:10.1186/1479-5868-10-116 Cite this article as: Ranasinghe et al.: Physical activity patterns among South-Asian adults: a systematic review. International Journal of Behavioral Nutrition and Physical Activity 2013 10:116. 23. Krishnan A, Shah B, Lal V, Shukla DK, Paul E, Kapoor SK: Prevalence of risk factors for non-communicable disease in a rural area of Faridabad district of Haryana. Indian J Public Health 2008, 52(3):117–124. y 24. Sugathan TN, Soman CR, Sankaranarayanan K: Behavioural risk factors for 24. Sugathan TN, Soman CR, Sankaranarayanan K: Behavioural risk factors for non communicable diseases among adults in Kerala, India. Indian J Med Res 2008, 127(6):555–563. non communicable diseases among adults in Kerala, India. Indian J Med Res 2008, 127(6):555–563. 25. Agrawal VK, Basannar DR, Sing RP, Dutt M, Abraham D, Mustafa MS: Coronary risk factors in a rural community. Indian J Public Health 2006, 50(1):19–23. g g Coronary risk factors in a rural community. Indian J Public Health 2006, 50(1):19–23. 26. Mittal M, Arora M, Bachhel R, Kaur N, Sidhu RS: Physical activity, indices of obesity and mean arterial blood pressure: Does place of living matters? rural vs urban. J Clin Diagn Res 2011, 5(5):1038–1042. 27. Haldiya KR, Mathur ML, Sachdev R: Lifestyle-related risk factors for cardiovascular disease in a desert population of India. Ranasinghe et al. International Journal of Behavioral Nutrition and Physical Activity 2013, 10:116 http://www.ijbnpa.org/content/10/1/116 Conclusions Curr Sci 2010, 99(2):190–195. 28. Agrawal R, Chaturvedi M, Singh S, Gupta SC: An epidemiological study of dietary and exercise habits as correlates of hypertension in persons aged 45 years and above in Agra District. Indian J Comm Health 2012, 24(2):91–96. 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: 29. Khuwaja AK, Kadir MM: Gender differences and clustering pattern of behavioural risk factors for chronic non-communicable diseases: Community-based study from a developing country. Chronic Illn 2010, 6(3):163–170. • Convenient online submission 30. Arambepola C, Allender S, Ekanayake R, Fernando D: Urban living and obesity: Is it independent of its population and lifestyle characteristics? Trop Med Int Health 2008, 13(4):448–457. 31. Katulanda P, Jayawardana R, Ranasinghe P, Rezvi Sheriff M, Matthews DR: Physical activity patterns and correlates among adults from a developing country: The Sri Lanka Diabetes and Cardiovascular Study. Public Health Nutr 2013, 16(9):1684–1692. 31. Katulanda P, Jayawardana R, Ranasinghe P, Rezvi Sheriff M, Matthews DR: Physical activity patterns and correlates among adults from a developing country: The Sri Lanka Diabetes and Cardiovascular Study. Public Health Nutr 2013, 16(9):1684–1692.
https://openalex.org/W4288349555	https://zenodo.org/records/3066814/files/2112ijsc03.pdf	English	null	Bayesian system reliability and availability analysis underthe vague environment based on Exponential distribution	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	10,929	Abstract Reliability modeling is the most important discipline of reliable engineering. The main purpose of this paper is to provide a methodology for discussing the vague environment. Actually we discuss on Bayesian system reliability and availability analysis on the vague environment based on Exponential distribution under squared error symmetric and precautionary asymmetric loss functions. In order to apply the Bayesian approach, model parameters are assumed to be vague random variables with vague prior distributions. This approach will be used to create the vague Bayes estimate of system reliability and availability by introducing and applying a theorem called “Resolution Identity” for vague sets. For this purpose, the original problem is transformed into a nonlinear programming problem which is then divided up into eight subproblems to simplify computations. Finally, the results obtained for the subproblems can be used to determine the membership functions of the vague Bayes estimate of system reliability. Finally, the sub problems can be solved by using any commercial optimizers, e.g. GAMS or LINGO. Keywords: Bayes point estimators, Vague environment, Nonlinear programming; System reliability, System availability, Exponential distribution, Precautionary loss function. Bayesian system reliability and availability analysis underthe vague environment based on Exponential distribution RaminGholizadeh1 , Aliakbar M. Shirazi2 and Maghdoode Hadian3 1Young Researchers Club, Ayatollah Amoli Branch, Islamic Azad University, Amol , Iran. 2Islamic Azad University, Ayatollah Amoli Branch, Mathematic Department,Iran. 3Young Researchers Club, Mashhad Branch, Islamic Azad University, Mashhad,Iran Islamic Azad University, Iran RaminGholizadeh1 , Aliakbar M. Shirazi2 and Maghdoode Hadian3 1Young Researchers Club, Ayatollah Amoli Branch, Islamic Azad University, Amol , Iran. 2Islamic Azad University, Ayatollah Amoli Branch, Mathematic Department,Iran. 3Young Researchers Club, Mashhad Branch, Islamic Azad University, Mashhad,Iran Islamic Azad University, Iran 2Islamic Azad University, Ayatollah Amoli Branch, Mathematic Department,Iran. 3Young Researchers Club, Mashhad Branch, Islamic Azad University, Mashhad,Iran Islamic Azad University, Iran International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 DOI : 10.5121/ijsc.2012.3103 1- Ramin.gholizadh@gmail.com 2- Zayrsil@gmail.com International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 analyzing system reliability by using interval-valued vague sets, and applied it for the reliability analysis of a Marine Power Plant. analyzing system reliability by using interval-valued vague sets, and applied it for the reliability analysis of a Marine Power Plant. Complex systems may have both kinds of uncertainty. Researchers have stated that probability theory can be used in concert with fuzzy set theory for the modeling ofcomplex systems Zadeh (1995), Barrett and Woodall (1997), Ross, Booker, and Parkinson (2003), and Singpurwalla and Booker (2004). Bayesian statistics provide a natural framework combining random and nonrandom uncertainty so fuzzy Bayesian methods are developed for the solutions of the reliability problems. Sellers and Singpurwalla (2008) addressed the reliability of multistate systems with imprecise state classification. On the other hand, Gholizadeh, Shirazi, Gildeh, and Deiri (2010), Gholizadeh, Shirazi, and Gildeh (2010), Görkemli and Ulusoy (2010), Huang, Zuo, and Sun (2006), Taheri and Zarei (2011), Viertl (2009), and Wu (2004, 2006) addressed the problem of imprecise data (approximately recorded failure time durations and number of failures) in reliability studies. Fuzzy Bayesian reliability method developed by Wu (2004, 2006).Taheri and Zarei (2011) modeled and evaluated Bayesian system reliability considering the vague environment. Our modeling approach is a generalization of Wu (2004, 2006) and Taheri and Zarei (2011). Vague environment is introduced in Section 2. Vague point estimator is represented in Section 3. In Section 4, the reliability and availability analysis via vague Bayesian method under squared error and precautionary loss functions for series system, parallel system and k-out-of-n system have been discussed is illustrated on an example. The computational procedures and examples are provided in order to clarify the theory discussed in this paper, and to give a possible insight for applying the vague set to Bayesian system reliability and availability. Conclusions and future extensions of this work are given in Section 6. 1. Introduction In the real world there are vaguely specified data values in many applications, such as sensor information. Fuzzy set theory has been proposed to handle such vagueness by generalizing the notion of membership in a set. Essentially, in a fuzzy set each element is associated with a point- value selected from the unit interval [0,1], which is termed the grade of membership in the set. A vague set, as well as an Intuitionistic fuzzy set, is a further generalization of a fuzzy set. Instead of using point-based membership as in fuzzy sets, interval-based membership isused in a vague set.Gau and Buehrer (1993) define vague sets. These sets are used by Shyi-Ming Chen and Jiann- Mean Tan (1994). Once the vague (intuitionistic fuzzy) set theory have been introduced, a number of authors investigated the reliability analysis in vague environments. Chen (2003) presented a method to analyze system reliability using the vague set theory, where the reliabilities of system components are represented by vague sets. Kumar et al. (2006) developed a method for DOI : 10.5121/ijsc.2012.3103 1- Ramin.gholizadh@gmail.com 2- Zayrsil@gmail.com 25 2. Vague environment Definition 1.A vague set in a universe of discourse is characterized by a true membership function, , and a false membership function, , as follows: ∶ →[0,1], ∶ →[0,1], and ( ) + ( ) ≤1, where ( ) is a lower bound on the grade of membership of derived from the evidence for , and ( ) is a lower bound on the grade of membership of the negation of derived from the evidence against . Suppose = { , , … }. A vague set of the universe of discourse can be represented by = ∑ [ ( ), 1 − ( )]/ , where 0 ≤ ( ) ≤1 − ( ) ≤1 and 1 ≤ ≤ . In other words, the grade of membership of is bounded to a subinterval [ ( ), 1 − ( )of [0, 1]. Thus, vague sets are a generalization of Fuzzy sets, since the grade of membership ( ) of in Definition 1 may be inexact in a vague set. We now depict a vague set in Fig. 1. Fig. 1.Membership Functions of a vague set. Fig. 1.Membership Functions of a vague set. Definition 2.The complement of a vague set is denoted by and is defined by 26 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 ( ) = ( ), 1 − ( ) = 1 − ( ). ( ) = ( ), 1 − ( ) = 1 − ( ). 1 − ( ) = 1 − ( ). Definition 3.A vague set is contained in another vague set , ⊆ , if and only if, ( ) ≤ ( ), 1 − ( ) ≤1 − ( ). ( ) ≤ ( ), 1 − ( ) ≤1 − ( ). ( ) ≤ ( ), 1 − ( ) ≤1 − ( ). Definition 4.Two vague sets and are equal, written as = , if and only if, ⊆ and ⊆ ; that is ( ) = ( ), 1 − ( ) = 1 − ( ). ( ) = ( ), 1 − ( ) = 1 − ( ). Definition 5.Let , be a vague set of . Then, we define -cuts and -cuts of as the crisp sets of given by = { ∶( ) ≥ }, ∈[0, 1], = ∶1 − ( ) ≥ , ∈[0, 1], = ∶1 − ( ) ≥ , ∈[0, 1], Definition 6.Let , be a vague set of ⊆ . We say that is convex if for all , ∈ and in [0,1], ( + (1 −) ) ≥min ( ), ( ) , 1 − ( + (1 −) ) ≥min 1 − ( ), 1 − ( ) . Definition 7.A vague set , of with continuous membership functions and is called a vague number if and only if and , for all , ∈(0, 1], are bounded closed intervals; i.e. = , = , We denote the class of all vague numbers by ( ). We denote the class of all vague numbers by ( ). Definition 8.Thevague number , is called a triangular vague number, if Definition 8.Thevague number , is called a triangular vague number, if 3. Vague point estimator With according to Taheri and Zarei (2011), Let X: Ω V( ).be a vague valued function. Then, is called to be a vague random variable if , and for all 0 ≤α , α ≤1, are (ordinary) random variables. Too, Let be a vague random variable whose distribution depends on a vague parameter . So, it could be said that , and are the parameters of distributions of (crisp) random variables , and , respectively. Our purpose is to estimate based on a Bayes approach. According to this approach, is considered as a vague random variable so that the crisp random variables , and have some distributions with parameters ( ) , … , ( ) , ( ) , … , ( ) , , … , , , … , , respectively. Concerning Definition 7, we can find the Bayes point estimator for each ∈ , . Consider ) 1 ( = { , }, { , } Then, contains all of the Bayes point estimators for each ∈ , . The same argument can be established for ∈ , . Then, contains all of the Bayes point estimators for each ∈ , . The same argument can be established for ∈ , . The vague Bayes point estimator for the vague parameter is defined to be a vague number , , where ) 2 ( ( ) = sup ∈[ , ] . ( ), 0 ≤ ≤1, ) 3 ( ( ) = sup ∈[ , ] . ( ), 0 ≤ ≤1, Definition 8.Thevague number , is called a triangular vague number, if Definition 8.Thevague number , is called a triangular vague number, if ( ) = − ( − ) ≤ ≤ − ( − ) ≤ ≤ 0 1 − ( ) = − − ≤ ≤ − − ≤ ≤ 0 27 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 Where ∈[1, ∞). We denote such a vague number by = ( , , , ) . Proposition 1.(Taheri and Zarei (2011); Resolution Identity for Vague Sets) Let , be a vague set of . Then ( ) ( ) Proposition 1.(Taheri and Zarei (2011); Resolution Identity for Vague Sets) Let , be a vague set of . Then ( ) = sup ∈[ , ] . ( ), 0 ≤ ≤1, ( ) = sup ∈[ , ] . ( ), 0 ≤ ≤1, ( ) = sup ∈[ , ] . ( ), 0 ≤ ≤1, ( ) = sup ∈[ , ] . ( ), 0 ≤ ≤1, [ ] ( ) = sup ∈[ ] . ( ), 0 ≤ ≤1, Where, (. ) is denoted for the indicator function. Where, (. ) is denoted for the indicator function. Remark 2. It is convenient to regard the Mellin transform as the moments of , i.e. Remark 2. It is convenient to regard the Mellin transform as the moments of , i.e. ( ; ) = ( ). Theorem 1. [25] Let , . . . , be independent random variables with ’s , . . . , respectively. Let ( ) be the of the random variable = ∏ . Then ( ; ) = ∏ ( ; ) . (5) With the help of Theorem1, we can obtain the posterior distribution of the system reliability from the posterior distribution of the component reliabilities. On the other hand, the Bayes estimator of the system reliability, under the squared error loss function, is the mean of the posterior distribution [21], so that we can use Eq. (4), for = 2, to obtain the Bayes estimator of the system reliability. 4. Vague Bayesian system reliability and availability In the Bayesian approach to system reliability, some prior distributions are assigned to the components of the system. Then, the posterior distribution of each component’s reliability can be found using the Bayes theorem. We can derive, therefore, the posterior distribution of the system reliability from the posterior distribution of each component reliability. The key point in this approach is that system reliability can be expressed as a product of independentrandom variables which corresponds to either component reliability (for series system) or to component unreliability (for parallel systems). To this end, we need to use the method of Mellin integral transform [2] described below. 28 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 uch a transform exists if ∫ \| ( )\| ( )is bounded for some > 0. Remark1. Based on the above definition, the inverse Mellin transform of ( ; ) is given by ( ) = ∫ ( , ) , where −∞and + ∞are complex values [2]. 4.1 Mellin transformation Definition 9. [2] Let be a non-negative random variable with pdf ( ). The Mellin transform of ,with respect to the complex parameter, is defined by ( ; ) = ∫ ( ) ( ) (4) Such a transform exists if ∫ \| ( )\| ( )is bounded for some > 0. 4.2 Bayesian approach to system reliability The fuzzy Bayesian interval estimate of reliability is calculated using the method proposed by Wu (2006). Let random variable be the th failure time of the th componentsand be the realization of this random variable, = 1, 2, . . . , , = 1, 2, . . . , . It is assumed that , = 1, 2, . . . , , are independent and identically distributed exponential random variables with parameter . is unknown; therefore, a gamma distribution with parameters and is assigned as prior distribution to quantify the uncertainty in . From previous experience we know the parameters of the prior distribution and .They can be interpreted as numbers of failures that occur in the duration for component since the expected value of gammadistribution is ⁄ . On the total operating time of , = ∑ , failures are observed for the component . Since the gamma distribution is a conjugate prior distribution for the exponential sampling distribution, the posterior distribution of is a gamma distribution with parameters ( + m )and ( + ). Reliability of the components for the duration ∗is ( ∗) = ∗and it is monotonically decreasing function of , so there is aone-to-one relationship between and . Therefore the unique inverse is λ = −ln r t∗ ⁄ so the distribution of can be determined fromthedistribution of as ( ) = ∗ = ∗ ∗, Where ( ) is either prior or posterior distribution of and ( ) is corresponding prior or posterior distribution of (Martz and Waller,1991). Assigning a gamma ( , ) prior distribution to corresponds to assigning a Negative-Log-Gamma ( , t∗ ⁄ ) priordistribution Where ( ) is either prior or posterior distribution of and ( ) is corresponding prior or posterior distribution of (Martz and Waller,1991). Assigning a gamma ( , ) prior distribution to corresponds to assigning a Negative-Log-Gamma ( , t∗ ⁄ ) priordistribution 29 29 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 to . Given the data, the posterior distribution of is a Negative-Log-Gamma ( + , ( + ) ∗ ⁄ ) distribution. Series system Consider a series system consisting of independent components. The system reliability is = ∏ .So from Eq. (5), the Mellin transform of the ( \| , )of system reliability is given by [ ( \| , ); ] = ∏ ( ) (6) [ ( \| , ); ] = ∏ ( ) (6) (6) Under a squared error loss function, the Bayes point estimate of the system reliability r isgiven as = [ \| , ] = [ ( \| , ); = 2] = ∏ (7) Under a squared error loss function, the Bayes point estimate of the system reliability r isgiven as = [ \| , ] = [ ( \| , ); = 2] = ∏ (7) Under a squared error loss function, the Bayes point estimate of the system reliability r isgiven as = [ \| , ] = [ ( \| , ); = 2] = ∏ (7) The Bayes point estimate of the system reliability under a precautionary loss function is: The Bayes point estimate of the system reliability under a precautionary loss function is: = ( [ \| , ]) = ( [ ( \| , ); = 3]) = ∏ (8) Now, suppose in a complex system the number of failures and failure times may be recorded imprecisely due to equipment and human errors. For such cases this imprecision also should be quantified in the calculations. Here vague set theory is used to quantify the uncertainty of imprecision. Failure rate, λ , reliability,r , and failure times,t , are taken as vague random variables. International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 Respectively, for all [ ]1,0 ∈  . According to the discussions in Section 3, let = { , }, { , } The above intervals contain all of the Bayes estimators for each ∈ ̃, ̃ . The same intervals can be established for ∈ ̃, ̃ . But, for α < have The above intervals contain all of the Bayes estimators for each ∈ ̃, ̃ . The same intervals can be established for ∈ ̃, ̃ . But, for α < have ̃≤̃, ̃≤̃, ̃ ≥̃, And so ̃≤̃ ≤ ̃ ≤̃. ̃≤̃, ̃≤̃, ̃ ≥̃, ̃≤̃ ≤ ̃ ≤̃. And so Therefore, and can be rewritten as Therefore, and can be rewritten as = ̃, ̃ (13) = ̃, ̃ (14) = ̃, ̃ (13) = ̃, ̃ (14) Now, using Proposition 1, the truth membership function and the false membership function of the vague Bayes estimate of ̃, denoted by ̃, are defined as follows Now, using Proposition 1, the truth membership function and the false membership function of the vague Bayes estimate of ̃, denoted by ̃, are defined as follows ̃( ) = ∈[ , ] . ̃ ( ), 0 ≤ ≤1 (15) ̃( ) = ∈[ , ] . ̃ ( ), 0 ≤ ≤1 (16) ems (15) (16) Parallel systems Series system It is assumed that prior distribution parameters are known from the previous experience so those and are taken as vague number (the parameter θ can be interpreted as the ‘pseudo’ number of and failures in a lifetime test of durationθ ‘pseudo’ time units).the Bayes point estimates of( ̃) , ( ̃) , ( ̃) and ( ̃) under a squared error loss function are given by = ∏ ( ) ( ) ( ) ( ) ( ) = ∏ ∑ ( ) ( ) ∑ ( ) ( ) ( ) , = ∏ ( ) ( ) ( ) ( ) ( ) = ∏ ∑ ( ) ( ) ∑ ( ) ( ) ( ) (9) = ∏ ( ) ( ) ( ) ( ) ( ) = ∏ ∑ ( ) ( ) ∑ ( ) ( ) ( ) (9) (9) = ( ) + ( ) ( ) + ( ) + ( ) ∑ ̃ ( ) + ( ) ∑ ̃ ( ) + ( ) + ( ) = ( ) + ( ) ( ) + ( ) + ( ) = ∑ ̃ ( ) + ( ) ∑ ̃ ( ) + ( ) + ( ) (10) (10) the Bayes point estimates of ̃ , ̃ , ̃ and ̃ under a precautionary loss function are given by 30 Parallel systems For a parallel system consisting of independent components, the system reliability is = 1 − ∏ (1 − )where is the component reliability of the th component. Equivalently, the ystemunreliability is the product of component unreliabilities = 1 − , i.e., = ∏ .The Bayes point estimate of system unreliability is given by For a parallel system consisting of independent components, the system reliability is = 1 − ∏ (1 − )where is the component reliability of the th component. Parallel systems Respectively, for all α ∈[0,1] the membership function of the vague Bayes point estimate of system reliability r is defined by the same way as discussed above. Parallel systems Equivalently, the ystemunreliability is the product of component unreliabilities = 1 − , i.e., = ∏ .The Bayes point estimate of system unreliability is given by y p y y g y [ \| , ] = [ \| , ] = 1 − + + + And And [Q \|m, ν] = E Q \|m , ν = 1 −2 ν + θ ν + θ + t + ν + θ ν + θ + 2t 31 31 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 The Bayes point estimate of the system reliability r under a squared error loss function is given by he Bayes point estimate of the system reliability r under a squared error loss function is given y The Bayes point estimate of the system reliability r under a squared error loss function is given by = [ \| , ] = [(1 − )\| , ] = 1 − [ \| , ] = 1 −∏ 1 − (17) The Bayes point estimate of the system reliability r under a precautionary loss function is given by y Under the vague assumptions as described above, the Bayes point estimates of ̃ , ̃ , ̃ and ̃ under a precautionary loss function are given by = 1 −2 1 − ( ) + ( ) ( ) + ( ) + ( ) + 1 −2 ( ) + ( ) ( ) + ( ) + ( ) ( ) + ( ) ( ) + ( ) + 2 ( ) 1 −2 1 − ( ) + ( ) ( ) + ( ) + ( ) + 1 −2 ( ) + ( ) ( ) + ( ) + ( ) ( ) + ( ) ( ) + ( ) + 2 ( ) (21) = 1 −2 1 − ( ) + ( ) ( ) + ( ) + ( ) + 1 −2 ( ) + ( ) ( ) + ( ) + ( ) ( ) + ( ) ( ) + ( ) + 2 ( ) 1 −2 1 − ( ) + ( ) ( ) + ( ) + ( ) + 1 −2 ( ) + ( ) ( ) + ( ) + ( ) ( ) + ( ) ( ) + ( ) + 2 ( ) (21) −2 ( ) + ( ) ( ) + ( ) + ( ) ( ) + ( ) ( ) + ( ) + 2 ( ) 2 1 − ( ) + ( ) ( ) + ( ) + ( ) −2 ( ) + ( ) + ( ) + ( ) + 2 1 −2 1 − ( ) + ( ) ( ) + ( ) + ( ) + 1 −2 ( ) + ( ) ( ) + ( ) + ( ) ( ) + ( ) ( ) + ( ) + 2 ( ) (21) + 1 −2 ( ) + ( ) ( ) + ( ) + ( ) ( ) + ( ) ( ) + ( ) + 2 ( ) (21) + 1 −2 ( ) + ( ) ( ) + ( ) + ( ) ( ) + ( ) ( ) + ( ) + 2 ( ) (21) (21) 32 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 Respectively, for all α ∈[0,1] the membership function of the vague Bayes point estimate of system reliability r is defined by the same way as discussed above. k-out-of-m system For a k-out-of-m system consisting of m independent and identical components, the system reliability is given by = ̅(1 −̅) That ̅is the reliability of the (common) component. The Bayes point estimate of system reliability r is the mean E[R\| , ] under the squared error loss function, and is given by = [ \| , ] = ̅(1 −̅) . k-out-of-m system ( ̅\| , ) = ∑ ∑ (−1) (23) The Bayes point estimate of system reliability at time , under a squared loss function, given by = ( [ \| , ]) = ∑ ∑ ∑ (−1) ( ) ( ) (24) Under the vague assumptions as described above, the Bayes point estimates of ( ̃) , ( ̃) , ( ̃) and( ̃) under a squared error loss function are given by Under the vague assumptions as described above, the Bayes point estimates of ( ̃) , ( ̃) , ( ̃) and( ̃) under a squared error loss function are given by ̃ = (−1) − + ( ) + + + ( ) ( ) ̃ = (−1) ( ) + + + ( ) ̃ = (−1) − + ( ) + + + ( ) ( ) (25) ̃ = (−1) − + ( ) + + + ( ) ( ) ̃ = (−1) − + ( ) + + + ( ) ( ) (26) (25) (26) 33 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 nder the vague assumptions as described above, the Bayes point estimates of ̃ , ̃ Under the vague assumptions as described above, the Bayes point estimates of ̃ , ̃ , ̃ and ̃ under a precautionary loss function are given by = (−1) − 2 −( + ) + ( ) + + + + ( ) ( ) ( ) = (−1) − 2 −( + ) + ( ) + + + + ( ) ( ) ( ) (27) ( ) ( ) = (−1) − 2 −( + ) + ( ) + + + + ( ) ( ) ( ) = (−1) − 2 −( + ) + ( ) + + + + ( ) ( ) ( ) (27) = (−1) − 2 −( + ) + ( ) + + + + ( ) ( ) ( ) = (−1) − 2 −( + ) + ( ) + + + + ( ) ( ) (28) ( ) (27) (28) Respectively, for all α ∈[0,1] the membership function of the vague Bayes point estimate of system reliability is defined by the same way as discussed above. 4.3 Bayesian approach to system availability Martz and Waller, (1991) presented the method to calculate the availability of a repairable system = ( ) ( ) + ( ) = + (29) (29) That X is the failure time and Y is the repair time. Assume X and Y are independent variables and have exponential distributions with parameters λ and μ respectively. Separate gamma distributions are assigned as prior distributions to λ and μ. To determine the availability of the system, the parameters of exponential distribution should be estimated. Vague Bayesian estimations under the squared error loss function by use Wu (2004) are given by = ( ) ( ) ( ) (30) = ( ) ( ) ( ) (31) = ( ) ( ) ( ) , = ( ) ( ) ( ) (30) = ( ) ( ) ( ) , = ( ) ( ) ( ) (31) (30) (31) (31) Vague Bayesian estimations under the precautionary loss function are given by = ( ) ( ) ( ) ( ) , = ( ) ( ) ( ) ( ) (32) = ( ) ( ) ( ) ( ) , = ( ) ( ) ( ) ( ) (33) (32) (33) 34 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 and are the prior distribution parameters of ; they are assumed to be known from previous experience.For the parameter of repair time distribution, vague Bayes point estimators under the squared error loss function are given in (34) and (35) = ( ) ( ) ( ) , = ( ) ( ) ( ) (34) = ( ) ( ) ( ) , = ( ) ( ) ( ) (35) (35) Vague Bayesian estimations under the precautionary loss function are given by = ( ) ( ) ( ) ( ) , = ( ) ( ) ( ) ( ) (36) = ( ) ( ) ( ) ( ) , = ( ) ( ) ( ) ( ) (37) (36) (37) and are the prior distribution parameters of ; they are assumed to be known from previous experience. 4.3 Bayesian approach to system availability Vague Bayes point estimators under the squared error loss function of availability interval bounds , , and are given by Vague Bayes point estimators under the squared error loss function of availability interval bounds , , and are given by Vague Bayes point estimators under the squared error loss function of availability interval bounds , , and are given by = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) , ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) (38) = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) , = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) (39) = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) , ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) (38) (38) = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) , = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) (39) = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) , = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) (39) (39) Vague Bayes point estimators under the precautionary loss function of availability interval bounds , , and are given by Vague Bayes point estimators under the precautionary loss function of availability interval bounds , , and are given by = ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ( ) = ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ( ) (40) (40) 35 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 = ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ( ) = ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ( ) (41) The availability of a series system consisting of independent components can be calculated with Eq. 4.3 Bayesian approach to system availability (42) (Martz and Waller, 1991) = ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ( ) = ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ( ) (41) The availability of a series system consisting of independent components can be calculated with Eq. (42) (Martz and Waller, 1991) = ∏ (42) = ∏ (42) (42) Inserting the vague Bayesian estimates of failure and repair rates into (42), vague Bayesian availability equations of a series system under squared error loss functions are given in (43) and (44) respectively = ∏ , = ∏ (43) = ∏ , = ∏ (44) (44) Vague Bayesian availability equations of a series system under precautionary loss functions are given by e Bayesian availability equations of a series system under precautionary loss functions are n by Vague Bayesian availability equations of a series system under precautionary loss functions are given by = ∏ , = ∏ (45) = ∏ , = ∏ (46) (46) Respectively, for all [ ]1,0 ∈  . According to the discussions in Section 3, let = { , }, { , } The above intervals contain all of the Bayes estimators for each ∈ , . The same intervals can be established for ∈ , . But, for α < have The above intervals contain all of the Bayes estimators for each ∈ , . The same intervals can be established for ∈ , . But, for α < have ≤ , ≤ , ≥ , ≤ , ≤ , ≥ , 36 36 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 And so And so ≤ ≤ ≤ . ≤ ≤ ≤ . ≤ ≤ ≤ . Therefore, and can be rewritten as Therefore, and can be rewritten as = , (47) = , (48) (47) Now, using Proposition 1, the truth membership function and the false membership function of the vague Bayes estimate of , denoted by , are defined as follows Now, using Proposition 1, the truth membership function and the false membership function of the vague Bayes estimate of , denoted by , are defined as follows ( ) = ∈[ , ] . ( ), 0 ≤ ≤1 (49) ( ) = ∈[ , ] . ( ), 0 ≤ ≤1 (49) ( ) = ∈[ , ] . ( ), 0 ≤ ≤1 (50) (49) ( ) = ∈[ , ] . ( ), 0 ≤ ≤1 (50) (50) The availability of a parallel system consisting of independent components can be calculated with Eqs. (51)– (53) (Martz and Waller, 1991) = ̅= 1 − (51) ̅= + (52) = 1 − + (53) (51) = 1 − + (53) (53) In the same way, inserting the vague Bayesian estimates of failure and repair rate into (50), Vague Bayesian availability equations of a parallel system under squared error loss functions are given in (53) and (54) respectively = 1 −∏ , = 1 −∏ (54) = 1 −∏ , = 1 −∏ (54) = 1 −∏ , = 1 −∏ (55) = 1 −∏ , = 1 −∏ (54) = 1 −∏ , = 1 −∏ (55) = 1 −∏ , = 1 −∏ (55) Vague Bayesian availability equations of a parallel system under precautionary loss functions are given by g y = 1 −∏ , = 1 −∏ (56) = 1 −∏ , = 1 −∏ (57) Respectively, for all α ∈[0,1] the membership function of the vague Bayes point estimate of system availability is defined by the same way as discussed above. Respectively, for all α ∈[0,1] the membership function of the vague Bayes point estimate of system availability is defined by the same way as discussed above. And so 37 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 For a k-out-of-m system consisting of m independent and identical components, the system availability is given by For a k-out-of-m system consisting of m independent and identical components, the system availability is given by = ∑ (1 − ) (58) = ∑ (59) = ∑ (59) Vague Bayesian availability equations of a -out-of- system under squared error loss functions is given by Vague Bayesian availability equations of a -out-of- system under squared error loss functions is given by g y Vague Bayesian availability equations of a parallel system under precautionary loss functions are given by Vague Bayesian availability equations of a parallel system under precautionary loss functions are given by 4 Computational procedures and example 4 Computational procedures and example we need to apply some computational techniques for evaluating the truth and false membership degrees of the vague Bayes point estimate as presented in Eq. (2) and Eq. (3) will be provided. From Eq. (1), we adopt the following notations p p p we need to apply some computational techniques for evaluating the truth and false membership degrees of the vague Bayes point estimate as presented in Eq. (2) and Eq. (3) will be provided. From Eq. (1), we adopt the following notations = [ ( ), ( )] = [ { ( ), ( )}, { ( ), ( )}] (64) = , = , , , (65) (64) Where Where ( ) = inf , ( ) = inf , ( ) = sup , ( ) = sup , = inf , = inf , = sup , = sup , From Eq. (1), the truth and false membership functions of the vague Bayes estimate , are give by From Eq. (1), the truth and false membership functions of the vague Bayes estimate , are given by ( ) = sup ∈[ , ] . ( ) = { : ( ) ≤ ≤ ( ), 0 ≤ ≤1 }, (66) ( ) = sup ∈[ , ] . ( ) = { : ( ) ≤ ≤ ( ), 0 ≤ ≤1 }, (66) (66) ( ) = sup ∈[ , ] . ( ) = : ≤ ≤ , 0 ≤ ≤1 . And so (67) (67) [ , ] Therefore, needs to be solved the following type of nonlinear programming problem 38 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 max min{ ( ), ( )} ≤ m { ( ), ( )} ≥ 0 ≤ ≤1 max min , ≤ m , ≥ 0 ≤ ≤1 Now, the following eight sub-problems have been considered Now, the following eight sub-problems have been considered Let and ∗be the objective values of subproblems − , and ∗, ∗, ∗ and ∗be the bj i l f b bl i l h Let and ∗be the objective values of subproblems − , and ∗, ∗, ∗ and ∗be the objective values of subproblems − , respectively. Assume that, objective values of subproblems − , respectively. Assume that, objective values of subproblems − , respectively. Assume that, j p p y ∗= ∗, ∗, ∗, ∗ and ∗= ∗, ∗, ∗, ∗ . Let ∗and ∗be the objective values of the original problems with optimal solution ∗and ∗respectively (in fact ∗= ∗, ∗= ∗). In according to Wu (2004), we have ∗= ∗and ∗= ∗, so to obtain the objective value ∗and ∗of the original nonlinear programming problems, it will be enough to just solve the eight sub-problems − and − . ∗= ∗, ∗, ∗, ∗ and ∗= ∗, ∗, ∗, ∗ . Let ∗and ∗be the objective values of the original problems with optimal solution ∗and ∗respectively (in fa ∗= ∗, ∗, ∗, ∗ and ∗= ∗, ∗, ∗, ∗ . Let ∗and ∗be the objective values of the original problems with optimal solution ∗and ∗respectively (in fact ∗= ∗, ∗= ∗). In according to Wu (2004), we have ∗= ∗and ∗= ∗, so to obtain the objective value ∗and ∗of the original nonlinear programming problems, it will be enough to just solve the eight sub problems − and − j g p p p y ∗= ∗, ∗= ∗). In according to Wu (2004), we have ∗= ∗and ∗= ∗, so to obtain the objective value ∗and ∗of the original nonlinear programming problems, it will enough to just solve the eight sub-problems − and − . Example 5.1. We consider a series-parallel system consisting of three independent components that have Exponential distributions, that structure as shown in Fig. 1. Fig. 2. The series–parallel system Fig. 2. And so The series–parallel system The follow data show that the failure times and repair times are assumed as vague numbers, since the failure times and repair times cannot be recorded precisely due to human errors, machine errors, or some unexpected situations. The simulated failure times and repair times are presented in Table 1. For example component 1, the first failure time and repair time are around 20 and 15 h, respectively.the second failure time and repair time are around30 and 20h. There are the same explanations for components 2 and 3. The prior distribution parameters (pseudo) of the failure rate and repair rate are presented in Table 2. 39 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 Where 3 , 4 , 5 , 6 , 8, 10 , 15 , 20 , 25, 30, 35 , 45and50are vague numbers with the following membership functions International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 Where 3 , 4 , 5 , 6 , 8, 10 , 15 , 20 , 25, 30, 35 , 45and50are vague numbers with the following membership functions ( ) = −1 4 1 ≤ ≤3 5 − 4 3 < ≤5 , 1 − ( ) = −1 2 1 ≤ ≤3 5 − 2 3 < ≤5 ( ) = −2 4 2 ≤ ≤4 6 − 4 4 < ≤6 , 1 − ( ) = −2 2 2 ≤ ≤4 6 − 2 4 < ≤6 ( ) = −3 4 3 ≤ ≤5 7 − 4 5 < ≤7 , 1 − ( ) = −3 2 3 ≤ ≤5 7 − 2 5 < ≤7 Now, we want to obtain the vague Bayesian system reliability and availability. Now, we want to obtain the vague Bayesian system reliability and availability. First of all, note that the -cuts and -cuts of the vague numbers3 ,4, 5 , 6 , 8, 10 , 15 , 20 , 25, 30 , 35 , 45and50 are And so First of all, note that the -cuts and -cuts of the vague numbers3 ,4, 5 , 6 , 8, 10 , 15 , 20 , 25, 30 , 35 , 45and50 are ( ) = −1 4 1 ≤ ≤3 5 − 4 3 < ≤5 , 1 − ( ) = −1 2 1 ≤ ≤3 5 − 2 3 < ≤5 ( ) = −2 4 2 ≤ ≤4 6 − 4 4 < ≤6 , 1 − ( ) = −2 2 2 ≤ ≤4 6 − 2 4 < ≤6 ( ) = −3 4 3 ≤ ≤5 7 − 4 5 < ≤7 , 1 − ( ) = −3 2 3 ≤ ≤5 7 − 2 5 < ≤7 Now, we want to obtain the vague Bayesian system reliability and availability. First of all, note that the -cuts and -cuts of the vague numbers3 ,4, 5 , 6 , 8, 10 , 15 , 20 , 25, 30 , 35 , 45and50 are Now, we want to obtain the vague Bayesian system reliability and availability. First of all, note that the -cuts and -cuts of the vague numbers3 ,4, 5 , 6 , 8, 10 , 15 , 20 , 25, 30 , 35 , 45and50 are 40 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 The vague Bayes point estimates of reliability under different loss functions of ̃, ̃, ̃and ̃ are obtained as follows (for all , ∈[0, 1]) The vague Bayes point estimates of reliability under different loss functions of ̃, ̃, ̃and ̃ are obtained as follows (for all , ∈[0, 1]) ( , [ , ]) ̃ = (15 + 10 ) + (25 + 10 ) + 140 (15 + 10 ) + (25 + 10 ) + 140 + 30 ( ) 1 −1 − (45 + 10 ) + (30 + 10 ) + 120 (45 + 10 ) + (30 + 10 ) + 120 + 30 ( ) 1 − (40 + 10 ) + (30 + 10 ) + 180 (40 + 10 ) + (30 + 10 ) + 180 + 30 2+(6+4 ) = 180 + 20 220 + 20 6+4 1 − 30 225 + 20 5+4 30 280 + 20 8+4 ̃ s = (25 −10 ) + (35 −10 ) + 140 (25 −10 ) + (35 −10 ) + 140 + 30 ( ) 1 − 1 − (55 −10 ) + (40 −10 ) + 120 (55 −10 ) + (40 −10 ) + 120 + 30 ( ) 1 − (50 −10 ) + (40 −10 ) + 180 (50 −10 ) + (40 −10 ) + 180 + 30 ( ) = 220 −20α 250 −20α 1 − 30 245 −20α 30 300 −20α ̃ s = 15 + 5 + 25 + 5 + 140 15 + 5 + 25 + 5 + 140 + 30 1 − 1 − 45 + 5 + 30 + 5 + 120 45 + 5 + 30 + 5 + 120 + 30 1 − (40 + 5α ) + (30 + 5α ) + 180 (40 + 5α ) + (30 + 5α ) + 180 + 30 ( ) = 180 + 10α 220 + 20α 1 − 30 225 + 10α 30 280 + 10α ̃ s = 25 −5 + 35 −5 + 140 25 −5 + 35 −5 + 140 + 30 1 − 1 − 55 −5 + 40 −5 + 120 55 −5 + 40 −5 + 120 + 30 ( , [ , ]) ̃ = (15 + 10 ) + (25 + 10 ) + 140 (15 + 10 ) + (25 + 10 ) + 140 + 30 ( ) 1 −1 − (45 + 10 ) + (30 + 10 ) + 120 (45 + 10 ) + (30 + 10 ) + 120 + 30 ( ) 1 − (40 + 10 ) + (30 + 10 ) + 180 (40 + 10 ) + (30 + 10 ) + 180 + 30 2+(6+4 ) = 180 + 20 220 + 20 6+4 1 − 30 225 + 20 5+4 30 280 + 20 8+4 ̃ s = (25 −10 ) + (35 −10 ) + 140 (25 −10 ) + (35 −10 ) + 140 + 30 ( ) 1 − 1 − (55 −10 ) + (40 −10 ) + 120 (55 −10 ) + (40 −10 ) + 120 + 30 ( ) 1 − (50 −10 ) + (40 −10 ) + 180 (50 −10 ) + (40 −10 ) + 180 + 30 ( ) = 220 −20α 250 −20α 1 − 30 245 −20α 30 300 −20α ̃ s = 15 + 5 + 25 + 5 + 140 15 + 5 + 25 + 5 + 140 + 30 1 − 1 − 45 + 5 + 30 + 5 + 120 45 + 5 + 30 + 5 + 120 + 30 1 − (40 + 5α ) + (30 + 5α ) + 180 (40 + 5α ) + (30 + 5α ) + 180 + 30 ( ) = 180 + 10α 220 + 20α 1 − 30 225 + 10α 30 280 + 10α ̃ s = 25 −5 + 35 −5 + 140 25 −5 + 35 −5 + 140 + 30 1 − 1 − 55 −5 + 40 −5 + 120 55 −5 + 40 −5 + 120 + 30 ̃ = (15 + 10 ) + (25 + 10 ) + 140 (15 + 10 ) + (25 + 10 ) + 140 + 30 ( ) 1 −1 − (45 + 10 ) + (30 + 10 ) + 120 (45 + 10 ) + (30 + 10 ) + 120 + 30 ( ) ̃ = (15 + 10 ) + (25 + 10 ) + 140 (15 + 10 ) + (25 + 10 ) + 140 + 30 ( ) 1 −1 − (45 + 10 ) + (30 + 10 ) + 120 (45 + 10 ) + (30 + 10 ) + 120 + 30 ( ) 1 − (40 + 10 ) + (30 + 10 ) + 180 (40 + 10 ) + (30 + 10 ) + 180 + 30 2+(6+4 ) = 180 + 20 220 + 20 6+4 1 − 30 225 + 20 5+4 30 280 + 20 8+4 (25 −10 ) + (35 −10 ) + 140 ( ) 1 1 − (50 −10 ) + (40 −10 ) + 180 (50 −10 ) + (40 −10 ) + 180 + 30 ( ) = 220 −20α 250 −20α 1 − 30 245 −20α 30 300 −20α ̃ s = 15 + 5 + 25 + 5 + 140 15 + 5 + 25 + 5 + 140 + 30 1 + 5α ) + (30 + 5α ) + 180 5α ) + (30 + 5α ) + 180 + 30 ( ) = 180 + 10α 220 + 20α 1 − 30 225 + 10α 30 280 + 10α ̃ s = 25 −5 + 35 −5 + 140 25 −5 + 35 −5 + 140 + 30 1 41 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 1 − 50 −10 + 40 −10 + 180 50 −10 + 40 −10 + 180 + 30 = 220 −10 250 −10 1 − 30 245 −10 30 300 −10 And ̃ = (180 + 20 ) (220 + 20 ) 1 −2 30 225 + 20 30 280 + 20 + 1 −2 195 + 20 225 + 20 195 + 20 255 + 20 1 −2 250 + 20 280 + 20 250 + 20 310 + 20 ̃ = (220 −20 ) (220 + 20 ) 1 −2 30 245 −20 30 300 −20 + 1 −2 215 −20 245 −20 215 −20 275 −20 1 −2 270 −20 300 −20 270 −20 330 −20 ̃ = 180 + 10 220 + 10 1 −2 30 225 + 10 30 280 + 10 + 1 −2 195 + 10 225 + 10 195 + 10 255 + 10 1 −2 250 + 10 280 + 10 250 + 10 310 + 10 ̃ = 220 −10 220 + 10 1 −2 30 245 −10 30 300 −10 + 1 −2 215 −10 245 −10 215 −10 275 −10 1 −2 270 −10 300 −10 270 −10 330 −10 The vague Bayes point estimates of availability under different loss functions of , , and are obtained as follows (for all , ∈[0, 1]) 1 − 50 −10 + 40 −10 + 180 50 −10 + 40 −10 + 180 + 30 = 220 −10 250 −10 1 − 30 245 −10 30 300 −10 And ̃ = (180 + 20 ) (220 + 20 ) 1 −2 30 225 + 20 30 280 + 20 + 1 −2 195 + 20 225 + 20 195 + 20 255 + 20 1 −2 250 + 20 280 + 20 250 + 20 310 + 20 ̃ = (220 −20 ) (220 + 20 ) 1 −2 30 245 −20 30 300 −20 + 1 −2 215 −20 245 −20 215 −20 275 −20 1 −2 270 −20 300 −20 270 −20 330 −20 ̃ = 180 + 10 220 + 10 1 −2 30 225 + 10 30 280 + 10 + 1 −2 195 + 10 225 + 10 195 + 10 255 + 10 1 −2 250 + 10 280 + 10 250 + 10 310 + 10 ̃ = 220 −10 220 + 10 1 −2 30 245 −10 30 300 −10 + 1 −2 215 −10 245 −10 215 −10 275 −10 1 −2 270 −10 300 −10 270 −10 330 −10 The vague Bayes point estimates of availability under different loss functions of , , and are obtained as follows (for all , ∈[0, 1]) And And And ̃ = (180 + 20 ) (220 + 20 ) 1 −2 30 225 + 20 30 280 + 20 + 1 −2 195 + 20 225 + 20 195 + 20 255 + 20 1 −2 250 + 20 280 + 20 250 + 20 310 + 20 ̃ = (220 −20 ) (220 + 20 ) 1 −2 30 245 −20 30 300 −20 + 1 −2 215 −20 245 −20 215 −20 275 −20 1 −2 270 −20 300 −20 270 −20 330 −20 And ̃ = (180 + 20 ) (220 + 20 ) 1 −2 30 225 + 20 30 280 + 20 + 1 −2 195 + 20 225 + 20 195 + 20 255 + 20 1 −2 250 + 20 280 + 20 250 + 20 310 + 20 ̃ = (220 −20 ) (220 + 20 ) 1 −2 30 245 −20 30 300 −20 + 1 −2 215 −20 245 −20 215 −20 275 −20 1 −2 270 −20 300 −20 270 −20 330 −20 ̃ = 180 + 10 220 + 10 1 −2 30 225 + 10 30 280 + 10 + 1 −2 195 + 10 225 + 10 195 + 10 255 + 10 1 −2 250 + 10 280 + 10 250 + 10 310 + 10 ̃ = 220 −10 220 + 10 1 −2 30 245 −10 30 300 −10 + 1 −2 215 −10 245 −10 215 −10 275 −10 1 −2 270 −10 300 −10 270 −10 330 −10 ̃ = 180 + 10 220 + 10 1 −2 30 225 + 10 30 280 + 10 + 1 −2 195 + 10 225 + 10 195 + 10 255 + 10 1 −2 250 + 10 280 + 10 250 + 10 310 + 10 The vague Bayes point estimates of availability under different loss functions of , , are obtained as follows (for all , ∈[0, 1]) The vague Bayes point estimates of availability under different loss functions of , , and are obtained as follows (for all , ∈[0, 1]) The vague Bayes point estimates of availability under different loss functions of , , and are obtained as follows (for all , ∈[0, 1]) A = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ∗{1 − ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) = + 1 − + + A = ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ∗{1 − ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) + ( ) ( ) ( ) = + 1 − + + A = + ∗{1 − 42 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 + + = + 1 − + + A = + ∗{1 − ( ) + + = + 1 − + + And A = ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) ∗ 1 − ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) A = ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) ∗ 1 − ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) A = + ∗ 1 − + + A = + ∗ 1 − + + International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 + + = + 1 − + + A = + ∗{1 − ( ) + + = + 1 − + + And A = ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) ∗ 1 − ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) A = ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) ∗ 1 − ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) ( )( ) ( ) ( )( ) ( ) + ( )( ) ( ) A = + ∗ 1 − + + A = + ∗ 1 − + + Therefore, we just need to solve subproblems and .We use the method provided in 5 to solve these problems. 6. Conclusion We developed Bayesian approach to system reliability analysis in the vague environment, Taheri and Zarei (2011), for system availability under different loss function based on Exponential distribution. In order to evaluate the truth and false membership degrees of the vague Bayes estimate, a nonlinear programming problem was solved. The vague Bayesian approach to system reliability and availability is a generalized version of the fuzzy Bayesian reliability and availability, and it is also an extension of the conventional Bayesian system reliability and availability. For future research, distributions other than exponential distribution can be used for failure and repair times of system. In addition to vague data in the analysis, vague multi state reli- reliability can be considered. International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 43 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 (i) if < 0.21687, then we solve the following problem, using a suitable software (LINGO): ( ) (i) if < 0.21687, then we solve the following problem, using a suitable software (LINGO): ( ) = ∈[0, 1]: ( ) = ( ) = ̃ = 180 + 20 220 + 20 1 − 30 225 + 20 30 280 + 20 ≤ 1 ̃( ) = ∈[0, 1]: = = ̃ = 180 + 10α 220 + 20α 1 − 30 225 + 10α 30 280 + 10α ≤ = ∈[0, 1]: = = ̃ = 180 + 10α 220 + 20α 1 − 30 225 + 10α 30 280 + 10α ≤ (ii) if > 0.21687, then we solve the following problem: (ii) if > 0.21687, then we solve the following problem: (ii) if > 0.21687, then we solve the following problem: (ii) if > 0.21687, then we solve the following problem: (ii) ̃( ) = ∈[0, 1]: ( ) = ( ) = = 180 + 20 220 + 20 1 − 30 225 + 20 30 280 + 20 ≥ 1 − ̃( ) = ∈[0, 1]: = = = 180 + 20 220 + 20 1 − 30 225 + 20 30 280 + 20 ≥ Therefore we now can obtain the membership degree for any given Bayes point estimate of vague system reliability ̃by evaluating the above formulas. Above conclusion is defined by the same way for system availability. International Journal on Soft Computing ( IJSC ) Vol.3, No.1, February 2012 For = 0.5yields ̃ . = 1 − = 0.21687 = ̃ . , i.e. ̃(0.21687) = 0.5. on the other hand, α = 1 yields r = 0.21687 = r , i.e. 1 −f (0.21687) = 1. Sinc V = [0.27851,0.29998], we are just interested in considering the system reliability ∈ applying the Supplemental Procedure Wu (2004) we have for ∈ Therefore, we just need to solve subproblems and .We use the method provided in Section 5 to solve these problems. For = 0.5yields ̃ . = 1 − = 0.21687 = ̃ . , i.e. ̃(0.21687) = 0.5. on . the other hand, α = 1 yields r = 0.21687 = r , i.e. 1 −f (0.21687) = 1. Since V = [0.27851,0.29998], we are just interested in considering the system reliability ∈ . By applying the Supplemental Procedure Wu (2004), we have, for ∈ . [4] Gau W.L. and Buehrer D.J., Vague sets, IEEE Trans. Systems Man Cybernet. 23(2) (1993) 610-614. [3] Chen S. M. and Tan J. 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[8] Huang H., Zuo M. J. & Sun Z., Bayesian reliability analysis for fuzzy lifetime data. Fuzzy Sets and Systems, 157(12) (2006), 1674–1686. [9] Kumar A., Yadav P.S. and Kumar S., Fuzzy reliability of a marine power plant using interval valued vague sets, International Journal of Applied Science and Engineering, 4 (2006) 71–82. [10] Martz HF, Waller RA, Bayesian reliability analysis. Wiley, New York (1982). [11] Ross, T. J., Booker, J. M., & Parkinson, W. J. (2003). Fuzzy logic and probability applications bridging the gap. Philadelphia: ASA-SIAM. [12] Sellers K. F. &Singpurwalla N. D., Many-valued logic in multistate and vague stochastic systems. International Statistical Review, 76(2) (2008), 247–267. [13] Singpurwalla N. D. & Booker J. B., Membership functions and probability measures sets. Journal of the American Statistical Association, 99(467) (2004), 867–877. [14] Springer MD, Thompson WE. The distribution of products of independent random variables. 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https://openalex.org/W2886326894	https://pressto.amu.edu.pl/index.php/rpeis/article/download/12509/12884	Polish	null	Rzeczpospolita Polska dobrem wspólnym wszystkich obywateli	Ruch Prawniczy, Ekonomiczny i Socjologiczny	2,018	cc-by	5,012	2 Zob. wyczerpujące opracowanie na temat zasad podstawowych w Konstytucji z 1997 r. Za- sady podstawowe Polskiej Konstytucji, Warszawa 1998. Co charakterystyczne, w opracowaniu tym nie omawia się zasady zawartej w art. 1 Konstytucji. * Artykuł za zgodą Wydawcy opiera się na tekście autora Rzeczpospolita Polska dobrem wspólnym wszystkich obywateli, który ukazał się w pracy zbiorowej Sądownictwo administracyjne gwarantem wolności i praw obywatelskich 1980–2005, pod red. J. Górala, R. Hausera, J. Trzciń- skiego, Wyd. NSA, Warszawa, s. 452-460. RUCH PRAWNICZY, EKONOMICZNY I SOCJOLOGICZNY Rok LXXX – zeszyt 1 – 2018 RUCH PRAWNICZY, EKONOMICZNY I SOCJOLOGICZNY Rok LXXX – zeszyt 1 – 2018 1 Zob. np. R. Sowiński, Interes publiczny – dobro wspólne. Wartości uniwersalne jako kate- gorie kształtujące pojęcie administracji, w: Z. Niewiadomski, Z. Cieślak (red.), Prawo do dobrej administracji, Warszawa 2003. wspólnym wszystkich obywateli, który ukazał się w pracy zbiorowej Sądownictwo administracyjne gwarantem wolności i praw obywatelskich 1980–2005, pod red. J. Górala, R. Hausera, J. Trzciń- skiego, Wyd. NSA, Warszawa, s. 452-460. 1 Zob. np. R. Sowiński, Interes publiczny – dobro wspólne. Wartości uniwersalne jako kate- gorie kształtujące pojęcie administracji, w: Z. Niewiadomski, Z. Cieślak (red.), Prawo do dobrej administracji, Warszawa 2003. 2 Zob. wyczerpujące opracowanie na temat zasad podstawowych w Konstytucji z 1997 r. Za- sady podstawowe Polskiej Konstytucji, Warszawa 1998. Co charakterystyczne, w opracowaniu tym nie omawia się zasady zawartej w art 1 Konstytucji 3 Literatura prawnicza dotycząca zagadnienia art. 1 Konstytucji, a więc Rzeczypospolitej Polskiej jako dobra wspólnego wszystkich obywateli, jest jak dotąd bardzo uboga. Poza wzmian- kami na ten temat w podręcznikach do nauki prawa konstytucyjnego można by wskazać następu- jące pozycje: „Rzeczpospolita dobrem wspólnym wszystkich obywateli” (wystąpienie Prezydenta Aleksandra Kwaśniewskiego w Trybunale Konstytucyjnym), „Państwo i Prawo” 2005, z. 6; B. Le- waszkiewicz-Petrykowska, Dobro wspólne versus konstytucyjne prawa i wolności jednostki, w: Konferencja Trybunału Konstytucyjnego Rzeczypospolitej Polskiej i Sądu Konstytucyjnego Repub- liki Litewskiej (Śreniawa 26–29.VI.2002), „Studia i Materiały” 17, 2003; A. Piechowiak, Filozoficz- ne podstawy rozumienia „dobra wspólnego”, „Kwartalnik Filozoficzny” 2003, nr 2; R. Sowiński, op. cit.; A. Szpor, Odwaga służenia dobru wspólnemu, w: Z. Niewiadomski, Z. Cieślak (red.), op. cit. RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* I. Niniejsze opracowanie jest wkładem do dyskusji na temat normatyw- nego znaczenia art. 1 Konstytucji i propozycją metodologii badań nad konsty- tucyjną zasadą, która głosi, że „Rzeczpospolita Polska jest dobrem wspólnym wszystkich obywateli”. Dotychczasowy punkt wyjścia w dyskusji na ten temat jest błędny przede wszystkim dlatego, że rozważa się pojęcie dobra wspólnego w kontekście pojęć synonimicznych, takich jak dobro ogółu, interes społeczny, interes publiczny, bez uwagi, że art. 1 Konstytucji pojęcie dobra wspólnego łączy z państwem, inaczej mówiąc, że art. 1 państwo nazywa dobrem wspólnym. I po drugie dla- tego, że próbuje się za pomocą pojęć ustawowych1 opisać treść pojęcia konsty- tucyjnego, choć wiadomo, że pojęcia konstytucyjne mają autonomiczny sens i wobec ustaw nadrzędny. Artykuł 1 Konstytucji RP, który stanowi, że „Rzeczpospolita Polska jest wspólnym dobrem wszystkich obywateli” zamieszczony w rozdz. I: Rzecz- pospolita, wyraża podstawową zasadę ustroju naszego państwa. Jest on po- mieszczony wśród takich zasad, jak: zasada państwa prawnego (art. 2), zasada zwierzchnictwa narodu (art. 4), zasada legalności i praworządności (art. 7), zasada, według której Konstytucja jest najwyższym prawem i że jej przepi- sy są bezpośrednio stosowane (art. 8). Wyliczone tu zasady nie wyczerpują wszystkich zawartych w rozdz. I Konstytucji2. Owo wyliczenie jest potrzebne, aby powiedzieć, że treść normatywna wszystkich spośród wymienionych zasad została w literaturze prawa konsty- tucyjnego opisana albo dlatego, że sama zasada została w miarę jasno wyrażo- na w Konstytucji (np. art. 7), albo dlatego, że orzecznictwo Trybunału Konsty- tucyjnego, Sądu Najwyższego i Naczelnego Sądu Administracyjnego – pozwo- liło opisać treść tej zasady (np. art. 2 czy art. 8 Konstytucji). 24 Janusz Trzciński Rzecz przedstawia się zgoła inaczej, gdy idzie o art. 1 Konstytucji. Nie ma dotąd ani w ramach prawa konstytucyjnego3, ani w orzecznictwie sądowym prób zdekodowania treści normatywnej zasady w nim wyrażonej. Zakładam jednak, że tak jak inne zasady ustrojowe wyrażone w naszej Konstytucji także zasada, według której Rzeczpospolita jest dobrem wspólnym wszystkich obywateli, ma swoją treść, i że art. 1 Konstytucji ma określony wa- lor prawny, i to nie tylko dlatego, że jest zamieszczony w tekście Konstytucji.i Przy braku definicji legalnej pojęcia „dobro wspólne” punktem wyjścia od- powiedzi na pytanie o treść zasady zawartej w art. 1 musi być analiza także innych przepisów Konstytucji. Otóż obok art. RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* 1 pojęcie dobra wspólnego w kontekście Rzeczypospolitej użyte jest dwukrotnie we wstępie do Konstytucji: w zdaniu, że „wszyscy oby- watele Rzeczypospolitej […] równi w prawach i w powinnościach wobec dobra wspólnego – Polski […] ustanawiamy Konstytucję Rzeczypospolitej Polskiej” oraz w zdaniu „Wszystkich, którzy dla dobra Trzeciej Rzeczypospolitej tę Kon- stytucję będą stosowali, wzywamy, aby czynili to, dbając o zachowanie przy- rodzonej godności człowieka, jego prawa do wolności i obowiązku solidarności z innymi, a poszanowanie tych zasad mieli za niewzruszoną podstawę Rze- czypospolitej Polskiej”, a także w art. 25 ust. 3 Konstytucji: „Stosunki między państwem a kościołami i innymi związkami wyznaniowymi są kształtowane na zasadach poszanowania ich autonomii oraz wzajemnej niezależności każ- dego w swoim zakresie, jak również współdziałania dla dobra człowieka i do- bra wspólnego” oraz w art. 82 Konstytucji: „obowiązkiem obywatela polskiego jest wierność Rzeczypospolitej Polskiej oraz troska o dobro wspólne”. Cytowa- ne przepisy Konstytucji użyte razem nie ułatwiają zdekodowania treści i zna- czenia prawnego art. 1 Konstytucji, choć pewne refleksje w oczywisty sposób się nasuwają: a) dobrem wspólnym jest Rzeczpospolita Polska, Polska – a więc państwo polskie. Ta konstatacja jest ważna dla propozycji badań nad zagadnieniem za- wartym w art. 1 Konstytucji, która musi uwzględniać konstytucyjną koncepcję państwa, b) dobro wspólne jako wartość konstytucyjna ujęte jest w Konstytucji w ob- szarze zasad ustroju państwa oraz w obszarze wolności, praw i obowiązków jednostki i ma służyć wszystkim obywatelom, c) zasada, że Rzeczpospolita Polska (Polska) jest dobrem wspólnym, jest w systematyce Konstytucji przed innymi zasadami ustrojowymi, w tym przed zasadą demokratycznego państwa prawnego. c) zasada, że Rzeczpospolita Polska (Polska) jest dobrem wspólnym, jest w systematyce Konstytucji przed innymi zasadami ustrojowymi, w tym przed zasadą demokratycznego państwa prawnego. 25 Rzeczpospolita Polska dobrem wspólnym wszystkich obywateli Uogólniając powyższe spostrzeżenia, należy powiedzieć, że gdy mówimy o dobru wspólnym w kontekście art. 1 Konstytucji, to mówimy o państwie, o jednostce, a przede wszystkim o relacji pomiędzy tymi podmiotami i że rela- cja ta nie redukuje się do płaszczyzny wolności, praw i obowiązków jednostki i przyczyn oraz metod ich ograniczania. II. Dekodując treść normatywną art. 1 Konstytucji, należy pomocniczo sięgnąć do polskiej praktyki konstytucyjnej, a także do dyskusji w Komisji Konstytucyjnej ZN4 nad pojęciem państwa jako „dobra wspólnego” i wyrażo- nych przy tej okazji poglądów. W trakcie debaty konstytucyjnej zwrócono uwagę, że pojęciem dobra wspólnego posługiwała się Konstytucja Polski z 1935 r. W art. 1 stanowiła, że „Państwo Polskie jest wspólnym dobrem wszystkich obywateli”. 4 Istotny fragment tej dyskusji cytuje: A. Piechowiak, op. cit., s. 10. 5 Zob. ibidem, s. 9–10. 6 Ibidem, s. 9. 7 Zob. ibidem, s. 9–10. 8 Zob. o tym J. Trzciński, Tendencje i kierunki rozwojowe europejskiego i konstytucjonalizmu, w: Z. Czeszejko-Sochacki (red.), Konstytucja Federalna Szwajcarskiej Konfederacji i Konstytucja Rzeczypospolitej Polskiej z 1997 r., Białystok 2001, s. 27–28. 7 Zob. ibidem, s. 9–10. RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* Na tle tak brzmiącej formuły dostrzega się zasadniczą różnicę pomiędzy art. 1 Konstytu- cji z 1997 r. a art. 1 Konstytucji z 1935 r. Różnica ta uzewnętrznia się w kolejno- ści wyrazów formuły „wspólne dobro” (1935) w odróżnieniu od „dobro wspólne” (1997)5. „Wykładnia gramatyczna terminu »wspólne dobro« każe uznać prymat państwa jako całości przed jednostką i położyć akcent na obowiązki obywateli wobec państwa. Wspólne dobro to coś, co jest wspólnym celem, celem działania wszystkich członków danej grupy, w tym przypadku obywateli”6. Co się tyczy Konstytucji z 1997 r., to należy uznać pogląd, że formuła „dobro wspólne” zasadniczo różni się od formuły „wspólne dobro” i daje wy- raz prymatowi jednostki przed państwem7. Innymi słowy, daje wyraz innej koncepcji praw i wolności jednostki będącej pochodną określonej koncepcji państwa. Analiza Konstytucji z 1997 r. prowadzi do wniosku, że państwo polskie jest polityczną organizacją społeczeństwa, w której system organów władzy (organów państwa) jest zaledwie elementem struktury państwa, a nie elementem definiującym państwo. Jest to koncepcja państwa, w któ- rym obywatel i jego organizacje są istotnym elementem współdefiniującym państwo8. Przyjęta w art. 1 Konstytucji formuła „dobra wspólnego” i pośrednio wy- nikająca z niej koncepcja państwa charakteryzuje koncepcję praw i wolności przyjętą w naszej Konstytucji i jest jednocześnie jej elementem. Jest to bez wątpienia indywidualistyczna koncepcja praw jednostki. Rozstrzyga w ten sposób pytanie: jednostka dla państwa czy państwo dla jednostki – na rzecz służebnej roli państwa wobec obywateli z zachowaniem oczywiście tych war- tości, które stanowią o bycie państwa, wartości, o których przede wszystkim mowa w art. 5 Konstytucji, który stanowi, że „Rzeczpospolita Polska strze- że niepodległości i nienaruszalności swojego terytorium, zapewnia wolności 26 Janusz Trzciński i prawa człowieka i obywatela oraz bezpieczeństwo obywateli, strzeże dzie- dzictwa narodowego oraz zapewnia ochronę środowiska, kierując się zasadą zrównoważonego rozwoju”. i prawa człowieka i obywatela oraz bezpieczeństwo obywateli, strzeże dzie- dzictwa narodowego oraz zapewnia ochronę środowiska, kierując się zasadą zrównoważonego rozwoju”. Ale nawet i te wartości w konkretnych sytuacjach mogą być przecież kon- frontowane z wartościami i prawami jednostki – albo przez odniesienie do art. 2, albo przez odniesienie do art. 31 ust. 3 Konstytucji, a więc do zasady proporcjonalności ograniczania praw. III. Dotychczasowe uwagi uprawniają do zgłoszenia następującej tezy me- todologicznej, będącej uogólnieniem konkluzji płynących z interpretacji tych postanowień Konstytucji, które używają pojęcia dobra wspólnego na określe- nie Rzeczypospolitej. 9 Zob. B. Lewaszkiewicz-Petrykowska, op. cit. 11 Art. 31: „1. Wolność człowieka podlega ochronie prawnej. 2. Każdy jest obowiązany sza- nować wolności i prawa innych. Nikogo nie wolno zmuszać do czynienia tego, czego prawo mu nie nakazuje. 3. Ograniczenia w zakresie korzystania z konstytucyjnych wolności i praw mogą być ustanawiane tylko w ustawie i tylko wtedy, gdy są konieczne w demokratycznym państwie dla jego bezpieczeństwa lub porządku publicznego, bądź dla ochrony środowiska, zdrowia i mo- ralności publicznej, albo wolności i praw innych osób. Ograniczenia te nie mogą naruszać istoty wolności i praw”. Art. 61 ust. 3: „Ograniczenie prawa, o którym mowa w ust. 1 i 2, może nastąpić 10 O związku tych zasad, a w szczególności zasady solidarności z zasadą dobra wspólnego wyrażoną w art. 1 Konstytucji, zob. Wystąpienie Prezydenta Aleksandra Kwaśniewskiego, op. cit. Zob. też J. Kurys, Sprawiedliwość społeczna. Szkice ze współczesnej teorii konstytucjonalizmu i praktyki polskiego prawa ustrojowego, Kraków 2004. RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* Otóż uważam, że przyjęta koncepcja Rzeczypospolitej Polskiej jako „dobra wspólnego” wszystkich obywateli nie tylko pozostaje we wzajemnym związku z koncepcją państwa przyjętą w Konstytucji i z koncepcją wolności i praw jednostki, ale także wyznacza obszary regulacji konstytucyj- nych mające podstawowe znaczenie dla opisania treści formuły „Rzeczpospoli- ta Polska jest dobrem wspólnym wszystkich obywateli”. Zaliczyć do nich nale- ży te, które dla zachowania ładu konstytucyjnego mają podstawowe znaczenie: ży te, które dla zachowania ładu konstytucyjnego mają podstawowe znaczen a) obszar wolności, praw i obowiązków jednostki, który opisuje nie tylko status jednostki w państwie, ale także opisuje obowiązki władzy (państwa) wobec jednostki, b) obszar funkcjonowania instytucji życia publicznego. Idzie tu o wybór optymalnego modelu ustroju państwa z punktu widzenia zachowania ładu konstytucyjnego mającego znaczenie dla statusu jednostki, b) obszar funkcjonowania instytucji życia publicznego. Idzie tu o wybór optymalnego modelu ustroju państwa z punktu widzenia zachowania ładu konstytucyjnego mającego znaczenie dla statusu jednostki, c) obszar stanowienia prawa będącego podstawowym instrumentem reali- zacji wartości konstytucyjnych. c) obszar stanowienia prawa będącego podstawowym instrumentem reali- zacji wartości konstytucyjnych. Propozycja, aby rozważań nad zagadnieniem Rzeczypospolitej Polskiej jako dobra wspólnego nie ograniczać, jak się to zwykło czynić, jedynie do pierwszego z wymienionych obszarów, a więc obszaru wolności i praw jednost- ki w zderzeniu z konstytucyjnie określonym interesem państwa9, lecz aby je rozciągnąć na obszary definiujące władzę państwową – bierze się z przekona- nia, że formuła dobra wspólnego „działa w obie strony”. Oznacza to, że dbałość o państwo jako dobro wspólne, będące demokratycznym państwem prawnym realizującym zasady sprawiedliwości społecznej (art. 2 Konstytucji), obciąża nie tylko obywatela, ale że obywatel ma także prawo oczekiwać, iż władza publiczna tak będzie działać, aby Rzeczpospolita także przez obywateli mogła być uznana za dobro wspólne. IV. Podstawowym warunkiem uznania „dobra” (czyt. państwa) za „dobro wspólne” jest takie ukształtowanie instytucji praw, wolności i obowiązków jednostki, które zapewni jednostce realizację przyznanych jej konstytucyjnie praw. Wydaje się, przy wszystkich zastrzeżeniach, jakie tu można zgłosić, że takimi prawami stanowiącymi o istocie instytucji wolności i praw w ramach koncepcji dobra wspólnego są: 27 Rzeczpospolita Polska dobrem wspólnym wszystkich obywateli a) zasada poszanowania godności ludzkiej stanowiącej źródło wolności i praw człowieka i obywatela (art. 30 i wstęp do Konstytucji), a) zasada poszanowania godności ludzkiej stanowiącej źródło wolności i praw człowieka i obywatela (art. RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* 30 i wstęp do Konstytucji), b) zasada równości praw i równości w prawie deklarująca, że wszyscy są równi wobec prawa i że wszyscy mają prawo do równego traktowania przez władze publiczne (art. 32), b) zasada równości praw i równości w prawie deklarująca, że wszyscy są równi wobec prawa i że wszyscy mają prawo do równego traktowania przez władze publiczne (art. 32), c) zasada sprawiedliwości społecznej (art. 2), której zastosowanie ma zna- czenie przede wszystkim w obszarach praw ekonomicznych i socjalnych, c) zasada sprawiedliwości społecznej (art. 2), której zastosowanie ma zna- czenie przede wszystkim w obszarach praw ekonomicznych i socjalnych, d) zasada solidarności (solidaryzmu społecznego – wstęp do Konstytucji i art. 20), która rzuca także światło na rozumienie zarówno zasady równości jak i sprawiedliwości społecznej10, d) zasada solidarności (solidaryzmu społecznego – wstęp do Konstytucji i art. 20), która rzuca także światło na rozumienie zarówno zasady równości jak i sprawiedliwości społecznej10, e) prawo do sądu (art. 45 ust. 1 Konstytucji) jako gwarancja realizacji praw jednostki. Wytypowane powyżej prawa, które mają, moim zdaniem, zasadnicze zna- czenie dla oceny instytucji wolności i praw jednostki, charakteryzują się tym, że zabezpieczają możliwość korzystania przez jednostkę z jej praw z uwzględ- nieniem praw innych osób, ale jednocześnie pozwalają – ze względu na solidar- ność, równość i sprawiedliwość społeczną – prawa jednostki krępować, właśnie ze względu na zachowanie ładu konstytucyjnego w obrębie dobra wspólnego. Głębokość korzystania z wolności i praw jednostki w ramach „dobra wspól- nego” wyznaczają dwa czynniki, których ocena nie może pozostać bez znacze- nia dla opisu instytucji praw i wolności w ramach RP jako „dobra wspólnego”. Są to instytucje obowiązków obywatela, które są swojego rodzaju kontrybucją na rzecz ogółu społeczeństwa (art. 82–86) oraz art. 31 ust. 3 zezwalający na ograniczenie wolności i praw jednostki. Zagadnienie podstawowych obowiązków jednostki, które z woli Konstytucji uznane są za konieczne do wprowadzenia dla powodzenia „dobra wspólnego” uznanego w tym kontekście za zapewnienie pewnych zadań publicznych w in- teresie wszystkich obywateli, uregulowane jest w art. 82 do 86 Konstytucji. Konstytucja zalicza do nich: – obowiązek wierności Rzeczpospolitej oraz troskę o dobro wspólne (art. 82), – obowiązek wierności Rzeczpospolitej oraz troskę o dobro wspólne (art. 82), obowiązek przestrzegania prawa RP (art 83) – obowiązek ponoszenia ciężarów i świadczeń publicznych (art. 84), – obowiązek ponoszenia ciężarów i świadczeń publicznych (art. 84), – obowiązek obrony ojczyzny (art. 85), – obowiązek ponoszenia ciężarów i świadczeń publicznych (art. 10 O związku tych zasad, a w szczególności zasady solidarności z zasadą dobra wspólnego wyrażoną w art. 1 Konstytucji, zob. Wystąpienie Prezydenta Aleksandra Kwaśniewskiego, op. cit. Zob. też J. Kurys, Sprawiedliwość społeczna. Szkice ze współczesnej teorii konstytucjonalizmu i praktyki polskiego prawa ustrojowego, Kraków 2004. 11 Art. 31: „1. Wolność człowieka podlega ochronie prawnej. 2. Każdy jest obowiązany sza- nować wolności i prawa innych. Nikogo nie wolno zmuszać do czynienia tego, czego prawo mu nie nakazuje. 3. Ograniczenia w zakresie korzystania z konstytucyjnych wolności i praw mogą być ustanawiane tylko w ustawie i tylko wtedy, gdy są konieczne w demokratycznym państwie dla jego bezpieczeństwa lub porządku publicznego, bądź dla ochrony środowiska, zdrowia i mo- ralności publicznej, albo wolności i praw innych osób. Ograniczenia te nie mogą naruszać istoty wolności i praw”. Art. 61 ust. 3: „Ograniczenie prawa, o którym mowa w ust. 1 i 2, może nastąpić 12 Np. art. 68 ust. 3: „Władze publiczne są obowiązane do zapewnienia szczególnej opieki zdrowotnej dzieciom, kobietom ciężarnym, osobom niepełnosprawnym i osobom w podeszłym wie- ku” albo art. 71 ust. 1: „Państwo w swojej polityce społecznej i gospodarczej uwzględnia dobro ro- dziny. Rodziny znajdujące się w trudnej sytuacji materialnej i społecznej, zwłaszcza wielodzietne i niepełne, mają prawo do szczególnej pomocy ze strony władz publicznych”. I choć adresatem tych postanowień jest przede wszystkim „państwo”, to przecież jest w nich zawarte minimum praw dla obywatela. Zob. o tym J. Trzciński, Komentarz do art. 68 Konstytucji, w: L. Garlicki (red.), Konsty- tucja Rzeczypospolitej Polskiej. Komentarz, t. 3, Warszawa 2003. wyłącznie ze względu na określone w ustawach ochronę wolności i praw innych osób i podmiotów gospodarczych oraz ochronę porządku publicznego, bezpieczeństwa lub ważnego interesu gospo- darczego państwa”. Art. 22: „Ograniczenie wolności działalności gospodarczej jest dopuszczalne tylko w drodze ustawy i tylko ze względu na ważny interes publiczny”. 13 Mam tu na uwadze choćby formułę art. 2 Konstytucji nakazującą, aby Rzeczpospolita była państwem demokratycznym, lub art. 4 ust. 2, że: „naród sprawuje władzę przez swoich przedsta- wicieli lub bezpośrednio”. 15 Na związek sprawnego działania aparatu państwowego z dobrem wspólnym zwraca też uwagę P. Winczorek w opinii z 3 lipca 2002 r. w sprawie interpretacji art. 203 Konstytucji RP w związku z art. 2 ustawy o NIK (zob. maszynopis, s. 2), a także TK w orzeczeniu K 24/02. 14 W istocie na gruncie prawa kompetencja łączy się nie z państwem, lecz z organami p stwa, czy szerzej – organami władzy publicznej. RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* 84), obowiązek obrony ojczyzny (art 85) – obowiązek dbałości o stan środowiska (art. 86). Inną formą ograniczenia wolności i praw przewidzianą w Konstytucji jest przede wszystkim art. 31 ust. 3 Konstytucji, ale także art. 61 ust. 3 i art. 2211. Inną formą ograniczenia wolności i praw przewidzianą w Konstytucji jest przede wszystkim art. 31 ust. 3 Konstytucji, ale także art. 61 ust. 3 i art. 2211. 28 Janusz Trzciński Analiza tych przepisów pokazuje, że jakkolwiek nie używają one sformułowa- nia, że ograniczenie może nastąpić ze względu na przesłankę „dobra wspólne- go”, to szczegółowe przesłanki art. 31 ust. 3, art. 61 ust. 3 i art. 22 w istocie tak są skonstruowane, że mogłyby wyczerpywać swoją treścią pojęcie dobra wspólnego. Należą do nich: ochrona bezpieczeństwa państwa i porządku pub- licznego, ochrona środowiska, ochrona zdrowia i moralności publicznej, ochro- na ważnego interesu gospodarczego państwa, ochrona wolności i praw innych osób, interes publiczny. Bez wątpienia cytowane tu przepisy art. 31 ust. 3, art. 22 i art. 61 ust. 3, a także art. 82–86 mają swój prosty związek z art. 1 Konstytucji; koncepcyjnie z niego wypływają i go opisują. V. Drugim z wyznaczonych przeze mnie obszarów materii konstytucyjnej mającym znaczenie dla zdefiniowania treści formuły, według której „Rzeczpo- spolita Polska jest dobrem wspólnym wszystkich obywateli”, jest obszar funk- cjonowania instytucji publicznych składających się na system organów władzy publicznej, a więc ważny element struktury państwa. Jeżeli prawdziwa jest teza, że państwo i jego organy mają określone obo- wiązki wobec obywateli opisane w Konstytucji12, a także wynikające z istoty de- mokratycznego państwa prawnego13, to minimalne postulaty realizacji dobra wspólnego przez organy władzy (w Konstytucji używa się formuły państwo)14 polegają na tym, że instytucje publiczne mają działać sprawnie15 i sprawiedli- wie w interesie społeczeństwa. Aby osiągnąć powyższe cele, powinny być zorga- nizowane demokratycznie16. Ten ostatni postulat oznacza możliwość wpływa- nia przez obywateli za pomocą przyznanych im środków i instytucji prawnych (np. referendum) na kształt i treść decyzji państwowych. Brak takich możliwo- ści prowadziłby do alienacji instytucji publicznych i do podważenia tezy o tym, że RP jest dobrem wspólnym wszystkich obywateli. Coś, co nie jest społecz- wyłącznie ze względu na określone w ustawach ochronę wolności i praw innych osób i podmiotów gospodarczych oraz ochronę porządku publicznego, bezpieczeństwa lub ważnego interesu gospo- darczego państwa”. Art. 22: „Ograniczenie wolności działalności gospodarczej jest dopuszczalne tylko w drodze ustawy i tylko ze względu na ważny interes publiczny”. 16 Realizacja postulatu trójpodziału władzy jest warunkiem koniecznym, ale nie wystarcza- jącym do zrealizowania tego celu. 19 Zob. wyroki TK: z 12 kwietnia 2000 r., K 8/98; z 30 stycznia 2001 r., K 17/00; z 10 październi- ka 2001 r., K 28/01; z 18 lutego 2003 r., K 24/02; z 25 listopada 2003 r., K 37/02; z 7 stycznia 2004 r., K 14/03. 17 Zagadnienie to jest niekiedy ujmowane jako zagadnienie „prawowitości” prawa, dla której stwierdzenia ważne jest, aby prawo było zgodne z interesem i preferencjami jednostek. Zob. W. Sadurski, Status jednostki w prawie: refleksje filozoficzno-prawne na temat prawowi- tości demokracji (tezy referatu), Konferencja na Wydziale Prawa i Administracji UW Warszawa, 25 czerwca 2005 r. (maszynopis). 18 Znane mi orzecznictwo SN nie odwołuje się wprost do art. 1 Konstytucji. NSA odwołał się do art. 1 Konstytucji w orzeczeniu z 12 kwietnia 2000 r., I SA/Ka 1740/98. W wyroku tym NSA dokonał interpretacji art. 9 k.p.a. przez pryzmat art. 1 Konstytucji. Oto teza tego wyroku wyjęta z uzasadnienia: „Obowiązek informowania i wyjaśniania stronom przez organ prowadzący postę- powanie całokształtu okoliczności faktycznych i prawnych toczącej się sprawy (art. 9 Kpa) powi- nien być rozumiany tak szeroko, jak to jest możliwe. Udowodnione naruszenie tego obowiązku po- winno być rozumiane jako wystarczająca podstawa do uchylenia decyzji, szczególnie wówczas, gdy urzędnik stwierdza (lub powinien stwierdzić), że strona zamierza podjąć działania wiążące się dla niej z niekorzystnymi skutkami, lub nawet ryzykiem wystąpienia podobnych skutków. W takim wypadku urzędnik ma wyraźny obowiązek w możliwie jasny sposób wyjaśnić całość okoliczności sprawy stronie i równie wyraźnie wskazać na ryzyko wiążące się z zaplanowanymi działaniami. Jest to jedyny odpowiadający zasadzie art. 1 Konstytucji sposób rozumienia art. 9 Kpa”. 19 Zob. wyroki TK: z 12 kwietnia 2000 r., K 8/98; z 30 stycznia 2001 r., K 17/00; z 10 październi- 25 czerwca 2005 r. (maszynopis). 18 Znane mi orzecznictwo SN nie odwołuje się wprost do art. 1 Konstytucji. NSA odwołał się do art. 1 Konstytucji w orzeczeniu z 12 kwietnia 2000 r., I SA/Ka 1740/98. W wyroku tym NSA dokonał interpretacji art. 9 k.p.a. przez pryzmat art. 1 Konstytucji. Oto teza tego wyroku wyjęta z uzasadnienia: „Obowiązek informowania i wyjaśniania stronom przez organ prowadzący postę- powanie całokształtu okoliczności faktycznych i prawnych toczącej się sprawy (art. 9 Kpa) powi- nien być rozumiany tak szeroko, jak to jest możliwe. Udowodnione naruszenie tego obowiązku po- winno być rozumiane jako wystarczająca podstawa do uchylenia decyzji, szczególnie wówczas, gdy urzędnik stwierdza (lub powinien stwierdzić), że strona zamierza podjąć działania wiążące się dla niej z niekorzystnymi skutkami, lub nawet ryzykiem wystąpienia podobnych skutków. W takim wypadku urzędnik ma wyraźny obowiązek w możliwie jasny sposób wyjaśnić całość okoliczności sprawy stronie i równie wyraźnie wskazać na ryzyko wiążące się z zaplanowanymi działaniami. Jest to jedyny odpowiadający zasadzie art. 1 Konstytucji sposób rozumienia art. 9 Kpa”. 17 Zagadnienie to jest niekiedy ujmowane jako zagadnienie „prawowitości” prawa, dla której stwierdzenia ważne jest, aby prawo było zgodne z interesem i preferencjami jednostek. Zob. W. Sadurski, Status jednostki w prawie: refleksje filozoficzno-prawne na temat prawowi- tości demokracji (tezy referatu), Konferencja na Wydziale Prawa i Administracji UW Warszawa, 25 czerwca 2005 r. (maszynopis). RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* 13 Mam tu na uwadze choćby formułę art. 2 Konstytucji nakazującą, aby Rzeczpospolita była państwem demokratycznym, lub art. 4 ust. 2, że: „naród sprawuje władzę przez swoich przedsta- wicieli lub bezpośrednio”. Rzeczpospolita Polska dobrem wspólnym wszystkich obywateli 29 nie akceptowane, trudno nazwać dobrem wspólnym. Na przykład nie będzie dobrem wspólnym system państwowy oparty na dyktaturze czy dominującej ideologii narzuconej społeczeństwu. nie akceptowane, trudno nazwać dobrem wspólnym. Na przykład nie będzie dobrem wspólnym system państwowy oparty na dyktaturze czy dominującej ideologii narzuconej społeczeństwu. VI. Trzecim obszarem mającym znaczenie dla identyfikacji treści formu- ły „Rzeczpospolita Polska jest dobrem wspólnym wszystkich obywateli” jest obszar reguł stanowienia prawa i treści samego prawa, które, aby mogło sku- tecznie być uważane za element dobra wspólnego, musi spełniać trzy warunki: a) używając formuły art. 1 Konstytucji: „wszyscy obywatele” (społeczeń- stwo) powinni identyfikować się z treścią prawa w stopniu, w jakim uwzględ- nia ono interesy społeczeństwa17 opisane w postanowieniach Konstytucji okre- ślających cele i zadania państwa (Rzeczypospolitej Polskiej), a) używając formuły art. 1 Konstytucji: „wszyscy obywatele” (społeczeń- stwo) powinni identyfikować się z treścią prawa w stopniu, w jakim uwzględ- nia ono interesy społeczeństwa17 opisane w postanowieniach Konstytucji okre- ślających cele i zadania państwa (Rzeczypospolitej Polskiej), b) społeczeństwo ma instytucjonalny wpływ na proces tworzenia prawa, tzn. że procedury stanowienia prawa uwzględniają postulat demokratyczności prawa, c) prawo musi być zgodne z Konstytucją, ustawą zasadniczą Rzeczypo- spolitej Polskiej, której powinna przysługiwać cecha względnej stabilności, ze względu na wartości, które formułuje. Wartościami tymi są demokratyczny ustrój państwowy i wolności i prawa jednostki. c) prawo musi być zgodne z Konstytucją, ustawą zasadniczą Rzeczypo- spolitej Polskiej, której powinna przysługiwać cecha względnej stabilności, ze względu na wartości, które formułuje. Wartościami tymi są demokratyczny ustrój państwowy i wolności i prawa jednostki. VII. Orzecznictwo sądowe18 w małym stopniu odwołuje się do art. 1 Kon- stytucji. Najliczniejsze ślady sięgania do zasady opisanej w art. 1 znajdziemy w orzecznictwie TK19. Jako wzorzec konstytucyjny nie występuje wprost w orzecznictwie TK, co może oznaczać, że wnioskujący lub skarżący nie widzą niezgodności po- szczególnych przepisów ustawy z art. 1 Konstytucji albo uważają, że art. 1 nie mógłby występować jako samodzielny wzorzec. Moim zdaniem nie można mieć wątpliwości co do normatywnego charakteru art. 1 Konstytucji i co do tego, 30 Janusz Trzciński czy art. 1 mógłby być wzorcem konstytucyjnym przy kontroli konstytucyjności prawa, może z wyjątkiem skargi konstytucyjnej. czy art. RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* 2 Konstytu- cji określającym Rzeczpospolitą Polską jako demokratyczne państwo prawne urzeczywistniające zasady sprawiedliwości społecznej, 31 Rzeczpospolita Polska dobrem wspólnym wszystkich obywateli d) wydaje się, że w obecnym stanie badań nad zagadnieniem art. 1 Konsty- tucji wszelkie uogólnienia szczegółowych uwag poczynionych powyżej byłyby przedwczesne przede wszystkim dlatego, że zbyt skąpa jest praktyka sądowa i trybunalska w tym względzie, a bez niej nie da się odpowiedzieć w pogłębiony sposób na pytanie o treść zasady, że „Rzeczpospolita Polska jest dobrem wspól- nym wszystkich obywateli”. prof. dr hab. Janusz Trzciński Uniwersytet Warszawski jtrzcinski@nsa.gov.pl prof. dr hab. Janusz Trzciński Uniwersytet Warszawski jtrzcinski@nsa.gov.pl RZECZPOSPOLITA POLSKA DOBREM WSPÓLNYM WSZYSTKICH OBYWATELI* 1 mógłby być wzorcem konstytucyjnym przy kontroli konstytucyjności prawa, może z wyjątkiem skargi konstytucyjnej. Występuje natomiast art. 1 i użyte w nim pojęcie „dobro wspólne” jako argument wzmacniający konkretne rozstrzygnięcia oparte na konkretnych wartościach konstytucyjnych, przede wszystkim – solidarności (solidaryzm), sprawiedliwości społecznej, równości, demokratycznym państwie prawnym. Zagadnienie „dobra wspólnego” w kontekście zasady solidarności spo- łecznej zostało użyte w wyroku TK z 30 stycznia 2001 r. (K 1700). Odwołując się do zasady solidarności społecznej, TK uznał, że nie tylko pracownicy, lecz także pracodawcy muszą ponieść pewne, określone koszty reformy pła- cowej (ubruttowienie wynagrodzeń pracowników) i że jest to zgodne z zasadą wyrażoną w art. 1 Konstytucji. Trybunał stwierdził, że wszyscy obywatele zarówno pracodawcy, jak i pracownicy są w stopniu odpowiednim do swo- ich możliwości zobowiązani do poświęcenia pewnych interesów własnych dla „dobra wspólnego”. Zagadnienie dobra wspólnego (art. 1) w kontekście zasady państwa praw- nego (art. 2) pojawia się w dwóch aspektach: w związku dobra wspólnego z zasadą sprawiedliwości społecznej, przez którą realizuje się zasada dobra wspólnego (K 8/98) i w związku z taką organizacją samorządu terytorialnego, która zapewni zdolność wykonywania zadań publicznych w interesie dobra wspólnego. Niekiedy też w uzasadnieniach wyroków TK dobro wspólne występuje w kontekście ograniczenia praw jednostki (np. ze względu na bezpieczeństwo państwa K 37/02), a w orzeczeniu K 14/03 mówi się o związku odpowiedniego modelu ustawy ze względu na zapewnienie praw wszystkich obywateli (a więc dobra wspólnego). VIII. Powyżej sformułowane uwagi, z których większość ma charakter uwag metodologicznych, tzn. pokazujących sposób podejścia do badań nad za- sadą wyrażoną w art. 1 Konstytucji, prowadzą mnie do następujących uogól- nień: a) próba rozumienia zasady „Rzeczpospolita Polska jest dobrem wspól- nym wszystkich obywateli” musi za punkt wyjścia w tle badań mieć koncepcję państwa zakodowaną w Konstytucji. „Dobro wspólne” jest pochodną koncep- cji państwa zarówno w płaszczyźnie koncepcji wolności i praw jednostki, jak i w płaszczyźnie organizacyjnej państwa (czyt. władzy publicznej), b) na płaszczyźnie konstytucyjnej dobro wspólne nie redukuje się do za- gadnienia ograniczenia wolności i praw jednostki w zestawieniu z interesem publicznym (interesem państwa). Zagadnienie należy widzieć także w płasz- czyźnie ustrojowej, w płaszczyźnie ładu konstytucyjnego, akceptowanego lub nie przez obywateli, c) z pojęciem „dobro wspólne” (Rzeczpospolita Polska) łączą się nie tylko obowiązki obywatela wobec państwa, ale i obowiązki państwa wobec obywa- tela. Ślady takiego rozumowania są wyraźnie zaznaczone w art. THE REPUBLIC OF POLAND – THE COMMON GOOD OF ALL CITIZENS S u m m a r y S u m m a r y The aim of this article is to propose a methodology for research into the constitutional prin- ciple of the common good (Article 1 of the Polish Constitution). The author takes the view that the basic condition for the recognition of a state as a common good is to ensure that the rights, freedoms and duties of an individual are shaped in such a way as to ensure that the individual’s constitutional rights can be exercised. The laws that constitute the essence of the institutions of the freedom and rights within the concept of the common good include: human dignity, equality in law, the principle of social justice, the principle of social solidarity, the right to a trial. The minimum requirements for authorities to pursue the common good are that public institutions act efficiently and fairly in the interest of society. Common good is a derivative of the concept of state regarding both, the concept of freedom and individual rights, and the organisational side of the state. On the constitutional level, the common good is not reduced to the issue of the limitation of individual freedoms and rights as compared to the public (State) interest. This issue should also be noted at the political level, the State’s constitutional order, whether accepted or not by the citi- zens. The concept of the common good is not only associated with the citizen’s duties towards the state, but also with the state’s duties towards the citizen. The closest context in which Article 1 of the Constitution should be read is Article 2 of the Constitution (the principle of a democratic state governed by law), since the two, taken together, define the Republic of Poland as a state.
https://openalex.org/W2808090886	https://europepmc.org/articles/pmc6015587?pdf=render	English	null	Synthetic biology in the German press: how implications of metaphors shape representations of morality and responsibility	Life sciences, society and policy	2,018	cc-by	9,856	© The Author(s). 2018 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. RESEARCH Open Access Döring Life Sciences, Society and Policy (2018) 14:14 https://doi.org/10.1186/s40504-018-0079-9 Synthetic biology in the German press: how implications of metaphors shape representations of morality and responsibility Martin Döring Correspondence: Doering@ metaphorik.de Institute of Geography, University of Hamburg, Bundesstraße 55, 20146 Hamburg, Germany Abstract: Synthetic biology (SynBio) represents a relatively young field of research which has developed into an important scientific endeavour. Characterised by a high degree of interdisciplinary work crossing disciplinary boundaries, such as biology, mathematics and engineering, SynBio has been, since its beginning, devoted to creating new biological functions, metabolic pathways or even minimal organisms. Although its often-articulated aim of developing new forms of life has so far not been archived, SynBio nowadays represents a well-established biotechnological approach and it has also attracted public concern, especially since Craig Venter’s work on Mycoplasma Mycoides JCVI-syn1.0. Taking these developments as a starting point, the paper empirically investigates the metaphorical representations of SynBio in two leading German media publications, the daily newspaper Die Frankfurter Allgemeine Zeitung and the weekly magazine Der Spiegel between 2000 and 2010. Using a novel combination of metaphor and co-occurrence analysis, the paper engages in a systematic examination of implicit moral implications inherent in linguistic images permeating this news coverage. It demonstrates a method of how media-metaphorical representations and their moral implications of SynBio could analytically be revealed and analysed. In doing so, it aims at contributing to empirical ethical analyses of the news coverage on SynBio in particular and offers an approach that methodologically adds to literature on responsible language use, which is emerging in science and technology studies and ethical analyses of new technologies. Keywords: Synthetic biology, Metaphor, Moral implications, German press coverage Synthetic biology: an emerging and consolidating field of research Synthetic Biology (SynBio) is a relatively young field of research (Endy 2005), closely linked to biotechnology, systems biology and metabolic engineering (Nesbet 2016). After 15 years of existence (De Lorenzo and Danchin 2008; Nisbet and Lewenstein 2002), it can now be regarded as a scientifically established discipline with its own journals, conferences, research centres and curricula at universities all over the world. The disciplinary rationale of SynBio is twofold, namely: “the design and construction of new biological parts, devices and systems as well as the redesign of existing parts” (Porcar and Peretó 2012: 79). Page 2 of 17 Döring Life Sciences, Society and Policy (2018) 14:14 Questions continue to be asked as to whether SynBio is a new scientific discipline in a biotechnological field of research where proteomics, systems biology (Döring et al. Synthetic biology in the German press: how implications of metaphors shape representations of morality and responsibility 2015) and metabolic engineering are comparable ambitious endeavours and whether Synbio’s engineering approach makes it a unique discipline or not (Keller 2002). Inter- estingly, studies from the field of the sociology of science and science and technology studies frame SynBio as a scientific discipline. Here, SynBio is conceived as a post-genomic engineering discipline that aims at making biology “less qualitative and descriptive and more quantitative and predictive” (Calvert and Fujimura 2009: 48). This shift, Calvert and Fujimura assume, exhibits a conceptual and methodological attempt to make biology a hard(er) science and develop a high degree of sophistication by in- cluding a quantitatively based rationale derived from computer science, mathematics and modelling. This has theoretical and methodological repercussions for biology, bio- technology, biomedical research, science-policy, the economy and society in general. The increasing scientific relevance of SynBio is also mirrored in the number of scien- tific publications found on Pubmed-Reminer (search term ‘Synthetic Biology’) which remained until 2008 at an average of approx. 60 papers per year, but has risen to 827 in 2017. The same holds true for scientific reviews – an important type of text written by experts reviewing research undertaken and assessing the future of a discipline. Here we find a steep increase between 2011 (57 reviews) and 2016 (231 reviews), only slowing down in 2017 to 198 reviews. This clearly indicates that a scientific narrative is woven around SynBio which “shapes the literature of a field into a story in order to enlist the support of readers to [...] [corroborate] this story” (Meyers 1991: 45). These numbers have, however, to be treated with caution as they do not indicate whether there is a sci- entific revolution taking place or whether rising figures, in terms of publication output, indicate a fashionable use of the term SynBio for strategic purposes. Furthermore, it should be stressed that many targets set by scientists working in the field of SynBio have not been achieved to date while others have materialised and attracted considerable scientific, political and public attention. This was the case in the development of Artemisinin, an anti-malaria drug extracted from engineered yeast and even more so in the production of the artificial organism Mycoplasma Mycoides JCVI-syn1.0 (Balmer and Herreman 2009: 221). Synthetic biology in the German press: how implications of metaphors shape representations of morality and responsibility Additionally, conceptual issues such as the principles of hierarchical abstraction or modularity (Serrano 2007: 1) or the integra- tion of the so-called chassis and the programme cause considerable problems (Dachin 2012). At a large press conference, Craig Venter publicly announced that the merging of syn- thetic sequences with sequences taken from another bacterium had been successful and proclaimed that the shift from biological reproduction of life, to the technological produc- tion of life, had been achieved by him and his research team (Cserer and Seiringer 2009). Such proclaimed scientific revolutions are in many cases accompanied by intensive media coverage and can lead to worries about the release of artificially generated life forms into the environment (Balmer and Martin 2008). Venter’s development of Mycoplasma Mycoides JCVI-syn1.0 was indeed accompanied by rapid and considerable media coverage semantically revolving around the metaphor of “playing god in Frankenstein’s footsteps” (van Belt 2009: 257). It was interesting to observe how the creation of a supposedly artifi- cial form of life caused considerable public concern about its possible ecological impact, but also how such an exceptional event was metaphorically and linguistically represented. Döring Life Sciences, Society and Policy (2018) 14:14 Page 3 of 17 Page 3 of 17 All these observations form the starting point of the present paper which aims at pro- viding a methodologically systematic and empirical analysis of moral implications in- herent in the metaphors that permeated the German newspaper coverage of SynBio in the seminal daily newspaper Die Frankfurter Allgemeine Zeitung and the renowned weekly magazine Der Spiegel. This article aims to methodologically and empirically complement existing philosophical and bioethical reflections in the areas of biotechno- logical innovation, biomedical developments (Nordgren 1998), systems biology (Döring et al. 2015), synthetic biology (Boldt 2016), and science and technology studies (Kom- duur et al. 2009). It differs from this literature insofar as it proposes a novel practical approach for studying metaphorical mapping processes from an ethical point of view. This is important because in the mapping process arguments and values are cognitively transferred from one domain of knowledge to another: the “production of certain [metaphorical] mappings is an act of ethical discourse” (Balmer and Herreman 2009: 221), which includes unnoticed or even hidden moral implications. Synthetic biology in the German press: how implications of metaphors shape representations of morality and responsibility This requires con- scious reflection in terms of a responsible use of metaphors which “are often used to help readers to connect to scientific results” (Kueffer and Larson 2014) and developments. In the following, I will first provide an overview of, and theoretically engage with, re- search undertaken on metaphor in linguistics and in the fields of science and technol- ogy studies with a focus on studies investigating images in the areas of genetics, genomics and synthetic and systems biology. Against this background, I will explain the approach taken for studying metaphors (Schmitt 2011). I will then explicate my method of analysing metaphorical mappings with the aid of a co-occurrence analysis. The art- icle will show how this analysis allows researchers to perform an empirically grounded analysis of hidden implications, moral or otherwise, in metaphorical mappings. Illus- trating this approach, I will analyse four main metaphorical concepts semantically per- meating the German news coverage. In the final section, I will summarise the theoretical, methodological and empirical implications of my findings and reflect on the fact that a qualitatively improved science communication can contribute to the re- sponsible use of metaphors. Metaphor and synthetic biology: theoretical dimensions for analysing moral implications Nowadays it is a truism to state that metaphors pervade science (Brown 2008) and are creatively applied in scientific thinking (Katherndahl 2014). Numerous scholars in dif- ferent areas of research have studied the constitutive role of metaphors for scientific thinking and the development of scientific concepts, theories and methods. The works by the philosophers Black (1962) and Blumenberg (1960) were among the first to develop coherent approaches and tools for analysing metaphors – not only – in scientific thought. Gentner and Jeziorski (1993) have investigated the formation of scien- tific work from a historical point of view tracing the conceptual steps taken from meta- phor to analogy. They reveal important structural mappings in the works of scientists, such as Sadi Carnot, Robert Boyle or Paracelus (Gentner and Jeziorski 1993: 448), show- ing how metaphorical mappings and analogies informed the development of scientific the- ories. Hesse, in turn, (1966; 1970) applied Max Black's (1962) and Ivor Richard’s (1936) Döring Life Sciences, Society and Policy (2018) 14:14 Page 4 of 17 interaction theory of metaphor and unravelled the explanatory power of metaphor in the context of the formation of analogical models in science. This research inspired Kay (2000) who investigated central metaphors in genetics such as “books”, “maps” and “information theory”, which all contributed to establishing the scientific rationale of molecular biology (Kay 1997). Later works by Maasen and Weingart (2000), Larson (2011) and Döring et al. (2015) are among the few who provide a discussion of different theories of metaphor, integrate them into the sociology of science and science and technology studies, and provide a methodological approach for exploring metaphorically driven knowledge dynamics in science. This also applies to the work of Nerlich and Hellsten (2005, 2011) who also performed comprehensive empirical research on metaphors in genetics, genomics and synthetic biology, especially news coverage. Even though a lot of research has been undertaken on different levels of media fram- ings, the production and reception of news in different countries (see for example Ancillotti and Eriksson 2016), a critical reflection, theoretical integration and methodo- logical elaboration of philosophical and philological research is still missing. This ap- plies in particular to metaphor theories proposed by Blumenberg (1960) Black (1962), Weinrich (1976), Lakoff and Johnson (1980), Lakoff (1987), Johnson (1987) and ex- tended by others (see Jäkel 1997: 141–146; Döring 2005: 25–122). Metaphor and synthetic biology: theoretical dimensions for analysing moral implications This can strongly constrain how we conceptualise the world but also open-up the possibility to question current conceptualisations and to develop alterna- tive ones (Lakoff and Johnson 1980: 96). Thus – and thirdly – metaphors are creative devices (Weinrich 1976: 175) for the production and shaping of meaning: they can be used to generate or explore alternative meanings that go beyond existing meanings (Lak- off and Turner 1989:80). This heuristic ability to restructure thought patterns (Jäkel 1997: 35) and knowledge dynamics applies to any kind of discourse. Fourth, thought patterns to a considerable extent consist of so-called conceptual metaphors. They should be under- stood as a cognitive deep structure aggregated from converging linguistic metaphors – to be understood as surface structures (Lakoff 1993: 244) – which can be analytically sum- marised in so-called idealised cognitive models (Lakoff 1987: 68), or models of thought (Weinrich 1976: 294). Finally, metaphors are based on a cognitive mapping process: “A metaphor [...] is a process by which we understand and structure one domain of experi- ence in terms of another domain of a different kind” (Johnson 1987: 15). Hence, more concrete meanings (source domain) are projected via a mapping upon an abstract domain (target domain) to make it semantically, cognitively and practically accessible. This map- ping (Weinrich 1976: 283) creates the possibility to analytically access and assess possible implications carried over from the source to the target domain. This can uncover “[…] im- plication complexes […]” (Black 1993: 28) enabling a wide range of possible associations which the focussing aspect of metaphor semantically compresses. Although compressed, implication complexes are important because the metaphorical mapping transfers mean- ing and connected moral values which require linguistic attention (Johnson 1993) and ethical inspection (Kaebnick and Murray 2013). All these aspects of metaphors are interesting in a context where a certain stagnation in metaphors used in the discourses about genetics, genomics and post-genomic re- search has been found (McLeod and Nerlich 2017: 6). So-called canonised metaphors such as “books”, “maps”, “blueprints”, “programmes” or a mechanistic imagery (Boudry and Pigliucci 2013) are still used to scientifically conceptualise and publicly convey dif- ferent sorts of meanings – and moral implications – about scientific issues, progress in research and their ethical, legal and social dimensions (Hellsten and Nerlich 2008; Ner- lich and Hellsten 2009; Nerlich et al. 2009). Metaphor and synthetic biology: theoretical dimensions for analysing moral implications Most of the social science research focusing on SynBio is rooted in media and com- munication sciences, philosophy and history. Hence, methods such as Grounded The- ory or approaches stemming from qualitative research are used to establish and analyse media corpora (Gschmeidler and Seiringer 2012) for the analysis of metaphors (Nerlich and Hellsten 2011). However, studies on the use and role of metaphor (Cserer and Seiringer 2009) often only partly engage with the theoretical dimensions of systematic research on metaphor (Schmitt 2011). This has led to analytical imprecision (Dabrock 2009), mixing linguistic and conceptual imagery and questions revolving around the relevance of certain types of metaphors in the news coverage. The criticism applies both to Keller’s (2002) sem- inal book on metaphors and models used in biology and to an article by Boudry and Pigliucci (2013), which provides an interesting, critical and historical investigation of engineering metaphors in SynBio without developing a basic notion of metaphor. The section on metaphor and worldviews in Boldt’s (2016) edited volume follows a compar- able rationale: the philosophical analyses undertaken here are interesting but a closer engagement with the theories of metaphor would have probably added precision to the ethical assessment of SynBio. Hellsten and Nerlich (2008) and O’Keefe et al. (2015) for example are among the few who perform a systematic analysis that not only includes the development of a compre- hensive media corpus and the application of social science methods, but also engage deeply with theories and methods research on metaphor. They rely on five aspects of metaphor which they argue are of analytical importance. First, metaphors are ubiqui- tous phenomena that pervade all discourses, be they scientific, political or public. They are a basic ingredients of human thinking and not just artistic or poetic decorations (Lakoff and Johnson 1980). In this respect conventionalised metaphors are more im- portant than creative ones as they semantically structure and shape everyday thinking and talking, often pass unnoticed but work unconsciously (Lakoff and Johnson 1980: Döring Life Sciences, Society and Policy (2018) 14:14 Page 5 of 17 Page 5 of 17 139). Second, metaphors possess a focusing function (Blumenberg 1960: 75). They high- light certain semantic aspects of a topic or discursive domain while hiding others (Jäkel 1997: 42). Metaphor and synthetic biology: theoretical dimensions for analysing moral implications This development also received criticism by synthetic biologists such as Porcar and Peretó (2016) or de Lorenzo (2011) (see also de Lorenzo (in press), this thematic series). They acknowledge the heuristic potentials and the semantic traps inherent in different kinds of metaphors. In their view, “meta- phorical biology” (Paton 1992) – that is a biological research conscious about its meta- phorical status – requires a critical inspection of the implications nestling in metaphors and conceptual images, to enable reflexive and responsible use of images in scientific thinking, practice and public communication. The metaphors of “writing”, “books”, “reading” or “blueprints” became conventiona- lised metaphorical reservoirs (Kueffer and Larson 2014: 720) through which they con- vey the image that the chemical structure of the DNA is information that can be read, understood and – to use another metaphor – be re-written, indeed edited. Going beyond such prior research, this paper develops a methodological approach that helps to partly reveal the metaphorical production and transmission of morality in Döring Life Sciences, Society and Policy (2018) 14:14 Page 6 of 17 the German news coverage. Studying the moral implications of metaphorical mappings in this way is important because “[...] the way we frame a given situation [or new tech- nology] will determine what we ought to do about it, and our semantic frames [...] are – at least to some extent – based on metaphor” (Johnson 1993: 52). Metaphors in synthetic biology: a method for analysing moral implications This procedure was followed by grouping the metaphorical expres- sions according to the mapping processes into higher-order conceptual metaphors. These conceptual metaphors represent generic structures that semantically permeate the news coverage and their recurrence bears a considerable impact on the overall meaning of the news coverage. Finally, the mappings in the linguistic metaphors under- lying the different conceptual metaphors were analysed. – for the example provided). Hence, the mappings of the whole corpus underwent an analysis that considered source and target domain as well as a reflection of the mapping processes involved. This procedure was followed by grouping the metaphorical expres- sions according to the mapping processes into higher-order conceptual metaphors. These conceptual metaphors represent generic structures that semantically permeate the news coverage and their recurrence bears a considerable impact on the overall meaning of the news coverage. Finally, the mappings in the linguistic metaphors under- lying the different conceptual metaphors were analysed. The novel aspect of the present approach resides in (a) examining the words surround- ing the words used in the source domain using Der Duden (the German equivalent of the Oxford English Dictionary) and (b) consulting the co-occurrence database Cyril-Belica (corpora.ids-mannheim.de) where each word in a large German language corpus is listed with its ‘profile’ of words with which it regularly co-occurs, that is, with which it is fre- quently used together. Using this database revealed a host of connotations defining the in- tangible semantic field and implication complexes of each metaphor. This enabled me not only to define the exact meaning of each word in the source domain but also provided the possibility to reveal some normally elusive implications. These were systematised and transferred into a list for each conceptual metaphor, with a particular focus on moral im- plications. As a final step, the implications of each conceptual metaphor were discussed in view of responsible language use in terms of whether they provide problematic or fruitful ways of engaging with SynBio (Kueffer and Larson 2014: 722). This involves making some subjective judgements. However, these are based on long-standing discussions with col- leagues about responsible language use in genomics and post-genomics (see Döring 2014). They are also grounded in what Nordgren (1998) calls imaginative ethics and where the key “that morality has to do with metaphors and imagination rather than with well-defined concepts and deduction” (p. 117). Metaphors in synthetic biology: a method for analysing moral implications A first step for analysing the news coverage consisted in establishing a corpus of news- paper articles about SynBio published in Die Frankfurter Allgemeine Zeitung and Der Spiegel. This was done using the LexisNexis news database for the period between 2000 and 2010 (Fig. 1). This period was chosen as it represents the emerging and consolidation phase of Syn- Bio and ends with the press conference given by Craig Venter on his newly designed artificial organism Mycoplasma Mycoides JCVI-syn1.0. The search term ‘Synthetische Biologie’ revealed an overall number of 41 articles for Die Frankfurter Allgemeine and 14 for Der Spiegel. All articles vary in length due to the category of print medium they appeared in and were mostly found in the news and the science section. After having established the data base, the next methodological step consisted in de- veloping a combined approach that merged linguistic (Jäkel 1997: 141–146) and science and technology studies’ (Maasen and Weingart 2000) perspectives of analysing meta- phor. The approach consisted in a first reading of all newspaper articles which helped to get a contextual overview over the structure of the media corpus. First impressions, authors, institutions mentioned, science-policy aspects, visual images, different text types and themes covered were noted in a ‘minute book’ and later on systematised in form of a thematic map. Against this structural and contextualising background, a close line-by-line reading revealed all linguistic metaphors occurring in the two newspapers. This procedure involved a first analysis of the mapping processes to secure the meta- phorical content of each metaphorical instantiation. Two hundred ninety-eight meta- phorical expressions were found in the newspaper coverage and transferred into a table where creative or idiosyncratic metaphors were deleted and the 265 metaphorical ex- pressions (he constructs protein molecules for genes, for example) and their mappings were re-analysed. This procedure enabled the exploration of the degree of Fig. 1 Number of articles appeared per year (kindly provided by Anne Brüninghaus) Döring Life Sciences, Society and Policy (2018) 14:14 Page 7 of 17 Page 7 of 17 metaphoricity of each metaphorical expression found and at the same helped to assess the source domains involved in the metaphorical mapping (building plans for proteins – for the example provided). Hence, the mappings of the whole corpus underwent an analysis that considered source and target domain as well as a reflection of the mapping processes involved. Metaphors in synthetic biology: a method for analysing moral implications In sum, this linguistically informed qualitative approach tries to pave the way for a more systematic and empirical way to reveal moral implications inherent in metaphors. In doing so, it aims at providing a small step towards a more grounded analysis of moral implications and their ethical problematisation. I will now turn to four prevailing conceptual metaphors derived from the 298 metaphorical expressions found in the media coverage, in which “metaphor works by applying to the principal subject a sys- tem of associated implications characteristic of the subsidiary subject [...]” (Black 1993: 28). Metaphors and morality in the German coverage of SynBio Metaphors and morality in the German coverage of SynBio The media discourse in Frankfurter Allgemeine Zeitung and Der Spiegel is mainly char- acterised by four overlapping conceptual metaphors: DOING SYNBIO IS CONSTRUCTING, DOING SYNBIO IS PLAYING A GAME, DOING SYNBIO IS PROGRAMMING and DOING SYNBIO IS WRITING AND EDITING A TEXT. In the following, each conceptual metaphor will be illus- trated by paradigmatic linguistic metaphors taken from the corpus. To start with, the most prominent conceptual metaphor found in the media corpus is DOING SYNBIO IS CONSTRUCTING. Here, the linguistic metaphors found are mainly in- formed by the verbs “bauen” (to build), as in the following example: Page 8 of 17 Döring Life Sciences, Society and Policy (2018) 14:14 1. Die Industrie brauche aauch künftig hervorragend ausgebildete Chemiker, um die für die Produktion von Substanzen notwendigen Moleküle “bauen” zu können. (FAZ 29.10.2002: 49) (Even in the future, industry needs excellently trained chemists who can “build” the necessary molecules for the production of substances) The verb “to build” appears here in the context of a newspaper article providing infor- mation about an early workshop for students studying SynBio. The metaphorical mapping inherent in the phrase “building molecules” (Moleküle bauen) is taken from the more general meaning of “bauen” and clearly applies the semantic field of building construction. The mapping applies what people know about building sites, bricklayers, bricks and all processes involved in house construction to the scientific fields of SynBio. In the quote, the chemists mentioned are – metaphorically speaking – bricklayers on molecular con- struction sites, and the analysis of “bauen” in the dictionary Der Duden reveals implica- tions of a deliberate and systematically plan carried out. The co-occurrence database reveals that the verb “bauen” is strongly correlated with all sorts of buildings and vehicles, which corroborates aspects of a structured and systematic approach which results in pro- ducing something. As building involves the use of vehicles, in the following quote an allusion to our every- day knowledge of vehicles is made through the use of the slang adjective “aufgemotzt” (pimping a car): 2. Wenn sie das Klassenziel erreichen, haben die Studenten eine Lebensform konstruiert, die es so nicht gab: einen synthetisch aufgemotzten Organismus […]. Metaphors and morality in the German coverage of SynBio (FAZ 07.03.2004: 65) (If they attain their group’s aim, the students have constructed a life form that didn’t exist before: a synthetically pimped organism) Here, the metaphorical mapping transfers semantic aspects of tuning cars to genetic- ally modified organisms and helps to convey a meaning of technical improvement as well as an aesthetical change regarding the appearance of the object. The textual con- text, however, restricts the semantic scope of the source domain to cars and car pro- duction – which could also be thought of as the metaphor of the chassis in SynBio – and communicates rather technical implications of production and improvement. This aspect is also corroborated by the findings in the co-occurrence database, in which the semantic field of “konstruieren” (to construct) mainly revolves around vehicles, clocks and –astonishingly – genetic engineering. This result indicates that the meaning of “konstruieren”, at least in the German language, has begun to connotate aspects of gen- etics and biotechnologies. Comparable features also emerge in the following case, where the previously men- tioned source domains of construction and vehicles are exploited in the nouns “Vehi- kel” (vehicle) and “genetische Konstrukte” (genetic constructs): 3. […] gibt es eine Alternative, bei der man gleich ganz auf natürliche Vehikel für genetische Konstrukte verzichtet. (FAZ 07.03.2004: 65) (there is an alternative where one can do completely without the natural vehicle in order to make genetic constructs) Döring Life Sciences, Society and Policy (2018) 14:14 Page 9 of 17 Page 9 of 17 Page 9 of 17 The noun “Vehikel” (vehicle) metaphorically maps the technical aspects of cars or other means of transport onto the domain of biological carriers, while the notion of genetic constructs refers to genetically engineered entities. Following Cyril-Belica, the metaphorical use of vehicle clearly entails a semantic dimension of car driving, motor- isation and traffic, while the notion of construct – as in the case of the verb “to con- struct” – inherits meaning dimensions revolving around biology and genetics. Meanings of building and constructing also have an impact on the use of other nouns. The metaphorical use of modules and bricks is quite frequent in the corpus, ei- ther in compounds such as “Genbausteine” (genetic building blocks) or “Bausteine” (building blocks). 4. Sie wollen nicht nur hier und dort ein Gen von Tier zu Tier transferieren oder auf gut Glück den einen oder anderen Genbaustein austauschen. Metaphors and morality in the German coverage of SynBio (Spiegel 01/2010: 113) (They don’t just want to transfer a gene here and there from animal to animal or randomly exchange this or that genetic building block) “Genbaustein” appears noticeably often and the notion of building block is clearly de- fined in Der Duden as stones for building. The entries found in Cyril-Belica, however, indicate that the meaning of building block has been affected by its metaphorical use in biology as frequent co-occurrences such as cell membrane, decoding or amino acid in- dicate. Hence, the language is beginning to change through what one might call ‘genet- ically modified metaphors’. While meanings of construction and vehicles are changing in German under the in- fluence of biology, SynBio is still dominated by meanings derived from ordinary uses of construction and engineering. But is the building or constructing of new forms of life in terms of cells or metabolic pathways really ‘engineering’? Proponents of responsible language use would question this easy mapping of construction and engineering onto SynBio and ask about the release of previously non-existent forms of life and its eco- logical consequences for bio security and bio safety, for example, as well as the deter- ministic connotations of engineering. The conceptual metaphor of DOING SYNBIO IS PLAYING A GAME is closely related to its partner DOING SYNBIO IS CONSTRUCTING. Both converge in the fact that the words of building bricks and toy blocks are semantically related. The way towards this relation- ship in the context of SynBio has been paved by the use of metaphors by Drew Endy for example: 5. Forscher haben begonnen, Mikroben nach dem Baukastenprinzip neu zusammenzusetzen: Die Kunst-Organismen sollen Rohstoffe produzieren… […]. (Spiegel 33/2006: 126) (Researchers have begun to put together microbes according to the construction kit principle: these artificial organisms are supposed to produce raw materials) Here, the metaphorical transfer of constructing has been mapped onto a specific chil- dren’s toy, the “Baukasten” (construction kit – in Germany normally consisting of coloured wooden blocks in a box). Der Duden explicitly refers in its entry to model kits to toy blocks, while the database Cyil-Belica strongly points towards the notion of Lego Döring Life Sciences, Society and Policy (2018) 14:14 Page 10 of 17 bricks as a toy. Hence, a new dimension of meaning enters the scene: playfulness which is conceptually linked with scientific work. Metaphors and morality in the German coverage of SynBio Such aspects are clearly mirrored in the use of diminutives such as “Bauklötzchen” (toy bricklets) that highlight converging aspects of small size (genetic entities) and children’s games: 6. Mit genetischen Bauklötzchen und synthetischen Molekülen wollen Forscher künstliche Bakterien und Viren konstruieren. (FAZ 07.03.2004: 65) (Using genetic toy bricks and synthetic molecules, researchers want to construct artificial bacteria and viruses) 6. Mit genetischen Bauklötzchen und synthetischen Molekülen wollen Forscher künstliche Bakterien und Viren konstruieren. (FAZ 07.03.2004: 65) (Using genetic toy bricks and synthetic molecules, researchers want to construct artificial bacteria and viruses) The metaphorical construct “genetic toy bricks” in the previous quote includes these aspects and implicitly links the childlike joy of playing with scientific creativity and tin- kering. These aspects converge with the Lego-metaphor, which conceptualises scientists working in SynBio as “Lego-Bauer” (Lego-builders) (Spiegel 01/2010: 113), while their targeted genetic or modified entities become Lego bricks. This metaphor goes back to Drew Endy, a well-known proponent of SynBio, and has been described by Der Spiegel as follows: 7. Als Kind begeisterte sich Endy für die unerschöpflichen Möglichkeiten der Lego- Steine, und Lego-Bauer ist er im Grunde geblieben. Nur dass er seine Werke nun aus anderen Bausteinen errichtet: Er bedient sich des Baukastens der Natur. (Spiegel 01/2010: 113) (As a child Endy was excited by the inexhaustible possibilities of Lego bricks; and he still remains a Lego builder. Of course, he now constructs his works of art out of different bricks: He uses the construction kit of nature) The quote tells the story of Endy’s fascination with the unlimited potentials of Lego bricks and elaborates on this analogy by metaphorically depicting him and fellow scien- tists as Lego-Builders (Lego-Bauer) who use nature as their construction kit (“Baukas- ten der Natur”), and ever since, this metaphor has been used to conceptualise newly developed genetic entities. These entities metaphorically became – in allusion to Wat- son and Cricks model of the double helix – coloured Lego bricks: 8. Bunten Legosteinen gleich werden dabei genetische Bauteile eingesetzt, um Bakterien wunschgemäß besondere Eigenschaften zu verleihen […]. (FAZ 27.01.2008: 61) (Similar to coloured Lego bricks, genetic construction pieces are used to endow bacteria with specific desired attributes) The metaphorical use of Lego led to the idea of a “Lego Biologie” (FAZ 12.11.2008: N1) which represents “synthetisches Bastelmaterial” (synthetic craft materials) (FAZ 26.05.2010: 29). Hence, the source domain of Lego and the metaphorical mapping functioned as an all-encompassing semantic reservoir to conceptualise genetic entities, the process of construction, the scientific discipline that focuses on this construction and the actors doing the construction work. A look in Der Duden shows that there is no entry for Lego while the database Cyril Belica exhibits co-occurrences such as “Spielzeughersteller” (toy manufacturer), Döring Life Sciences, Society and Policy (2018) 14:14 Page 11 of 17 Page 11 of 17 “Spielsachen” (toys; literally: ‘play things’) or “Baukasten” (toy kit). The semantic focus here is on playfulness, childhood play and creativity. 6. Mit genetischen Bauklötzchen und synthetischen Molekülen wollen Forscher künstliche Bakterien und Viren konstruieren. (FAZ 07.03.2004: 65) (Using genetic toy bricks and synthetic molecules, researchers want to construct artificial bacteria and viruses) The question, however, remains as to whether the development of artificial forms of life should be framed in this playful and eu- phemistic way. The connotations and implications of Endy’s metaphor brush over prob- lematic aspects of SynBio and convey ideas of innocent playing and tinkering with toys which, in the context of SynBio, should however be based on stringent bio security mea- sures. Hence, the metaphor opens up a network of implications that responsible language use should unmask and critically discuss. The third metaphorical concept used is the conceptual metaphor DOING SYNBIO IS PRO- GRAMMING. Here, images revolving around computers and software are mainly communi- cated through verbs such as “programmieren” (programming) and “umprogrammieren” (reprogramming). Biologists, geneticists or chemists become computer programmers, while cells and other biological entities such as metabolic processes are conceived as computers or software. Hence, interventions in cells and other biological entities are framed by map- pings derived from the source domain of information technology: 9. Von ihr erwartet man eine Vielzahl von biotechnischen Umbrüchen - beispielsweise Bakterien, die so programmiert werden, dass sie Kohle in Biogas umwandeln, oder Mikroben, die Kerosin produzieren. (FAZ 15.08.2009: 31) (We expect from it a multitude of biotechnical breakthroughs – for example bacteria which are programmed in such a way as to transform coal into biogas, or microbes that produce kerosene) 9. Von ihr erwartet man eine Vielzahl von biotechnischen Umbrüchen - beispielsweise Bakterien, die so programmiert werden, dass sie Kohle in Biogas umwandeln, oder Mikroben, die Kerosin produzieren. (FAZ 15.08.2009: 31) (We expect from it a multitude of biotechnical breakthroughs – for example bacteria which are programmed in such a way as to transform coal into biogas, or microbes that produce kerosene) Der Duden clearly lists an information technology focus on the verb “programmie- ren” (programming), and this is corroborated by the search on the co-occurrence data- base: the metaphorical mapping transmits an information technology focus. The same also holds true for the verb “umprogrammieren” (reprogramming), which refers to a change in the functioning of a biological entity: 10. Wenn dem so ist, wäre das eine gute Nachricht, denn Venter und sein Team sind gegenwärtig dabei, mit Forschungsgeldern von Exxon-Mobil eine fünf bis sieben Quadratkilometer große Algenfarm einzurichten, in der umprogrammierte Algen Biokraftstoff produzieren werden. 6. Mit genetischen Bauklötzchen und synthetischen Molekülen wollen Forscher künstliche Bakterien und Viren konstruieren. (FAZ 07.03.2004: 65) (Using genetic toy bricks and synthetic molecules, researchers want to construct artificial bacteria and viruses) (FAZ 15.08.2009: 31) (If this were true, it would be good news, as Venter and his team are currently engaged in creating an algae farm of seven square kilometres with money from Exxon-Mobil, in which re- programmed algae will produce biofuel) 10. Wenn dem so ist, wäre das eine gute Nachricht, denn Venter und sein Team sind gegenwärtig dabei, mit Forschungsgeldern von Exxon-Mobil eine fünf bis sieben Quadratkilometer große Algenfarm einzurichten, in der umprogrammierte Algen Biokraftstoff produzieren werden. (FAZ 15.08.2009: 31) (If this were true, it would be good news, as Venter and his team are currently engaged in creating an algae farm of seven square kilometres with money from Exxon-Mobil, in which re- programmed algae will produce biofuel) The semantic focus on information technology also affects the images used to con- ceptualise different levels of biological entities and their prescribed functions. In the following quote, for example, journalists go as far as to compare bacteria to universal Turing machines: 11. Es ist durchaus üblich, Bakterien mit universellen Turing-Maschinen zu vergle- ichen, Zellen sind dementsprechend Computer und DNA eine Software. (FAZ 21.05.2010: 33) (It’s absolutely common to compare bacteria with universal Turing machines; accordingly, cells are computers and DNA is software) Page 12 of 17 Döring Life Sciences, Society and Policy (2018) 14:14 Page 12 of 17 Hence, the metaphorical mapping envisages cells as computers and DNA as software. It cognitively provides the opportunity to act upon biological processes from a different conceptual angle than biologists have been accustomed to do. This interpretation is also mirrored in the patterns of IT-related co-occurrences in the Cyril-Belica database: Prevalent nouns such as “hardware”, “software”, “engineering” etc., tighten the semantic field and set a precise focus reconfiguring the semantics of genetics and biology. Computer programs do not only require reprogramming in general, they also need a clear description of how to execute the instructions of a computer programme. Here, the old metaphor of “schreiben” (writing) comes into play, but in the sense of writing programmes rather than letters on a piece of paper. From there, extrapolations are made to ‘guiding’ not only nature but also evolution: The metaphor strongly resonates with the images used in the news coverage about the decoding of the human genome. 6. Mit genetischen Bauklötzchen und synthetischen Molekülen wollen Forscher künstliche Bakterien und Viren konstruieren. (FAZ 07.03.2004: 65) (Using genetic toy bricks and synthetic molecules, researchers want to construct artificial bacteria and viruses) Now we are supposed to write it. This is how Venter reconfigures the success metaphor of the decipherment of the genome and the expectation that’s associated with it of making hereditary genetic material readable) 14. Bisher habe man den genetischen Code gelesen. Nun gelte es, ihn zu schreiben. So greift Venter die Erfolgsmetapher von der “Entschlüsselung” des Genoms und die damit verbundene Erwartung einer Lesbarmachung des Erbgutes auf. (FAZ 25.05.2010: 29) (Up to now we have been able to read the genetic code. Now we are supposed to write it. This is how Venter reconfigures the success metaphor of the decipherment of the genome and the expectation that’s associated with it of making hereditary genetic material readable) This quote brings together all these prevailing images and their well-known meta- phorical mappings. A close inspection of words like “decipherment” in Cyril-Belica proves that all these metaphors have nowadays been taken over by genetics as many co-occurrences with “Erbgut” (genotype), “Genom” (genome) or “Gen” (gene) indicate. Furthermore, these metaphors with their “genetische Buchstaben” (genetic characters) (FAZ 2701.2008), their “Textsalat[e]” (text salads) and their “Textfetzen” (scraps of text) (both Spiegel 01/2010: 110–111) paved the way for the shift from understanding to writing: 15. “Mit der DNA schreiben wir die Anleitung”, sagt George Church in seinem Eckbüro in der Abteilung für Genetik, “und programmieren die Zelle wie einen Computer. “(Spiegel 33/2006: 127) (“Using DNA we write the instruction”, says George Church in his corner office in the department of genetics,” and we programme the cell like a computer”) The source domain of writing enabled a conceptual shift from understanding to cre- ating. It was also flexible enough to be reused and re-metaphorised in metaphors of computational and engineered writing, re-writing and – metaphorically speaking – cre- ative writing. The last aspect in particular is mirrored in the final quote of this section, in which the aim of SynBio is expressed in “deren Genom neu geschrieben werden soll” (whose genome will have to be newly written): 16. Im Gegensatz zu der “Bottom-up”- Konstruktion geht dieser “Top-down”-Ansatz von einfachen Bakterien aus, deren Genom aber neu geschrieben werden soll. (Spiegel-Online 09.07. 2006) (In contrast with the “bottom-up” construction, this “top-down” approach starts with simple bacteria, whose genome is however supposed to be re-written) 16. Im Gegensatz zu der “Bottom-up”- Konstruktion geht dieser “Top-down”-Ansatz von einfachen Bakterien aus, deren Genom aber neu geschrieben werden soll. (Spiegel-Online 09.07. 6. Mit genetischen Bauklötzchen und synthetischen Molekülen wollen Forscher künstliche Bakterien und Viren konstruieren. (FAZ 07.03.2004: 65) (Using genetic toy bricks and synthetic molecules, researchers want to construct artificial bacteria and viruses) Though not explicitly mentioned, DNA is here envisioned as the basic program and the strong deterministic thinking implicated in it leads to the conclusion that even the evolution of humankind could be controlled one day. The network of implications associated with the writing metaphor found in Der Duden and in Cyril-Belica strongly connotes traditional meanings as well as elements nowadays typical of IT-technologies. But a future step is at hand for so-called “gene en- gineers”, namely “blind writing”, which does not require programmers but works on the basis of automated and autonomous algorithms: 13. Die Geningenieure von morgen sollen daher den Computer Zufallsauswahlen kombinieren lassen, schreiben quasi blind die Software synthetischer Organismen. (Spiegel-Online 27.12.2008) (The gene engineers of tomorrow are therefore supposed to let the computer combine random selections, to write quasi blindly the software of synthetic organisms) All in all, the metaphorical reservoir of IT-technologies frames biological processes, entities and their modification as a computational/technological endeavour. This con- ceptualisation is problematic as it focuses on the linear manipulation of life processes and, furthermore, transmits ideas of control of isolated products or processes which hide biological complexity. The narrowness of this conceptual metaphor therefore not only necessitates reflection when used, but also an explicit negotiation and assessment of its underlying assumption: Is doing biology really engineering, programming and reprogramming? The aforementioned aspects and implications partly converge with the conceptual metaphor of DOING SYNBIO IS READING AND WRITING which is the last metaphorical con- cept examined here. This well-known metaphor was used in the context of SynBio to Döring Life Sciences, Society and Policy (2018) 14:14 Page 13 of 17 Page 13 of 17 introduce a shift from reading to writing and indicates a conceptual and epistemic turning point from understanding to creation. Consequently, the metaphorical use of “entschlüsseln” (decoding), “lesen” (reading) and “Entschlüsselung” (deciphering) pre- vail in the first segment of the corpus because the development of SynBio was still on- going and the decoding of the human genome only happened a few years before. The journalist actually refers to the “success metaphor of ‘deciphering”: 14. Bisher habe man den genetischen Code gelesen. Nun gelte es, ihn zu schreiben. So greift Venter die Erfolgsmetapher von der “Entschlüsselung” des Genoms und die damit verbundene Erwartung einer Lesbarmachung des Erbgutes auf. (FAZ 25.05.2010: 29) (Up to now we have been able to read the genetic code. 6. Mit genetischen Bauklötzchen und synthetischen Molekülen wollen Forscher künstliche Bakterien und Viren konstruieren. (FAZ 07.03.2004: 65) (Using genetic toy bricks and synthetic molecules, researchers want to construct artificial bacteria and viruses) 2006) (In contrast with the “bottom-up” construction, this “top-down” approach starts with simple bacteria, whose genome is however supposed to be re-written) A look at both Der Duden and Cyril Belica shows that words like “reading” and “writ- ing” are still firmly rooted in the semantic field of books and texts. Their meanings have not yet extended to uses in genomics or post-genomics. The verb “entschlüsseln” Döring Life Sciences, Society and Policy (2018) 14:14 Page 14 of 17 Page 14 of 17 (deciphering), by contrast, has now acquired a strong semantic focus on genetics, geno- type and genes. This has implications for responsible language use, as it is almost im- possible to avoid these ‘metaphors’. They have become basic and almost unavoidable lexical and conceptual tools for talking about genomic and post-genomic innovation and technologies. To conclude, the analysis revealed four prevailing conceptual metaphors and the study of the implications nestling in the media-metaphorical mappings uncovered the different and sometimes converging arrays of moral implications inherent in these met- aphors. These are summarised in the following table (Fig. 2). Overall, the conceptual metaphors used by the German media highlight aspects of controlled engineering (Cserer and Seiringer 2009) on the one hand and playful, even innocuous, scientific work on the other. Although critical comments with regard to problems revolving around biosafety and biosecurity are occasionally mentioned in the news coverage, journalists (and the scientists they quote) only rarely address the pos- sible dangers, impacts and implications of this technology for biology and society. This can be seen as a lack of responsible thinking induced by irresponsible language use. The newsworthiness of a revolutionary technology and scientific discipline appears to determine the semantics of the news coverage between 2000 and 2010, while a respon- sible use of metaphors and their possible implications is simply lacking and this con- strains responsible thinking as well. Consequently, ethical, legal and social implications of SynBio stay in the background while positive visions of scientific progress abound, creating great expectations of biomedical remedies for modern diseases, combating hunger, making new types of fuel or simply promising genetically modified bacteria that can ‘eat’ ocean oil spills. The language of SynBio: between hidden moralities and responsibility Between 2000 and 2010, two seminal news outlets in Germany depicted Synbio mainly through the lens of four conceptual metaphors that create an unproblematic and technocratic image of SynBio. The semantic associations analysed with the help of Cyril-Belica revealed hidden moral implications inherent in the metaphorical mapping Fig. 2 Moral implications of media-metaphors Döring Life Sciences, Society and Policy (2018) 14:14 Page 15 of 17 Page 15 of 17 processes. The semantic analysis of the words used in the source domains hence proved to be useful as the target domains are not only semantically structured by them, but also inherit the implications and value complexes connected with the metaphorical use of the lexical items used. This not only shows how abstract entities or aspects are framed by concrete domains, cultural knowledge or experiences, but how the source domain resonates with intangible semantic fields and values, and projects its implica- tions onto the target domain. This hidden moral aspect of metaphor use has to date not systematically been studied in this way, neither in research on metaphor (Johnson 1993; Lakoff and Johnson 1999), nor in its role in science technology studies, the social study of science or the sociology of scientific knowledge. The theoretical and empirical potentials outlined here require further conceptual and empirical elaboration. This also applies to the method used. The findings of this paper support a call for more research into the responsible use of language in general and of metaphor in particular (Kueffer and Larson 2014). A technology assessment as metaphor assessment (Mambrey and Tepper 2000), as it has been carried out in this article with respect to media coverage, creates awareness of the moral implications of unconscious conceptual metaphors and can increase under- standing of how public opinions may be shaped by them. It can, of course, also facili- tate ethical reflection and negotiation in science and society. This would, however, require further innovation in terms of enabling participative processes, which avoid the deficit model of public understanding of science and in which scientists, journalists and audiences collaboratively assess the emerging implications of metaphors (Larson 2011). Indeed, this applies to scholars, policy makers, industry and the public who all are – willingly or unwillingly – involved in the project of SynBio. Competing interests Competing interests The author declares that he has no competing interests. p g The author declares that he has no competing interests. The language of SynBio: between hidden moralities and responsibility Whatever linguistic struc- tures or images are used, the language of SynBio and the social, ethical, legal and polit- ical associations it carries requires constant scrutiny because – to paraphrase Abraham Lincoln – you cannot escape ethical responsibility for technologies by evading their moral implications. Acknowledgements g This paper grew out of a symposium entitled ‘Metaphors, Synthetic Biology and Responsibility’ organised by Brigitte Nerlich and Carmen McLeod held at the University of Nottingham on 22 May 2017. I would like to thank Brigitte Nerlich and Carmen McLeod as well as the two anonymous reviewers for their valuable comments on earlier versions of this paper. Thanks also to Anne Brüninghaus (Hamburg) who helped me in generating Fig. 1. Funding G M g German Ministry of Science and Education, project “Towards a Holistic Conception of Life? Epistemic Presumptions and Socio-cultural Implications of Systems Biology”, funding code: 01GP0904. g German Ministry of Science and Education, project “Towards a Holistic Conception of Life? Epistemic Presumptions and Socio-cultural Implications of Systems Biology”, funding code: 01GP0904. Authors’ contributions Authors’ contributions The author read and approved the final manuscript. Ethics approval and consent to participate Not applicable. Publisher’s Note Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Page 16 of 17 Döring Life Sciences, Society and Policy (2018) 14:14 Received: 4 January 2018 Accepted: 22 May 2018 References Ancillotti M, Eriksson S. Synthetic biology in the press: media portrayal in Sweden and Italy. In: Hagen K, Engelhard M, Toepfer G, editors. Ambivalences of creating life: societal and philosophical dimensions of synthetic biology. Dordrecht: Springer; 2016. p. 141–56. 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https://openalex.org/W2788687768	https://re.public.polimi.it/bitstream/11311/1069327/4/Organizational%20goals%20-%20post-print.pdf	English	null	Organizational Goals: Antecedents, Formation Processes and Implications for Firm Behavior and Performance	International journal of management reviews	2,018	public-domain	12,019	Please cite as: Kotlar, J., De Massis, A., Wright, M., & Frattini, F. (2018). Organizational goals: antecedents, formation processes and implications for firm behavior and performance. International Journal of Management Reviews, 20, S3-S18. Abstract The existence of definite organizational goals is a longstanding and central premise in organization and management research, yet a reexamination of this body of knowledge is timely and long overdue. Many important aspects of organizational goals have received very fragmented attention and there has been little prior attempt to synthesize and compare the effects of these different goals on firm behavior and performance. We present a review of existing theoretical and empirical evidence on organizational goals, and develop an analytical framework emphasizing the variety of organizational goals, their attributes, antecedents and outcomes, the role of context and feedback loops. Drawing on this framework we set out an agenda for further research aimed at advancing current understanding of organizational goals and implications for firm behavior and performance. Josip Kotlar Lancaster University Management School Lancaster, LA1 4YX, United Kingdom j.kotlar@lancaster.ac.uk Alfredo De Massis Free University of Bozen-Bolzano Bolzano, 39100, Italy & Lancaster University Management School Lancaster, LA1 4YX, United Kingdom a.demassis@lancaster.ac.uk Mike Wright Imperial College Business School Exhibition Road, 46, London, SW7 and ETH, Zurich mike.wright@imperial.ac.uk Federico Frattini Politecnico di Milano 4/b Via Lambruschini, Milano, 20133 , Italy. federico.frattini@polimi.it Paper accepted for publication in the International Journal of Management Reviews Please cite as: Kotlar, J., De Massis, A., Wright, M., & Frattini, F. (2018). Organizational goals: antecedents, formation processes and implications for firm behavior and performance. International Journal of Management Reviews, 20, S3-S18. Introduction The existence of definite organizational goals is a central premise in organization and management research. It is more than 50 years since Cyert and March’s (1963) study on the behavioral theory of the firm and more than 30 years since Fama and Jensen’s (1983a, 1983b) work on the effects of differences in goals arising from variety of ownership and control. The existence of definite organizational goals is a central premise in organization and management research. It is more than 50 years since Cyert and March’s (1963) study on the behavioral theory of the firm and more than 30 years since Fama and Jensen’s (1983a, 1983b) work on the effects of differences in goals arising from variety of ownership and control. Several decades since the publication of these seminal studies, organizational goals continue to play a central role in management research (e.g., Argote & Greve, 2007; Fiegenbaum, Hart, & Schendel, 1996; Greve, 2003; Shinkle, 2012) and practice (e.g., Collins, 2017; Kaplan & Norton, 2007; Levinson, 2003). However, many important aspects of organizational goals, especially those related to their antecedents, the processes through which they are formed, and their organizational consequences, have received very fragmented attention, and are thus only loosely integrated in our understanding of organizational behavior and performance. This lack of understanding is given added emphasis by recent political and social trends from parties of both the left and right that call into question the purpose of the corporation and its role in addressing social and environmental challenges (Mayer, Wright, & Phan, 2017; Phan, Siegel & Wright, 2016; Several decades since the publication of these seminal studies, organizational goals continue to play a central role in management research (e.g., Argote & Greve, 2007; Fiegenbaum, Hart, & Schendel, 1996; Greve, 2003; Shinkle, 2012) and practice (e.g., Collins, 2017; Kaplan & Norton, 2007; Levinson, 2003). However, many important aspects of organizational goals, especially those related to their antecedents, the processes through which they are formed, and their organizational consequences, have received very fragmented attention, and are thus only loosely integrated in our understanding of organizational behavior and performance. Introduction This lack of understanding is given added emphasis by recent political and social trends from parties of both the left and right that call into question the purpose of the corporation and its role in addressing social and environmental challenges (Mayer, Wright, & Phan, 2017; Phan, Siegel & Wright, 2016; 2 Mitchell et al., 2016; Porter & Kramer, 2014). Thus, a reexamination of the concept of organizational goals, as well as their antecedents and consequences, is timely and long overdue. Given the importance of goal setting for predicting organizational behaviors and outcomes, it is essential to develop a more detailed and comprehensive understanding of the factors affecting firms’ decisions to pursue a specific set of goals. In this article, we present a review of existing theoretical and empirical evidence on organizational goals, and develop an analytical framework emphasizing the variety of goals pursued by business organizations, their antecedents, formation processes and outcomes, the role of context and feedback loops. We then provide an overview of the articles published in this special issue and conclude by proposing an agenda to inform future studies in this area. Given the importance of goal setting for predicting organizational behaviors and outcomes, it is essential to develop a more detailed and comprehensive understanding of the factors affecting firms’ decisions to pursue a specific set of goals. In this article, we present a review of existing theoretical and empirical evidence on organizational goals, and develop an analytical framework emphasizing the variety of goals pursued by business organizations, their antecedents, formation processes and outcomes, the role of context and feedback loops. We then provide an overview of the articles published in this special issue and conclude by proposing an agenda to inform future studies in this area. The Multiple Facets of Organizational Goals Organizational goals can be broadly defined as desired organizational outcomes that can be used to guide action and appraise organizational performance (e.g., Mohr, 1973) but distinct from measurable targets (March & Simon, 1958). There has been much debate about what actually constitutes an organizational goal, and different theoretical traditions offer different conceptualizations. In classic economic theory, firms were traditionally seen as monolithic actors that, under the assumption of perfect rationality, pursue a unitary goal of profit maximization. However, this perspective was critically extended over the years. For example, scholars have argued that “official” goals stated in vision and mission statements do not always accurately reflect what actually drives organizational behaviour (e.g., Perrow, 1961). Most notably, in the seminal book “A Behavioral Theory of the Firm”, Cyert and March (1963) noted that people have goals, but collectivities of people do not. Subsequently, researchers have put substantial effort at understanding the individual goals of those individuals who are most able to influence what the organization actually does, including 3 emphasis on major decision makers, the executive core, or the dominant coalition (Connolly, Conlon, & Deutsch, 1980). Cyert and March (1963) argued that organizational goals are not unitary and given, and emphasized that the firm is a coalition of shareholders, managers, employees, and other parties, each with their own goals. Thus, organizational goals are the result of continued bargaining and stabilization processes among relevant parties within the firm, leading to multiple dimensions along which organizational goals are set, including production, inventory, sales, market share, and profitability. Moreover, behavioral theory suggests that organizational goals influence firm behavior through processes of satisficing, search, adaptive learning, and sequential attention (Cyert & March, 1963; March & Simon, 1958). These basic insights had a tremendous impact on subsequent theory and research (Argote & Greve, 2007), resulting in a variety of related perspectives each putting emphasis on different dimensions and characteristics of organizational goals. Fiegenbaum et al. (1996) integrated major theoretical perspectives to form a three- dimensional matrix of organizational goals. Specifically, motivation theory (Latham & Yukl, 1975), prospect theory (Kahneman & Tversky, 1979) and the resource- based view of the firm (Barney, 1991) provide insights about the content of organizational goals, such as strategic inputs used to evaluate employees' performance, and strategic outputs used to evaluate the performance of managers. The Multiple Facets of Organizational Goals Industrial economics (Porter, 1980), resource dependence (Pfeffer & Salancik, 1978), and institutional theory (DiMaggio & Powell, 1983) perspectives, in turn, elucidate the terms of reference firms use to evaluate organizational goals in relation to the external environment, including competitors, customers, and other stakeholders. Finally, Fiegenbaum et al. (1996) underscore the importance of time as a further dimension of organizational goals, suggesting that they can be defined either in relation to the past accomplishments, as per corporate identity (Dutton & Dukerich, 1991) and 4 4 organizational learning (Levitt & March, 1988) perspectives, or in relation to future ambitions as per the strategic intent perspective (Hamel, Doz, & Prahalad, 1989). In addition to underlying the multi-dimensional nature of organizational goals, prior research also emphasizes that organizational goals are directly linked to goals at other levels of analysis, including individual, group and institutional levels. Finally, directly emanating from the behavioral theory of the firm is the notion that organizational goals are dynamic and change over time in response to changes in internal or external factors (Ansoff, 1979). Taken together, these theoretical perspectives and related research streams emphasize the primacy of organizational goals in directing firm behavior and performance, as well as the complexity arising from their multi-dimensional and dynamic nature. Because organizational goals are inherently complex, it is not surprising that the measurement of organizational goals remains an important and still largely unresolved issue for both management research and practice (Cameron, 2010; Richard et al., 2009; Venkatraman & Ramanujam, 1986). In this regard, Venkatraman and Ramanujam (1986) note that organizations have both financial and operational goals and highlight how measurement of organizational goals is constrained by availability of data, especially when it comes to operational goals, for which primary sources of data are needed, . In practice, the “balanced scorecard” approach (Kaplan & Norton, 2007) has long been used to address the complexity of dealing with multiple goals. Although the balanced scorecard provides a simplified tool to manage multiple organizational goals, this approach offers only a partial answer to managers’ need to understand how complex sets of goals interact to influence multiple outcomes. In sum, existing research on organizational goals is large and provides a wealth of insights for both management research and practice. The Multiple Facets of Organizational Goals In reviewing and synthesizing this body of research we aim for a more unified perspective that provides a comprehensive understanding of organizational goals and their role in business organizations, identifies 5 critical gaps in current research, and points to future research directions that can contribute to move this field forward. Such a comprehensive literature review is important as organizational goals have great practical and theoretical importance and also because assumptions about organizational goals represent, either implicitly or explicitly, the core assumptions on which organization and management research is built. Opening the “Black Box” of Organizational Goals We summarize existing research on organizational goals in Figure 1, which provides a comprehensive view of organizational goals in terms of their varying content and attributes, antecedents, and outcomes. Moreover, the model emphasizes the role of context as well as feedback loops that underlie the dynamic nature of organizational goals. Our analysis underscores the multi-level nature of organizational goals in relation to both their antecedents and outcomes. Figure 1 about here Variety of Organizational Goals: Goal Content and Attributes Variety of Organizational Goals: Goal Content and Attributes Among the variables representing the goals an organization may pursue, researchers have mostly focused on profitability (Greve, 2003), which has direct consequences for managers’ careers and job market value. But organizations often pursue other goals including productivity, sales, market share, and status (Baum et al., 2005). Indeed, research acknowledges the existence of a broad and very heterogeneous array of organizational goals that go beyond profit (e.g., Fiegenbaum et al. 1996). Cyert and March (1963) emphasized different dimensions of the organizational goal construct that reflect the focus of different coalitions within the firm. This early conceptualization was subsequently significantly expanded, specifically with the rise of research on corporate social responsibility, on the one hand, and institutional theory on the other, which respectively inform two main dimensions of organizational goals from prior literature. 6 First, organizational goals can be either financial or non-financial. Financial goals, particularly profit maximization, was long emphasized in early management literature, perhaps reflecting the nature of business during the industrial revolution in the nineteenth century, when firms operated in economic scarcity and were almost unaffected by any interference from the government (Chrisman & Carroll, 1984). The emphasis on profits as a unitary organizational goal was also legitimized by classical economic theory based on Adam Smith’s “invisible hand”, which would ensure that each individual actor’s behaviors, even if driven by self-interest, would lead to the promotion of the public good (Hay & Gray, 1974). During the 1980s, the emphasis on profits became encapsulated in the goal of maximizing shareholder value (Rappaport, 1986). For example, central to the economic “theory of the firm” is the notion that managers’ primary job is to maximize profit in order to create value for shareholders, thereby seeing any behavior that deviates from profit maximization as inherently inefficient, if not immoral (e.g., Fama, 1980; Jensen & Meckling, 1976). However, around this time the advent of the notion of corporate social responsibility led management scholars to begin to reevaluate the role of business in society and to recognize the broader contribution of business to overall societal welfare (Donaldson & Preston, 1995; Freeman, 1984). Governments gradually introduced a large number of socially motivated laws and regulations through which they increased their influence on business. Thus, the legitimacy of business organizations as an institution came into question, and firms started to consider a wider set of non-financial goals (Ansoff, 1979) reflecting their relation to broad societal issues. Variety of Organizational Goals: Goal Content and Attributes The corporate social responsibility literature identifies a large number of non- financial, or social goals, including fulfilling community needs, building positive labor relationships, improving public image, and good citizenship (Keim, 1978). These goals coexist, and sometimes conflict, with the organization’s financial goals. First, organizational goals can be either financial or non-financial. Financial goals, particularly profit maximization, was long emphasized in early management literature, 7 7 A second important content dimension of organizational goals relates to their sources, which can be either internal or external. Internal organizational goals reflect the interests of the coalitions within the firm, for example profit maximization is viewed as the ultimate financial interest of shareholders, whereas growth has typically been seen as a main focus of top management especially if they are not owners, productivity relates to the interests of operations and productivity managers, and quality reflects the interest of product management (cf. Cyert & March, 1963). Moreover, the literature on the role of founders and founding families in business also emphasizes the importance of internal non-financial goals, which reflect the interests and concerns of these parties, such as the ability to exercise family authority, act altruistically toward family members, fulfill desires for belonging, affect and intimacy, and the perpetuation of the family values and dynasty (Chrisman et al., 2012; Gómez-Mejía et al., 2007; Kotlar & De Massis, 2013). Research also acknowledges that firm’s coalitions and different individuals may have, respectively, manifold group and individual goals that concur with the emergence of goal diversity (e.g., Cyert & March, 1963). Moreover, a single individual may have multiple and conflicting goals at the same point in time, which may lead to self-control agency problems and influence firm behavior and performance (De Massis et al., 2016). Hence, goals can be heterogeneous across different firm’s coalitions and different individuals in the same organization as well as for a single individual. On the other hand, firms have multiple external stakeholders with heterogeneous A second important content dimension of organizational goals relates to their sources, which can be either internal or external. Variety of Organizational Goals: Goal Content and Attributes Internal organizational goals reflect the interests of the coalitions within the firm, for example profit maximization is viewed as the ultimate financial interest of shareholders, whereas growth has typically been seen as a main focus of top management especially if they are not owners, productivity relates to the interests of operations and productivity managers, and quality reflects the interest of product management (cf. Cyert & March, 1963). Moreover, the literature on the role of founders and founding families in business also emphasizes the importance of internal non-financial goals, which reflect the interests and concerns of these parties, such as the ability to exercise family authority, act altruistically toward family members, fulfill desires for belonging, affect and intimacy, and the perpetuation of the family values and dynasty (Chrisman et al., 2012; Gómez-Mejía et al., 2007; Kotlar & De Massis, 2013). Research also acknowledges that firm’s coalitions and different individuals may have, respectively, manifold group and individual goals that concur with the emergence of goal diversity (e.g., Cyert & March, 1963). Moreover, a single individual may have multiple and conflicting goals at the same point in time, which may lead to self-control agency problems and influence firm behavior and performance (De Massis et al., 2016). Hence, goals can be heterogeneous across different firm’s coalitions and different individuals in the same organization as well as for a single individual. On the other hand, firms have multiple external stakeholders with heterogeneous goals, including institutions and regulatory bodies, the community, customers and the media (e.g., Freeman, 1984). External or institutional sources of organizational goals are particularly emphasized by institutional theorists (e.g., DiMaggio and Powell, 1983). Examples of external goals include status, rankings, and certifications, among many others. Interestingly, profit maximization is both an internal and external goal as, on the one hand, it responds to 8 the firm’s internal coalitions focus on obtaining side payments (Cyert & March, 1963), while on the other hand it reflects the interests of the firm’s shareholders who aim at increasing their wealth (Fama, 1980). Besides the two main dimensions of organizational goals discussed above, prior research has identified a number of attributes that characterize organizational goals. Perhaps the most widely examined attribute of organizational goals is goal difficulty, which is a central element of goal setting theory and is defined as the probability that a goal can be achieved given current situational characteristics and resources (Locke & Latham, 1990). Variety of Organizational Goals: Goal Content and Attributes Besides the two main dimensions of organizational goals discussed above, prior research has identified a number of attributes that characterize organizational goals. Perhaps the most widely examined attribute of organizational goals is goal difficulty, which is a central element of goal setting theory and is defined as the probability that a goal can be achieved given current situational characteristics and resources (Locke & Latham, 1990). Moreover, goals differ in terms of novelty, or the degree to which the paths for achieving a given goal are known given current capabilities (Sitkin et al., 2011). When goals are both extremely difficult and novel, they are defined as stretch goals (Gary et al., 2017; Kerr & Landauer, 2004). Another important attribute of organizational goals is goal specificity (Locke & Latham, 2002), also known as explicitness (Hollenbeck, Williams, & Klein, 1989), which relates to the completeness of knowledge about what organizational outcomes would fulfill a desired goal (Hirst, 1987). Finally, research differentiates goals in terms of their duration, which defines the time horizons used to evaluate a goal (Loewenstein & Thaler, 1989). Moreover, goals differ in terms of novelty, or the degree to which the paths for achieving a given goal are known given current capabilities (Sitkin et al., 2011). When goals are both extremely difficult and novel, they are defined as stretch goals (Gary et al., 2017; Kerr & Landauer, 2004). Another important attribute of organizational goals is goal specificity (Locke & Latham, 2002), also known as explicitness (Hollenbeck, Williams, & Klein, 1989), which relates to the completeness of knowledge about what organizational outcomes would fulfill a desired goal (Hirst, 1987). Finally, research differentiates goals in terms of their duration, which defines the time horizons used to evaluate a goal (Loewenstein & Thaler, 1989). The content dimensions of organizational goals and their attributes discussed above collectively define the variety of organizational goals, representing the central element of the model presented in Figure 1. Antecedents of Organizational Goals Antecedents of Organizational Goals Questions of what factors influence organizational goals and what are the mechanisms through which these factors operate have been addressed in a large body of research encompassing diverse theoretical perspectives and levels of analysis. Researchers have increasingly highlighted the need to understand the processes and mechanisms through which 9 organizations can pursue a definite set of goals despite the different interests of their individual members. But, notably, Argote and Greve (2007) observed that despite the emphasis posed in Cyert and March’s (1963) behavioral theory on conflicts of interest and internal bargaining among organizational members and coalitions thereof, the theoretical and empirical work exploring how organizational goals are formed is not nearly as refined as work done at the aggregate level. Scholars in the behavioral theory tradition have suggested that organizational goals reflect the power of individual organizational members, organization units (Pearce & DeNisi, 1983), or social groups (e.g., families; Kotlar & De Massis, 2013). The mechanisms through which individual goals influence organizational goals have been substantiated by research in social psychology and political science, which has shown that individuals form coalitions to pursue their goals (Pearce & DeNisi, 1983), as well as the processes and mechanisms that characterize group decision making (Hinsz, Tindale, & Vollrath, 1997). At the organizational level, several factors influence organizational goals such as firm age and size (Short & Palmer, 2003) and firm resources (Audia & Greve, 2006). Organizations that differ in terms of industrial sector, size, ownership type, governance, or market position, among other characteristics, pursue diverse organizational goals. For example, service industry firms are likely to place profitability before size goals, whereas manufacturing firms may follow the opposite approach (Greve, 2008). The upper echelon literature (e.g., Miller, Burke, & Glick, 1998) has focused on the role of the CEO, the board of directors (BoD), and the top management team (TMT). Concerning the CEO, there is significant evidence that CEO goals depend critically on the proportion of firm shares they own (e.g., Martin, Gomez-Mejia, & Wiseman, 2013). When considering the BoD and the TMT, scholars have particularly emphasized their demographic, structural and cognitive diversity (Glick, Miller, & Huber, 1993), the level of consensus 10 (Dess, 1987) or cohesion (Pitcher & Smith, 2001), the goal congruence between parties such as between the CEO and the TMT (Colbert et al., 2008) or between family and non-family coalitions (Kotlar & De Massis, 2013; Patel & Cooper, 2014). Antecedents of Organizational Goals The theory of stakeholder salience provides valuable insights about the mechanisms through which the goals of organizational coalitions influence organizational goals, particularly emphasizing factors such as power, urgency and legitimacy (Agle, Mitchell, & Sonnenfeld, 1999). Finally, prior research has identified antecedents of organizational goals at the institutional level, including the firm’s industry and general economic conditions (Gooding, Goel, & Wiseman, 1996). Relatedly, researchers in the institutional theory literature have emphasized how practices and structures that represent legitimate ways of operating in an industry influence organizational goals through mechanisms of adoption (DiMaggio & Powell, 1983), in turn driven by the perception that a given practice and structure relates positively to firm performance (Barreto & Baden-Fuller, 2006; Davis & Greve, 1997). Outcomes of Organizational Goals Prior research has emphasized the consequences of organizational goals at different levels of analysis. At the individual level, goal setting and human motivation theories link organizational goals to organizational members’ motivation and commitment (Gollwitzer, 1996), as well as to a variety of individual performance indicators such as task performance (Mitchell & Silver, 1990), organizational citizenship behavior (Podsakoff et al., 2000) or creativity (Shalley, 1991). Common in this research is the notion that a key mechanism through which organizational goals influence individual outcomes relates to the incentives and rewards provided to organizational members (Locke & Latham, 2002). Research on the outcomes of organizational goals at the group level has been based on similar theoretical positions and produced similar results. For example, prior studies have linked goal difficulty and specificity to collective motivation and commitment, group 11 behavior (Lant, 1992) as well as group performance (O'Leary-Kelly, Martocchio, & Frink, 1994). The outcomes of organizational goals at the organizational level have been a major subject in strategic management research, where scholars have examined the impact of organizational goals on firms’ strategic behavior, including strategic risk taking (Bromiley, 1991), search (Chen & Miller, 2007), organizational learning (Ben-Oz & Greve, 2012) and, ultimately, firm performance. Behavioral theorists agree that organizations embrace strategic and operational change by introducing new products, entering new markets, engaging in M&As, and increasing R&D investments when they observe declining performance relative to their selected goals (Audia & Greve, 2006; Chrisman & Patel, 2012). behavior (Lant, 1992) as well as group performance (O'Leary-Kelly, Martocchio, & Frink, 1994). Antecedents of Organizational Goals The outcomes of organizational goals at the organizational level have been a major subject in strategic management research, where scholars have examined the impact of organizational goals on firms’ strategic behavior, including strategic risk taking (Bromiley, 1991), search (Chen & Miller, 2007), organizational learning (Ben-Oz & Greve, 2012) and, ultimately, firm performance. Behavioral theorists agree that organizations embrace strategic and operational change by introducing new products, entering new markets, engaging in M&As, and increasing R&D investments when they observe declining performance relative to their selected goals (Audia & Greve, 2006; Chrisman & Patel, 2012). Context Another recurrent topic in existing research on organizational goals is the role of context. Several scholars have emphasized the importance of external factors such as environments and institutions for organizational goals, including environmental instability (Gooding et al., 1996) and expectations from relevant institutions (Mezias, Chen, & Murphy, 2002). However, we know relatively little about the influence of multiple contexts on the antecedents, variety and outcomes of organizational goals. In an attempt to integrate the role of context in our model, we focus on four broad categories of contexts. First, the micro context refers primarily to individual choice and the local environment where decisions are made (Dopfer, Foster, & Potts, 2004), and includes, among other factors, cognition, attention, and logics of individual organizational members (e.g., Locke, 2000). These contexts are likely to shape individual goals and influence the way organizational goals are both formed and implemented in organizations. Second, the meso context refers to the organization-level factors inside the firm, such as ownership and governance, generation of firm leadership, and resources, which are likely to influence the selection of organizational goals and create organizational-level boundaries for the 12 implementation of organizational goals into action. The macro context includes the sector in which the firm operates, the cultural institutions in which the firm is embedded, the existing legal frameworks in a given society or state, and the technologies available to measure and predict the accomplishment of organizational goals. Finally, the chrono context refers to the factors that lead to evolutionary or punctuated changes in the institutional or organizational contexts affecting the organizational goals of firms. For instance, disruptive events along the life of an organization or the sector in which it operates, such as business exits, successions, waves of mergers and acquisitions, shifts in technological paradigms, may determine changes in the organizational goals (Decker & Mellewigt, 2007; Kotlar & De Massis, 2013). The behaviors of the stakeholders in family firms are influenced by the contexts in F db k l Finally, a significant body of research points to the important role of feedback loops that originate from the outcomes of organizational goals and dynamically influence their antecedents and content (Shinkle, 2012). This research shows that firms continuously evaluate their performance against past performance or the performance of other firms (Iyer & Miller, 2008; Mishina et al., 2010). Context Moreover, the behavioral agency model combines insights from prospect theory and agency theory to explain how discrepancies between aspiration and performance influence decision making through mechanisms of problem framing and loss aversion (Harris, Johnson, & Souder, 2013). The evaluation of performance relative to goal aspirations is further complicated by the existence of multiple goals that are not always independent from one another. Multiple organizational goals may have additive effects, jointly influencing a single outcome, as well as interactive effects, such that the accomplishment of one goal may lower or increase the saliency of another goal, following hierarchical rules (Greve, 2008). When organizational decision makers respond to performance feedback regarding different goals, such responses may follow a sequential attention (Ethiraj & Levinthal, 2009; Greve, 2008) or a simultaneous 13 attention logic (Labianca, Fairbank, Andrevski, & Parzen, 2009; Shinkle, 2012). The former envisages that firms shift their attention as particular goals become more or less salient (March & Shapira, 1992). Goals are weakly correlated and/or conflicting, and goal conflict is resolved with temporal differentiation, or sequential attention, where the accomplishment of one goal requires giving up others (Ethiraj and Levinthal, 2009). Under such circumstances, decision makers face “mixed gambles” and their decisions depend primarily on their consideration of goals in relation to current and prospective wealth (Martin, Gomez-Mejia, & Wiseman, 2013), defined either in financial or non-financial terms (Kotlar et al., 2017). Nevertheless, organizational decision makers often pay simultaneous attention to multiple goals that are not necessarily uncorrelated or conflicting (Gaba & Joseph, 2013; Greve, 2008; Labianca, Fairbank, Andrevski, & Parzen, 2009). As goals are often hierarchically-related to each other such that intermediate goals may be instrumental to accomplishing the higher- level goal of profit maximization (Ethiraj and Levinthal, 2009), performance feedback on multiple goals can occur simultaneously, calling decision makers to jointly formulate organizational responses (March & Simon, 1958). In this circumstance, distribution of attention in response to performance feedback regarding hierarchically-related goals can be regulated by different rules (Ocasio, 1997) such as, for instance, priority-based or causality- based attention rules. Papers in the Special Issue In this section, we situate the papers in the special issue within the over-arching framework of Figure 1. As summarized in Table 1, each literature review focuses on a different topic in relation to organizational goals, draws from different theoretical perspectives, and develops new arguments in response to existing research gaps. We discuss the insights offered by these articles in relation to our theoretical model about the antecedents, variety and outcomes of organizational goals. 14 Greve and Teh (2017) examine and integrate the complementary insights provided by research using the behavioral theory of the firm and institutional theory to advance a behavioral theory of organizational responses to external goals. Specifically, they provide a comprehensive review of these two theoretical traditions, emphasizing their strengths and uncovering their respective limitations, and showing how the insights from each perspective can inform the other to provide a more complete understanding of both internal and external sources of organizational goals. The review identifies three main mechanisms through which external goals enter the organization internally. First, firms adopt external goals by creating a supportive dominant coalition that is responsive to institutional pressures. Interestingly, the authors note that the political support for pursuing external goals is stronger when external goals can be linked to other internal goals such as profitability, and can be influenced positively or negatively by external actors. Second, firms are more likely to adopt external goals when members of the dominant coalition see these goals as side payments to important stakeholders outside the firm. Third, external goals can become institutionalized, especially when firms deal with environmental uncertainty and the adoption of an external goal legitimizes the firm in its environment. Taken together, this paper points to the complementary gaps in the behavioral theory of the firm and institutional theory, making way for new studies at the intersection between these two theoretical perspectives. Greve and Teh (2017) examine and integrate the complementary insights provided by research using the behavioral theory of the firm and institutional theory to advance a behavioral theory of organizational responses to external goals. Specifically, they provide a comprehensive review of these two theoretical traditions, emphasizing their strengths and uncovering their respective limitations, and showing how the insights from each perspective can inform the other to provide a more complete understanding of both internal and external sources of organizational goals. Papers in the Special Issue The review identifies three main mechanisms through which external goals enter the organization internally. First, firms adopt external goals by creating a supportive dominant coalition that is responsive to institutional pressures. Interestingly, the authors note that the political support for pursuing external goals is stronger when external goals can be linked to other internal goals such as profitability, and can be influenced positively or negatively by external actors. Second, firms are more likely to adopt external Foss and Linder (2017) criticize the tendency of management scholars to ascribe goals to supra-individual entities such as organizational groups or firms, and to treat these entities as a unitary actor, and thus propose a “microfoundation” perspective aimed at decomposing the collective concept of organizational goals in terms of their lower-level constituent components. Thus, they examine organizational goals as a multilevel concept, and review antecedents and consequences of organizational goals across micro and macro levels of analysis. This review leads the authors to develop a microfoundational framework that 15 explains how organizational goals form and how subsequently they affect organizational outcomes, providing valuable insights for future research that examines the cross-level processes underlying the antecedents and outcomes of organizational goals. In doing so, the authors also identify major theoretical perspectives that inform the micro-level factors (namely, social cognitive theory, theory of mind, elaboration likelihood model, social projection theory, mediating hierarchs and goal-framing theory) and macro-level factors (prospect theory, cognitive biases, individual-level goal setting theory, agency theory, and self-determination theory). William, Pieper, Kellermanns, & Astrachan (2017) provide a systematic review of the growing literature on family business goals, thus contributing new insights about the role of the meso context on organizational goals. Specifically, the authors note that family ownership and management are a common trait of most firms worldwide, and emphasize the role of family-centered goals as a building block of what constitutes a family business as well as a main driver of differences in behavior and performance between family and non-family firms. By reviewing the literature, they identify antecedents, characteristics, related outcomes, moderators and feedback loops that are unique to the family business context. In terms of antecedents, the authors underscore family presence, role of individual family members, founder influence and leadership, family history and culture, socioemotional wealth, organizational identity, succession intentions, and national culture and ethnicity. Papers in the Special Issue The review also informs our understanding of the characteristics of family firm goals in terms of the number and heterogeneity of such goals. The outcomes of family-centered goals include family member relationships, socioemotional wealth, governance, management, and strategy. Then, the review identifies several moderators of the relationship between goal antecedents, goal characteristics, and goal outcomes, including founder centrality, succession, and family involvement in management. Finally, the article clarifies the key elements that characterize 16 16 feedback loops in relation to organizational goals, such as communication and interaction, conflict, community reputation, and the balanced scorecard. Taken together, this review article provides a holistic look at family business goal literature that has the potential to inspire new research directions and advance the understanding of goals in the wider management literature. Gagné (2017) focuses on the consequences of organizational goals for the behavior of organizational members, addressing specifically the question of what mechanisms enable firms to transform organizational goals into individual goals and behaviors. The article draws on the theory of action phases to explain how organizations and employees commit to organizational goals and successfully pursue them. Moreover, it integrates insights from self- determination theory to explain how organizational members develop the motivation and commitment to pursue organizational goals. The theoretical model developed from these perspectives emphasizes specific phases through which organizational goals influence organizational members’ behaviors, namely mission and strategic goal development, organizational level implementation, intentions in the form of strategic plans and dynamic capabilities, individual goal internalization, and implementation plans at the individual level. These goal phases are, in turn, influenced by the attraction, selection, socialization and management of organizational members. In sum, this model helps organize existing research on the individual-level outcomes of organizational goals, and guides future research in the area of behavioral strategy by integrating motivational considerations into strategic management models. Gagné (2017) focuses on the consequences of organizational goals for the behavior of organizational members, addressing specifically the question of what mechanisms enable firms to transform organizational goals into individual goals and behaviors. The article draws on the theory of action phases to explain how organizations and employees commit to organizational goals and successfully pursue them. Moreover, it integrates insights from self- determination theory to explain how organizational members develop the motivation and commitment to pursue organizational goals. Papers in the Special Issue The theoretical model developed from these perspectives emphasizes specific phases through which organizational goals influence organizational members’ behaviors, namely mission and strategic goal development, organizational level implementation, intentions in the form of strategic plans and dynamic capabilities, individual goal internalization, and implementation plans at the individual level. These goal phases are, in turn, influenced by the attraction, selection, socialization and management of organizational members. In sum, this model helps organize existing research on the individual-level outcomes of organizational goals, and guides future research in the area of behavioral strategy by integrating motivational considerations into strategic management models. Table 2 about here While multiple goals are recognized, there is a need for further research into their nature and drivers across all levels. Much research has taken goals as given, yet we need to know more about where goals come from. Research is needed to explore the relationship between the nature of goals and outcomes in order to assess whether organizations are performing well or not. Such research is also important in assessing performance outcome differences between different types of firms. Important research questions arise concerning whether goals are driven endogenously or exogenously or through some combination of both. The variety of exogenous contextual factors are acknowledged (Zahra & Wright, 2011), yet we have little systematic evidence on the differential impact of each of these contextual factors on organizational goals. For example, there may be conflicts between the goals of shareholders and managers and new mechanisms that change ownership and control may determine changes in these goals (e.g. executive stock options, private equity buyouts). There may also be conflicts between the goals of multiple principals and agents. Control by customer-owners in mutual firms diverts organizational goals from profits and growth toward creating customer welfare. Again, in firms characterized by a major overlap between ownership and control (e.g., family firms) managers lose incentive to pursue growth and profitability goals, and prioritize non-economic goals (Gómez-Mejía et al. 2007). A number of research questions also emerge in relation to the outcomes of organizational goals. Most importantly, addressing the conceptual gap between organizational goals and firm performance needs more attention in future research. If we define performance as achievement of goals, most of the performances reported in empirical studies must be classified only as outcomes because these studies did not specify the goals to be achieved; did not clarify the contexts within which the goals are to be achieved; and/or did not assess performance in terms of the extent to which the outcomes have achieved the goals. Further While multiple goals are recognized, there is a need for further research into their nature and drivers across all levels. Much research has taken goals as given, yet we need to know more about where goals come from. Research is needed to explore the relationship between the nature of goals and outcomes in order to assess whether organizations are performing well or not. Such research is also important in assessing performance outcome differences between different types of firms. Future Research Directions An agenda for future research based on the elements in the framework of organizational goals developed in Figure 1 is summarized in Table 2. 17 Table 2 about here Table 2 about here Important research questions arise concerning whether goals are driven endogenously or exogenously or through some combination of both. The variety of exogenous contextual factors are acknowledged (Zahra & Wright, 2011), yet we have little systematic evidence on the differential impact of each of these contextual factors on organizational goals. For example, there may be conflicts between the goals of shareholders and managers and new mechanisms that change ownership and control may determine changes in these goals (e.g. executive stock options, private equity buyouts). There may also be conflicts between the goals of multiple principals and agents. Control by customer-owners in mutual firms diverts organizational goals from profits and growth toward creating customer welfare. Again, in firms characterized by a major overlap between ownership and control (e.g., family firms) managers lose incentive to pursue growth and profitability goals, and prioritize non-economic goals (Gómez-Mejía et al. 2007). A number of research questions also emerge in relation to the outcomes of organizational goals. Most importantly, addressing the conceptual gap between organizational goals and firm performance needs more attention in future research. If we define performance as achievement of goals, most of the performances reported in empirical studies must be classified only as outcomes because these studies did not specify the goals to be achieved; did not clarify the contexts within which the goals are to be achieved; and/or did not assess performance in terms of the extent to which the outcomes have achieved the goals. Further 18 research questions relate to the impact of organizational goals on outcomes at the individual, group, and institutional levels. Moreover, it will be important for future studies to consider links between organizational goals and outcomes across both internal/external and financial/non-financial goals dimensions. Finally, a major area for further research concerns how organizational goals adapt to feedback loops regarding organizational outcomes and contextual changes. In particular how, at various levels, changes to goals are or are not implemented. Moreover, prior research has largely focused on feedback regarding a single performance goal, namely firm profitability (Shinkle, 2012), thereby replacing March and Simon’s (1958) seminal notion that multiple goals coexist in organizations (Gaba & Joseph, 2013; Gavetti et al., 2012). Thus, we are left with a gap in understanding of conditions leading decision makers to embrace different rules to guide attention and responses to performance feedback regarding multiple hierarchically-related goals. Table 2 about here Taken together, our theoretical framework and the articles published in this special issue have important implications for practice as well as for future research. In particular, an important issue concerns the need for organizations to define performance in relation to the goals they are aiming to achieve. Similarly, analysts need to assess performance in terms of the extent to which the outcomes have achieved the organization’s goals. Also, our proposed framework can constitute a background policy document for policy makers. The goals of business organizations are being paid increasing attention in the public domain, and our study facilitates understanding of the various types of goals and their attributes, and can support policy makers in their decisions on how to build a system of supporting initiatives for organizational goals in line with the social and environmental challenges they aim to address. Conclusion Conclusion This paper starts from acknowledging the need for a reexamination of the vast theoretical and empirical research on organizational goals. Through a review of the literature, 19 we develop an analytical framework that is helpful to synthetize theoretical and empirical research on organizational goals, by focusing on the variety in type of goals an organization may pursue, the goal attributes, antecedents and outcomes, the contextual influences on goal setting, and the feedback loops through which the achievement of goals influences their formation and organizational decision making. 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References Summary of the Special Issue Articles 25 Title Authors Topic Theory Key arguments Goal selection internally and externally: A behavioral theory of institutionalization Greve & Teh The role of institutional environment in providing organizational goals Behavioral theory of the firm Institutiona l theory Organizational goals have both internal and external sources Review of internal and external sources of organizational goals and their selection Mechanisms through which goals enter the organization internally: • Supportive Dominant Coalition • Side Payments to Important Stakeholders • Goal Institutionalization Review of complementary gaps in the behavioral theory of the firm and institutional theory Microfoundations of organizational goals: A review and new directions for future research Foss & Linder Microfoundatio ns of organizational goals Cross-level aspects of organizational goals Multi- theoretical approach Organizational goals are both independent and dependent variable Review of main antecedents and consequences of organizational goals in extant research Review of cross-level (macro-to-micro and micro-to-macro) processes linking organizational goals to its antecedents and consequences. Review of main theories related to research questions: • Micro: social cognitive theory; theory of mind; elaboration likelihood model; social projection theory; mediating hierarchs; goal-framing theory • Macro: prospect theory; cognitive biases; individual-level goal setting theory; agency theory; self- determination theory Family firm goals and their effects on strategy, family, and organizational behavior: A review and research agenda William, Pieper, Kellerma nns, & Astracha n The antecedents, characteristics and outcomes of organizational goals in family firms. Multi- theoretical approach Family ownership and management create family-centered goals that cause differences in behavior and performance between family and non-family firms. Identifies antecedents, characteristics and outcomes of family business goals, as well as moderating factors in the relationship between goal antecedents, goal characteristics, and goal outcomes and the role of feedback loops. • Antecedents of family firm goals include family presence, role of individual family members, founder influence and leadership, family history and culture, socioemotional wealth, organizational identity, succession intentions, and national culture and ethnicity. • Characteristics of family firm goals include the number and heterogeneity of goals. • Outcomes of family firm goals include family member relationships, socioemotional wealth, governance, management, and strategy. • Moderators include founder centrality, succession, and family involvement in management. • Feedback loops are characterized by communication and interaction, conflict, community reputation, and the balanced scorecard. References From strategy to action: Transforming organizational goals into organizational behavior Gagné How organizational goals are transformed into organizational behavior Human motivation theories Advances Motivational Model of Organizational Goal Pursuit where a social context (organizational goals) influences individual conditions (motivation), which in turn influence individual action, which influences social outcomes (organizational success). Provides a conceptual map of the organizational goals construct across literatures. Examines the links between organizational-level goal setting, the internalization of organizational goals by satisfying individual psychological needs, employees’ implementation intentions, incentives and individual employees’ outcomes. Proposes a process for transforming strategy into action based on exercises and practices. 25 Figure 1. Organizational Goals: Variety, Antecedents, Outcomes and Context Outcomes of Organizational Goals CONTEXT Bargaining Antecedents of Organizational Goals Meso context: Ownership and governance Generations Resources  Internal goals External goals Growth Production Quality Individual level: Organizational Members Institutional level: Practices and structures VARIETY OF ORGANIZATIONAL GOALS Financial goals Non- financial goals Status Rankings Certifications Control Identity Emotions Profit Goal attributes: Difficulty, novelty, specificity, duration Macro context: Sector Culture & Law Technology  Chrono context: Time & life cycle Disruptions (succession, exit, M&A)  Individual level: Motivation & commitment Individual performance Organizational level: Firm behavior Firm performance Micro context: Cognition Attention Logics Incentives and rewards Adoption Strategic decision-making Organizational level: Upper echelon (CEO, BoD, TMT) Planning Group level: Organizational coalitions Group salience Group level: Collective motivation & commitment Group behavior Group performance Social interaction Goal content: Feedback loops: Sequential vs. simultaneous attention logic; Attention rules (e.g., priority-based, causality-based); Mixed gambles Chrono context: Time & life cycle Disruptions (succession, exit, M&A)  VARIETY OF ORGANIZATIONAL GOALS Individual level: Organizational Members Control Identity Emotions Growth Production Quality Group level: Organizational coalitions Emotions Profit Financial goals Non- financial goals Planning Status Rankings Certifications Institutional level: Practices and structures Adoption External goals Goal attributes: Difficulty, novelty, specificity, duration 26 Table 2. Some Future Research Questions on Organizational Goals Individual level Group level Organizational level Institutional level Variety of organizational goals How are heterogeneous goals prioritized/reconciled by individuals and organizations? Who takes the initiative in resolving conflicting goals between different organizational members? How do number and variety of organizational members affect goal types? To what extent do different organizational coalitions and groups have multiple [and conflicting] goals? How do these differ according to different types of groups? References To what extent can changes in ownership structure and different types of new owners (next generation family; private equity/VC; public markets; SWFs. etc.) change organizational goals? How do multiple goals interact? What are the mutual relationships between financial and non-financial, and external and internal goals? What are the attributes of different types of goals? How does the heterogeneity of goals vary across ownership types, sectors, etc.? To what extent is there isomorphism regarding organizational goals? To what extent do different institutional actors create a need for organizations to address multiple [and conflicting] goals? Does the number and heterogeneity of institutions influence the types of organizational goals? If yes, how? Antecedents of organizational goals Are there other mechanisms, in addition to bargaining and/or stabilization, through which organizational members can influence individual goals? Does the formation of individual goals depends on individuals’ characteristics? How do goals formation processes differ when different types of individuals are involved? How do organizational members, organizational coalitions and institutions influence each other’s goals, and how do they interact in influencing collective and organizational goals? Are there other mechanisms, in addition to salience, through which organizational coalitions influence organizational goals? How does the organization’s upper echelon influence the number and diversity of organizational goals, and the processes through which goals are formed? How do organizations implement their goals once they have been defined? How does the organization identify decisions and allocate tasks that must be made to pursue the goals? How do organizational goals vary between similar firms in different institutional contexts? To what extent do capital market actors influence the nature of organizational goals and their timescale? Outcomes of organizational goals How do the outcomes of organizational goals influence individual behavior such as commitment, retention, turnover? What incentives and rewards system are better suited to motivate individuals within the organization? Do these mechanisms vary among different types of individuals and depending on different types of goals? How do the outcomes of organizational goals influence group behavior such as commitment, conflict, leadership, turnover? What social interaction mechanisms are better suited to determine collective motivation, behavior and performance? Do these mechanisms vary depending on the types of goals? To what extent do profits and observed performance reflect the achievement of organizational goals? How do financial/non-financial and internal/external goals influence non-financial and external/internal goals, and vice-versa? References How does the content of organizational goals interact with goal attributes in determining performance at the individual, group, and organizational level? What are the organizational constraints on goal implementation? How does planning processes change depending on goal content and attributes? How may the pursuit of organizational goals create pressures that determine changes at the institutional level? What’s the difference between purpose and goals? Have these two concepts different implications at institutional level? Is purpose a precursor of goals? What are the implications of corporate purposes for policy and decision making? Institutional level Organizational level 27 How do contextual factors such as institutional policies influence group goals and subsequent behavior relating to remuneration and job satisfaction? What are the consequences for organizations and coalitions within organizations? Do contextual factors such as resources, generation of leadership control and ownership or governance configurations change the processes through which collective goals are formed? How and why? Do contextual factors such as resources, generation of leadership control and ownership or governance configurations affect goal setting and strategic planning processes? How and why? How does context influence the relationships between the antecedents and the variety of organizational goals? How does context influence organizational goals and the relationships between organizational goals and their antecedents/outcomes? Do organizational goals, goal formation processes and their consequences changes across firms from different industries? How do firms with different ownership and/or governance structures differ in how they form organizational goals and their antecedents/consequences How do technologies influence the measurement and achievement of organizational goals? For instance, how can social networks, big data and analytics be used to collect and interpret data about goal achievement and the to predict the achievement of organizational goals? How does the social, legal and cultural environment influence organizational goals and the relationships between organizational goals and their antecedents/outcomes? How do institutional and political notions of the purpose of the corporation affect the nature of organizational goals? To what extent does the tax regime influence short-term vs long-term organizational goals? In relation to the chrono-context, how do global and national crises, stages of economic development or situational events such as succession and exit affect organizational goals? How do organizational culture and values (e.g., collectivism vs. individualism) and structure (e.g., power distance) influence goals at different levels? What facilitates/constrains adaptation of group level goals in relation to organizational feedback? References How do group goals constrain the ability of organizations to adapt to feedback/ How do firms use feedback to update their goals? How does feedback influence the role of individual organizational members, coalitions, upper echelons, and institutions? To what extent do organizations change or evolve their goals as a consequence of feedback mechanisms? When does organizational goal change occur? How is goal change related to organizational life-cycles? How does goal change occur? What factors facilitate and constrain such change? To what extent? What attention logics/rules work better in specific circumstances? When, and why? How do organizations adapt goals in relation to institutional change? How and through which mechanisms does the pursuit of organizational goals determine changes in established institutions? For instance, how does the pursuit of social goals in businesses influence the establishment of new norms and regulations which, in turn, affect organizational behavior? The role of context How do contextual factors such as institutional policies (e.g., executive remuneration, pensions, and job tenure) influence individual goals relating to remuneration and job satisfaction? What are the consequences for individuals’ motivation, commitment and performance? And the consequences for organizations? How do contextual factors such as institutional policies influence group goals and subsequent behavior relating to remuneration and job satisfaction? What are the consequences for organizations and coalitions within organizations? Do contextual factors such as resources, generation of leadership control and ownership or governance configurations change the processes through which collective goals are formed? How and why? Do contextual factors such as resources, generation of leadership control and ownership or governance configurations affect goal setting and strategic planning processes? How and why? The role of feedback loops Under what conditions do decision makers embrace different logics and rules to guide attention and responses to performance feedback regarding multiple hierarchically-related goals? What response patterns do they follow? Who takes the initiative in the response process? How do decision-makers respond to incomplete, delayed and ambiguous feedback on organizational goals? What facilitates/constrains adaptation of group level goals in relation to organizational feedback? How do group goals constrain the ability of organizations to adapt to feedback/ m How does the social, legal and cultural environment influence organizational goals and the relationships between organizational goals and their antecedents/outcomes? How do institutional and political notions of the purpose of the corporation affect the nature of organizational goals? References To what extent does the tax regime influence short-term vs long-term organizational goals? In relation to the chrono-context, how do global and national crises, stages of economic development or situational events such as succession and exit affect organizational goals? How do organizational culture and values (e.g., collectivism vs. individualism) and structure (e.g., power distance) influence goals at different levels? l How do organizations adapt goals in relation to institutional change? How and through which mechanisms does the pursuit of organizational goals determine changes in established institutions? For instance, how does the pursuit of social goals in businesses influence the establishment of new norms and regulations which, in turn, affect organizational behavior? How does the social, legal and cultural environment influence organizational goals and the relationships between organizational goals and their antecedents/outcomes? How does the social, legal and cultural environment influence organizational goals and the relationships between organizational goals and their antecedents/outcomes? How does context influence the relationships between the antecedents and the variety of organizational goals? How does context influence the relationships between the antecedents and the variety of organizational goals? How does context influence the relationships between the antecedents and the variety of organizational goals? How does context influence organizational goals and the relationships between organizational goals and their antecedents/outcomes? How do institutional and political notions of the purpose of the corporation affect the nature of organizational goals? Do organizational goals, goal formation processes and their consequences changes across firms from different industries? To what extent does the tax regime influence short-term vs long-term organizational goals? How do firms with different ownership and/or governance structures differ in how they form organizational goals and their antecedents/consequences In relation to the chrono-context, how do global and national crises, stages of economic development or situational events such as succession and exit affect organizational goals? How do organizational culture and values (e.g., collectivism vs. individualism) and structure (e.g., power distance) influence goals at different levels? How do firms use feedback to update their goals? How does feedback influence the role of individual organizational members, coalitions, upper echelons, and institutions? What attention logics/rules work better in specific circumstances? When, and why? 28
W2464850821.txt	https://bsd.biomedcentral.com/track/pdf/10.1186/s13293-016-0084-8	en		Male and female hypertrophic rat cardiac myocyte functional responses to ischemic stress and β-adrenergic challenge are different	Biology of sex differences	2,016	cc-by	8,721	Bell et al. Biology of Sex Differences (2016) 7:32 DOI 10.1186/s13293-016-0084-8 RESEARCH Open Access Male and female hypertrophic rat cardiac myocyte functional responses to ischemic stress and β-adrenergic challenge are different James R. Bell1†, Claire L. Curl1†, Tristan W. Harding1, Martin Vila Petroff2, Stephen B. Harrap1 and Lea M. D. Delbridge1,3* Abstract Background: Cardiac hypertrophy is the most potent cardiovascular risk factor after age, and relative mortality risk linked with cardiac hypertrophy is greater in women. Ischemic heart disease is the most common form of cardiovascular pathology for both men and women, yet significant differences in incidence and outcomes exist between the sexes. Cardiac hypertrophy and ischemia are frequently occurring dual pathologies. Whether the cellular (cardiomyocyte) mechanisms underlying myocardial damage differ in women and men remains to be determined. In this study, utilizing an in vitro experimental approach, our goal was to examine the proposition that responses of male/female cardiomyocytes to ischemic (and adrenergic) stress may be differentially modulated by the presence of pre-existing cardiac hypertrophy. Methods: We used a novel normotensive custom-derived hypertrophic heart rat (HHR; vs control strain normal heart rat (NHR)). Cardiomyocyte morphologic and electromechanical functional studies were performed using microfluorimetric techniques involving simulated ischemia/reperfusion protocols. Results: HHR females exhibited pronounced cardiac/cardiomyocyte enlargement, equivalent to males. Under basal conditions, a lower twitch amplitude in female myocytes was prominent in normal but not in hypertrophic myocytes. The cardiomyocyte Ca2+ responses to β-adrenergic challenge differed in hypertrophic male and female cardiomyocytes, with the accentuated response in males abrogated in females—even while contractile responses were similar. In simulated ischemia, a marked and selective elevation of end-ischemia Ca2+ in normal female myocytes was completely suppressed in hypertrophic female myocytes—even though all groups demonstrated similar shifts in myocyte contractile performance. After 30 min of simulated reperfusion, the Ca2+ desensitization characterizing the male response was distinctively absent in female cardiomyocytes. (Continued on next page) * Correspondence: lmd@unimelb.edu.au † Equal contributors 1 Department of Physiology, University of Melbourne, Melbourne, Victoria, Australia 3 Cardiac Phenomics Laboratory, Department of Physiology, University of Melbourne, Melbourne, Victoria 3010, Australia Full list of author information is available at the end of the article © 2016 The Author(s). 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. 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. Bell et al. Biology of Sex Differences (2016) 7:32 Page 2 of 15 (Continued from previous page) Conclusions: Our data demonstrate that cardiac hypertrophy produces dramatically different basal and stress-induced pathophenotypes in female- and male-origin cardiomyocytes. The lower Ca2+ operational status characteristic of female (vs male) cardiomyocytes comprising normal hearts is not exhibited by myocytes of hypertrophic hearts. After ischemia/reperfusion, availability of activator Ca2+ is suppressed in female hypertrophic myocytes, whereas sensitivity to Ca2+ is blunted in male hypertrophic myocytes. These findings demonstrate that selective intervention strategies should be pursued to optimize post-ischemic electromechanical support for male and female hypertrophic hearts. Keywords: Cardiomyocyte, Sex/gender, Cardiac hypertrophy, Ischemia/reperfusion, Stress response Background For both women and men, cardiovascular disease is the leading cause of death and disability [1, 2]. Cardiac hypertrophy is the most potent cardiovascular risk factor after age [3], and importantly, relative mortality risk linked with cardiac hypertrophy is greater in women [4]. There is a growing clinical recognition that myocardial cardiac stress responses, including neurohumoral activation, exhibit inherent sex difference [5]. Ischemic heart disease is the most common form of cardiovascular pathology for men and women [6]. Ischemic events occur earlier in men, and ischemia-related arrhythmia and sudden cardiac death incidence are higher in men. In contrast, while younger women have a lower risk of ischemic heart disease, in the event of a myocardial infarct, they experience higher mortality and rate of heart failure, even though reperfusion status is equivalent [7, 8]. Women with ischemic heart disease have higher rates of hospitalization than men [9]. The essential question of whether the mechanisms underlying ischemic heart disease in women differ from men remains unanswered [10]—and the cognate issue of whether ischemia coincident with hypertrophic comorbidity has differing gender aetiology and outcome has not been addressed. The importance of achieving a more detailed understanding of basic mechanisms of sex difference in pathophysiologic processes, which have particular relevance to cardiovascular disease and health demography, has been emphasized [11]. The value of sex-inclusive quality preclinical data to inform large-scale clinical studies has also been recently highlighted in the context of unexpected findings delivered by the RELAX trial—which sildenafil (a phosphodiesterase 5 inhibitor) did not show a beneficial effect in the treatment of heart failure with preserved ejection fraction [12]. In retrospect, it became apparent that this outcome might have been predicted if the rationale for the trial had not been reliant on data from male-only human and animal studies [13, 14]. Experimentally, important insights have been gained in relation to sex-specific differentials in the impacts of cardiac hypertrophy and of exposure to ischemic insult—yet still, knowledge is lacking [15, 16]. The extent of hypertrophic remodeling in female rodents in response to loading stress is generally attenuated, yet exogenous oestrogen and oestrogen receptor (oestrogen receptor α (ERα)) agonists have been shown to increase mortality in mice subjected to aortic constriction or to infarction [17–19]. Generally, female hearts are less susceptible to post-ischemic contractile dysfunction, findings which primarily derive from experiments involving animal models where function is not compromised by preexisting pathologies [15, 20–22]. We have previously established that the intrinsic resistance of isolated female hearts to ischemic insult is abrogated in a setting of primary cardiac hypertrophy [23]. Most fundamentally, protection against hypertrophy and ischemia-driven arrhythmogenesis and contractile dysfunction is determined by cardiomyocyte Ca2+ regulatory status. Whilst limited sex-specific information is available at the myocyte level, in vitro findings suggest differential female and male contractile/Ca2+ handling characteristics in simulated ischemia and also sex differences in the responsiveness of myocytes to manipulation of adrenergic activation [24, 25]. Specifically, it is reported that during simulated ischemia, only female cardiomyocytes exhibit a reduction in Ca2+ transient amplitude (vs non-ischemic control) and that in reperfusion, female cardiomyocytes are hyper-contractile (vs males) and more likely to survive the ischemia/ reperfusion challenge [24]. Sex differences in adrenergic responses apparently reflect reduced cyclic adenosine monophosphate (cAMP) levels in female myocytes (vs male), contributing to lower Ca2+ levels and internal Ca2+ store flux responses [25]. Importantly, no previous cardiomyocyte functional studies have interrogated the role of pre-existing hypertrophic pathology in determining the sex-specific responses to ischemic injury—a highly relevant clinical scenario of comorbidity [26, 27]. In this study, utilizing an in vitro experimental approach, our goal was to examine the proposition that responses of male and female cardiomyocytes to stress stimuli (β-adrenergic activation and simulated ischemia/ Bell et al. Biology of Sex Differences (2016) 7:32 reperfusion insult) may be differentially affected by the presence of pre-existing cardiac hypertrophy. Using a purpose-derived rat model of normotensive cardiac hypertrophy, the hypertrophic heart rat (HHR), a range of cardiomyocyte morphologic and electromechanical functional studies was performed using microfluorimetric techniques. We have previously reported premature mortality in the HHR (vs normal heart rat (NHR)), with ex vivo HHR exhibiting normal systolic function and an increased vulnerability to ischemia/reperfusion dysfunction/injury [23, 28]. The present study now demonstrates that cardiac hypertrophy produces dramatically different pathophenotypes in female- and maleorigin cardiomyocytes exposed to ischemia. These findings suggest implications for sex-specific refinement of reperfusion therapeutic interventions. Methods Animals Animals were obtained from established colonies of the HHR and the control NHR, maintained at the Biological Research Facility at the University of Melbourne, Australia, and derived as reported [29]. Briefly, these rats originated from the F2 progeny of a cross between the Fischer 344 and spontaneously hypertensive rat (SHR). Normotensive offspring were selected for either enlarged or normal heart size by echocardiography over successive generations to achieve genetic stability. Telemetry, tail-cuff plethysmography and direct intra-arterial recording methods have been used to determine the normotensive status of these animals [29]. Experiments were conducted and animals handled in the manner specified by the NHMRC/CSIRO/ACC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1997) and the EU Directive 2010/63/EU for animal experiments, with approval and oversight by the University of Melbourne Animal Ethics Committee. Cardiomyocyte isolation procedure Ventricular myocytes were isolated from NHR and HHR rats, as described [30]. Briefly, animals were weighed, killed by decapitation under deep halothane anaesthesia, hearts were excised, trimmed, buffer washed and blotted prior to taking weight measurement by tare. Hearts were retrogradely perfused with bicarbonate-buffered Ca2+-free Krebs (in millimolars: 118 NaCl, 4.8 KCl, 1.2 KH2PO4, 1.2 MgCl2, 25 NaHCO3, 11 D-glucose) at 73 mmHg for 3 min at 37 °C. Cardiomyocytes were then perfused with type II collagenase (50 mg/ml, 295 U/mg; Worthington Biochemical Corporation, NJ, USA) to enable heart digestion. The heart was removed from the cannula, and the ventricular cardiomyocytes dispersed in Krebs/collagenase solution (in millimolars: 146.2 NaCl, 4.7 KCl, 0.4 NaH2PO4-H2O, 1.1 MgSO4-7H2O, 10 (4-(2- Page 3 of 15 hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 11 D-glucose) supplemented with trypsin inhibitor (25 μg/ml; Sigma-Aldrich, MO, USA) to inhibit residual collagenase activity. For each heart, 100 rod-shaped and regularly striated cardiomyocytes were selected at random for length and width measurement at ×400 magnification using an inverted light microscope and calibrated eye piece as previously described [31]. Cardiomyocyte volume was derived from the product of cell length and width, multiplied by 7.59 × 10−3 pl/μm2 [32]. Cardiomyocyte Ca2+ handling and twitch analysis Isolated myocyte Ca2+ levels and shortening performance were measured [30]. Cell suspensions were incubated with the Ca2+ fluorescent dye, fura2-AM (2.5 μM, 20 min, 25 °C; Invitrogen). Myocytes were superfused with 2 mM Ca2+-HEPES buffer (in millimolars: 146.2 NaCl, 4.7 KCl, 0.4 NaH2PO4H2O, 1.1 MgSO47H2O, 10 HEPES, 11 glucose, pH 7.4) and stimulated to establish steady state contractile performance at 4 Hz (5 min, 37 °C). Cardiomyocyte twitch properties were assessed by video-based edge detection (IonOptix, MA, USA). The indices used to describe twitch cycle were twitch amplitude normalized to diastolic cell length (twitch amplitude percentage) and maximal rate of shortening and lengthening (mrs and mrl, respectively; μm/s, normalized to diastolic cell length). Myocyte Ca2+ signals were measured by microfluorimetry (360:380 nm fluorescence ratio, 1000 Hz sampling; IonOptix, MA, USA) [30]. Fluorescence signals were corrected for background. The indices used to describe the Ca2+ transient were amplitude (F360:380) and the time constant of decay (tau, ms; time constant measured from curve fitted from 10 % below the peak level to baseline of transient). Cardiomyocyte twitch and Ca2+ fluorescent ratio properties were measured off-line using IonWizard (IonOptix) and were determined by average of ten steady state transients for each myocyte. Ca2+-shortening phase loops were constructed for individual activation cycles by plotting Ca2+ level vs myocyte shortening throughout the shortening and lengthening phases of the cycle. During the relaxation (i.e. lengthening) phase, the Ca2+ level at half (50 %) relaxation may be interpreted as an index of myofilament Ca2+ sensitivity [33]. Adrenergic challenge protocol To assess the response of male and female NHR and HHR cardiomyocytes to an adrenergic challenge, myocytes stabilized for 5 min in 2 mM Ca2+-HEPES buffer were superfused with 10 nM isoproterenol for 5 min. Previous dose range and time-course studies confirmed this was an optimal time point to allow a maximal shortening and amplitude Ca2+ response to reach a new steady state [34]. Bell et al. Biology of Sex Differences (2016) 7:32 Page 4 of 15 (and consequently volume) dimension (Table 1 and Fig. 1, †p < 0.05; Fig. 1b–d). These data confirm a robust hypertrophic phenotype in female and male HHR. Simulated ischemia/reperfusion protocol To assess the response of male and female NHR and HHR cardiomyocytes to an ischemia and reperfusion challenge, cardiomyocytes were superfused with a modified HEPES buffer (in millimolars: 136 NaCl, 8 KCl, 0.4 NaH2PO4-H2O, 1.1 MgSO4-7H2O, 2.0 CaCl2, 10 HEPES, 10 lactate, pH 6.8, 100 % N2 gas saturation) for 20 min to simulate conditions associated with ischemia, including hypoxia, hypercapnia, acidosis, substrate deprivation and lactate accumulation [30, 35, 36]. At the end of the 20-min superfusion with the simulated ischemia solution, the perfusate was switched back to the basal 2-mM Ca2+-HEPES buffer for a further 30 min. Cardiomyocytes were electrically stimulated (4 Hz) throughout the ischemia/reperfusion protocol. Cardiomyocyte basal performance—sex differences suppressed with hypertrophy Basal NHR cardiomyocyte contractile performance was lower in female cardiomyocytes compared with males (Fig. 2a–d, Additional file 1: Table S1 p < 0.05). This lower female contractility was marked in normal female myocytes (approximately 50 % lower of the percentage shortening values vs male NHR), though time to peak shortening was particularly slow in female HHR cardiomyocytes (Additional file 1: Table S1). Lower contractility in normal female myocytes was associated with a trend for lower Ca2+ transient amplitude and systolic Ca2+ (Fig. 2e, f, Additional file 1: Table S1). No sex difference was observed in NHR cardiomyocyte diastolic Ca2+ levels or the time constant of Ca2+ transient decay (Fig. 2g, h). Lower contractility was not apparent in female HHR cardiomyocytes relative to male (Fig. 2b). As for NHR, overall Ca2+ levels did not differ between male and female HHR myocytes (Fig. 2e–h). These data show that basal contractile performance in nonhypertrophic male and female cardiomyocytes is fundamentally different, but this sex differential is lost when myocytes exhibit pathological hypertrophy. Statistics Data are presented as mean ± SEM. Differences between groups were assessed by two-way ANOVA and with repeated measures as appropriate. Myocyte survival curves were assessed by log-rank Mantel-Cox test. Data were considered significant at p < 0.05. All statistical calculations were performed using SPSS V.22.0 (IBM Corp, NY, USA). Statistical annotation used throughout the study to designate significance level: #, strain effect; , sex effect; †, sex-strain interaction. Post hoc analysis was performed on data in which a sex-strain interaction was evident, with significant differences in post hoc analysis represented in the figures (§, p < 0.05, male vs female of same strain) directly above the relevant figure bar or data point. Augmented HHR Ca2+ activator response to β-adrenoceptor stimulation in male cardiomyocytes absent in female cardiomyocytes β-Adrenoceptor agonist challenge in vitro elicited a substantial increase in cardiomyocyte contractility (Fig. 3a, Additional file 2: Table S2). The magnitude of this response was similar in male and female cardiomyocytes, but more pronounced in the presence of hypertrophy (Fig. 3b–d, M NHR 178.9 ± 16.1 %, F NHR 213.5 ± 32.1 %, M HHR 282.9 ± 36.3 %, F NHR 300.7 ± 25.2 %, #p < 0.05). Sex differences in the Ca2+ response to β-adrenoceptor stimulation were evident. The extent of isoproterenolinduced increase in Ca2+ transient amplitude was selectively blunted in female HHR myocytes compared with males (Fig. 3e, †p < 0.05). This sex-specific response was not observed in non-hypertrophic (NHR) myocytes. Results Cardiac and cardiomyocyte hypertrophy in male and female HHR Female body and heart weights were significantly lower than strain and age-matched NHR and HHR values (Table 1). Heart weight and cardiomyocyte dimensions were smaller in female NHR (Table 1 and Fig. 1, respectively), yet cardiac weight index (CWI; g/kg) was greater compared with male NHR. In HHR, cardiac and cardiomyocyte hypertrophy was most pronounced in female with augmented CWI associated with more substantial myocyte enlargement in width Table 1 Age and body/heart weights of male and female NHR and HHR Parameter NHR HHR Male Female Male Female Age (weeks) 14.6 ± 0.7 (15) 13.3 ± 0.3 (13) 14.8 ± 1.0 (14) 13.7 ± 0.6 (13) Body weight (g) 352 ± 7.9 (15) 180 ± 5.5# (13) 299 ± 10.2## (14) 171 ± 4.7## (13) Heart weight (mg) 1410 ± 28.5 (15) 882 ± 27.7# (13) 1514 ± 85.8 (14) 1202 ± 69.2#,## (13) CWI (mg/g) 4.02 ± 0.1 (15) # 4.91 ± 0.1 (13) #sex p < 0.05; ##strain p < 0.05, mean ± SEM, n = hearts or animals in brackets for each group ## 5.12 ± 0.3 (14) 7.04 ± 0.4#, ## (13) Bell et al. Biology of Sex Differences (2016) 7:32 Page 5 of 15 Fig. 1 Cardiomyocyte hypertrophy in male and female HHR. a Representative images of cardiomyocytes from male and female NHR and HHR hearts. b–d Augmented CWI associated with more substantial myocyte enlargement in width (and consequently volume) dimension in female vs male HHR (sex p < 0.05; #strain p < 0.05; †sex-strain interaction; §post hoc male vs female p < 0.05, mean ± SEM. Each data point mean of n = 50 cells from each of eight hearts/group, total 400 cells/gp) Diastolic Ca2+ levels were stable with isoproterenol challenge in all treatment groups (Fig. 3f). A more rapid transient decay (decrease in tau) was evident in hypertrophic male and female cardiomyocytes, consistent with a trend for increased maximum rate of lengthening (Fig. 3d, g). These data demonstrate that the Ca2+ response to β-adrenergic stimulation is different in male and female hypertrophic cardiomyocytes—the accentuated response in HHR male myocytes is not achieved in female myocytes. Selective Ca2+ elevation in female myocytes at end ischemia abrogated with hypertrophy Myocyte contractile performance and Ca2+ levels were tracked throughout the time-course of a simulated ischemia/reperfusion challenge (sample time-compressed Bell et al. Biology of Sex Differences (2016) 7:32 Page 6 of 15 Fig. 2 Basal sex differences in cardiomyocyte performance suppressed with hypertrophy. a Representative records of myocyte shortening in male and female NHR and HHR cardiomyocytes. b–d Basal twitch amplitude (% shortening), maximum rate of shortening/lengthening are lower in female cardiomyocytes compared with males—an effect that is accentuated in NHR compared with HHR. e No sex difference was observed in Ca2+ transients, though a trend for lower Ca2+ transient amplitude was evident in female NHR cardiomyocytes (f). No sex difference was observed in cardiomyocyte diastolic Ca2+ levels or the time constant of Ca2+ transient decay (g, h). (sex p < 0.05; p < 0.05; mean ± SEM; n = 8–13 cells from four to five hearts/group) record, Fig. 4a, Additional file 2: Table S2). At the commencement of ischemia, all cell groups demonstrated an abrupt decrease in twitch amplitude and a small increase in resting length. This reduction in twitch amplitude reached a plateau within the first 5 min of simulated ischemia (Fig. 4b), with no significant difference between groups in the extent of twitch amplitude reduction at end ischemia (Fig. 4c). Ca2+ transient responses to simulated ischemia were very different in male and female cardiomyocytes (Fig. 4d, †p < 0.05). In contrast to males, female NHR cardiomyocytes exhibited a substantial increase in amplitude Ca2+ at the end of ischemia (relative to pre-ischemic basal; M NHR 104.8 ± 13.3 %, F NHR 186.2 ± 35.9 %, p < 0.05), which was not associated with a concomitant increase in shortening (Fig. 4e). This marked and selective elevation of endischemia Ca2+ in female NHR was totally abrogated with hypertrophy (HHR, Fig. 4e, M HHR 73.1 ± 9.5, F HHR 66.8 ± 10.9 %). Despite a significantly lower Ca2+ signal in hypertrophic cardiomyocytes (Fig. 4e, #p < 0.05), the Bell et al. Biology of Sex Differences (2016) 7:32 Page 7 of 15 Fig. 3 β-Adrenoceptor stimulation Ca2+response blunted in hypertrophic female HHR cardiomyocytes. a Representative cardiomyocyte shortening trace (central portion time compressed) showing isoproterenol (10 nM) exposure-elicited response of rapid and sustained increase in cell twitch. b–d The extent of increase in shortening and maximum rate of shortening was similar in male and female cardiomyocytes, but more pronounced in the presence of pathological hypertrophy. Rate of lengthening was unchanged. e The extent of isoproterenol-induced increase in Ca2+ transient amplitude was selectively blunted in female HHR myocytes. f Diastolic Ca2+ levels were stable with isoproterenol challenge in all treatment groups, whilst tau (g) was significantly faster in HHR of both sexes (#strain p < 0.05; †sex-strain interaction; §post hoc male vs female p < 0.05, mean ± SEM, n = 9–11 cells from four to five hearts/group) Bell et al. Biology of Sex Differences (2016) 7:32 Fig. 4 (See legend on next page.) Page 8 of 15 Bell et al. Biology of Sex Differences (2016) 7:32 Page 9 of 15 (See figure on previous page.) Fig. 4 Selective Ca2+ elevation in female myocytes at end ischemia is abrogated with hypertrophy. a Representative cardiomyocyte shortening record shows modulated diastolic length and twitch amplitude in simulated ischemia and reperfusion (time compressed). b All groups show a similar pattern of shortening response to simulated ischemia. c No significant difference between groups was detected in the extent of change in twitch reduction at end ischemia. d Ca2+ transient responses during the time-course of simulated ischemia. e Female NHR cardiomyocytes exhibited a substantial increase in amplitude Ca2+ at the end of ischemia which was not evident in female hypertrophic myocytes. f Diastolic Ca2+ increased throughout ischemia in male HHR cardiomyoctes. g At end ischemia, increased diastolic Ca2+ in male HHR was absent in female HHR myocytes. (sex p < 0.05; #strain p < 0.05; †sex-strain interaction; §post hoc male vs female p < 0.05; mean ± SEM; n = 7–14 cells from 6 to 11 hearts/group) shortening responses between male and female HHR were not different (Fig. 4c, p = ns). A sex-strain interaction was evident for diastolic Ca2+ during simulated ischemia (Fig. 4f, g). An increase in diastolic Ca2+ in male HHR cardiomyocytes was not evident in female HHR, which remained at near-basal levels of diastolic Ca2+ (Fig. 4g). This contrasted with NHR myocytes, with a trend for greater diastolic Ca2+ at the end of simulated ischemia compared with males. Myofilament Ca2+ desensitization in reperfused hypertrophic male myocytes is absent in females During the simulated reperfusion phase of the protocol, myocytes of all groups most typically exhibited a rapid decrease in resting length (i.e. a diastolic contracture response) with decrement in twitch amplitude, followed by gradual return to a new steady state in the extent of myocyte shortening, albeit diastolic relaxation state was only partially regained (Fig. 4a). Myocyte attrition (acute necrosis/oncosis) was observed throughout both the ischemic and reperfusions phases of the protocol (Fig. 5a). In males, there was a trend for hypertrophic myocytes (HHR) to be more resistant to cell death during the simulated ischemia/reperfusion challenge compared with normal myocytes (NHR), though this did not reach significance (Fig. 5a). A strain trend was not apparent in female myocytes. All groups showed full recovery from ischemic diastolic contracture, with resting cardiomyocyte length lower at end reperfusion compared with the preischemic basal level (Fig. 5b). The extent of this reduction was greatest in male NHR cardiomyocytes and significantly greater than in female NHR cardiomyocytes. This sex difference was not apparent when cardiomyocytes were hypertrophic. During the first 5 min of reperfusion, spontaneous contractions were evident in male NHR (one of eight cardiomyocytes) and HHR (four of nine cardiomyocytes) cardiomyocytes, contrasting with a lack of spontaneous contractions in both female NHR and HHR cardiomyocytes, At the end of the 30-min simulated reperfusion challenge, surviving male and female cardiomyocytes from normal and hypertrophic hearts exhibited very different recovery parameters (Fig. 5c, †p < 0.05). Male HHR cardiomyocytes demonstrated equivalent shortening to male NHR controls, yet amplitude Ca2+ was significantly increased. This directly contrasted with female HHR cardiomyocytes, where shortening was significantly increased compared with female NHR controls, yet amplitude Ca2+ was not different. Phase loop analyses were performed to assess individual myocyte Ca2+-shortening relationships (Fig. 5d). Male, but not female, HHR cardiomyocytes exhibited a large decrease in myofilament Ca2+ sensitivity, with an approximate two- to threefold increase in Ca2+ levels at 50 % myocyte relaxation compared with male NHR and female NHR and HHR cardiomyocytes (Fig. 5e, †p < 0.05). These data demonstrate that, in contrast to hypertrophic male myocytes, myofilament Ca2+ responsiveness in hypertrophic female cardiomyocytes was not characterized by ‘desensitization’. Thus, a major sex-specific stress response phenotype difference emerges between female and male hypertrophic cardiomyocytes when subjected to simulated ischemia and reperfusion challenge. Discussion This investigation provides new evidence that male and female hearts with an underlying pathological hypertrophy respond differently to cellular stresses. Using the novel HHR model (a model of normotensive intrinsic hypertrophy derived through a multigenerational program of selective breeding), our findings document pronounced cardiac and cardiomyocyte enlargement in females—including cardiomyocyte dimensional increase in female HHR equivalent to male HHR. Under basal conditions, a lower twitch amplitude in female myocytes was prominent in normal (NHR) but not in hypertrophic (HHR) myocytes. The cardiomyocyte Ca2+ responses to β-adrenergic challenge differed in hypertrophic male and female cardiomyocytes, with the accentuated response in males abrogated in females—even while contractile responses were similar. In simulated ischemia, the Ca2+ transient responses of hypertrophic male and female myocytes were markedly different—though again, all groups demonstrated similar shifts in myocyte contractile performance. A prominent and selective elevation of end-ischemia Ca2+ in female NHR was completely suppressed in female hypertrophic myocytes. Similarly, after the 30-min simulated reperfusion, male and female cardiomyocytes evinced different Ca2+ response patterns Bell et al. Biology of Sex Differences (2016) 7:32 Page 10 of 15 Fig. 5 Myofilament Ca2+ desensitization in hypertrophic male myocytes at the end of reperfusion is absent in females. a Cardiomyocyte survival plots throughout simulated ischemia and reperfusion. b Resting myocyte length decreased to a greater extent in male NHR cardiomyocytes compared with females, with this sex difference absent in the HHR. c At end reperfusion, male HHR cardiomyocytes maintain contractility yet exhibit significantly increased Ca2+. Female HHR myocytes show augmented contractility with substantially lower transient Ca2+. d Phase loop analysis shows male, but not female, HHR cardiomyocytes exhibit a modified Ca2+-shortening relationship. e Male HHR cardiomyocytes exhibit significantly lower level of myofilament Ca2+ sensitivity, with an approximate two to threefold increase in Ca2+ levels at 50 % myocyte relaxation. (sex p < 0.05; #strain p < 0.05; †sex-strain interaction; §post hoc male vs female p < 0.05; mean ± SEM; n = 5–8 cells from four to six hearts/group) indicating an absence of the Ca2+ desensitization characterizing the male response. Our data demonstrate that cardiac hypertrophy produces dramatically different basal and stress-induced pathophenotypes in female- and male-origin cardiomyocytes. The lower Ca2+ operational status characteristic of female (vs male) cardiomyocytes comprising normal hearts is not exhibited by myocytes of hypertrophic hearts. After ischemia/reperfusion, availability of activator Ca2+ is suppressed in female hypertrophic myocytes, whereas sensitivity to Ca2+ is blunted in male hypertrophic myocytes. These findings demonstrate that selective intervention strategies should be pursued to optimize post-ischemic electromechanical support for male and female hypertrophic hearts. Normal female-male cardiomyocyte contractile performance differential is eliminated with concomitant hypertrophy The present study assessed male/female cardiomyocyte function and stress responses in a novel pathological hypertrophic HHR strain (vs control NHR strain). Significantly lower body weight in female rats is consistent with previous reports [23, 37–39], as are our findings that cardiomyocyte dimensions are smaller in normal females (vs male NHR) [24, 40–42]. This male/ female cell size difference is not always evident and may be dependent on animal strain, aging/disease context and the experimental conditions under which myocyte dimensions are measured (e.g. resting Ca2+ concentration) [37, 43]. The possibility that there is a fundamental Bell et al. Biology of Sex Differences (2016) 7:32 relationship between cardiomyocyte size and electromechanical characteristics which may partly reflect a sex-dependent size-scaling effect merits future investigation. The present finding that under standardized basal conditions, normal female cardiomyocytes contract at a lower operating level than male myocytes is consistent with our earlier report (subsequently confirmed by others) [16, 39]. This feature of female cardiomyocyte performance suggests a relatively ‘economical’ basal metabolic state. There are discrepancies within the literature regarding the mechanisms driving this female phenotype, though changes in Ca2+ transporter function are undoubtedly implicated. While differences in protein expression levels of various Ca2+ transporters have been reported between males and females in some contexts [15, 30], oestrogen regulation of the activity of some key Ca2+ handling effectors is likely to be of most functional importance [15, 44–48]. These previous studies assessing Ca2+ transporter expression/activity have utilized a series of different species (e.g. mouse, rat, guinea pig) and animal ages, which may explain variability in findings. We have recently shown that expression of the sarcoplasmic reticulum (SR) Ca2+ release channel (RyR2) does not differ in male/female Sprague Dawley (SpD) rats (12– 16 weeks) [30], similar to that reported in 5–10month-old C57Bl/6 mice [25]. This contrasts to previous studies in SpD and Wistar rats (age not stated), reporting an increased expression of the SR Ca2+ release channel (RyR2) [49, 50]. We also showed no difference in sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB) expression in male/female SpD rats, consistent with previous reports [49]. Directly relevant to the present study, we have shown that expression of RyR2, SERCA2a and PLB was not different in female HHR and NHR ventricular homogenates [30]. Our data indicate that it is the regulation of Ca2+ transporter activity, and not the expression, that differs in males/females. In particular, we have shown that basal Ca2+/calmodulindependent kinase II (CaMKII) phosphorylation (and hence, CaMKII-mediated phosphorylation of RyR2 and PLB) is lower in females, a mediator which exerts major influence on numerous Ca2+ transporter properties [30]. Estrogenic influence on cardiomyocyte Ca2+ handling includes voltage-gated Ca2+ current suppression, internal Ca2+ store release modulation and myofilament Ca2+ sensitivity alteration [25, 46, 51–59]. The sex-specific distinguishing characteristics in contractile phenotypes in non-hypertrophic myocytes can be partially attributed to a Ca2+-dependent mechanism, but likely non-Ca2+ dependent estrogenic actions (cell energetics/gene expression) are involved [5, 60] as the extent of contractility performance difference between males and females was more prominent than the Ca2+ transient amplitude differences. Page 11 of 15 Hypertrophy is linked with accentuated response to β-adrenoceptor stimulation in male, but not female myocytes Sex-specific aspects of altered β-adrenoceptor response in cardiac disease pathogenesis are not well characterized. Experimentally, evidence indicates that the β-adrenergic response is less pronounced in healthy cardiac tissues/myocytes of females compared with males under some circumstances. We and others have previously shown that Ca2+ transients are smaller in female cardiomyocytes (vs males) treated with isoproterenol [39, 61–63]. These male/female differences do not appear due to changes in β-adrenergic expression/density [64, 65], and suppressed responsiveness in females is more likely attributable to downstream signaling intermediates (i.e. adenylyl cyclase/PKA/cAMP) [25, 62]. We did not observe male/female differences in the extent of increase in twitch or Ca2+ transient amplitude in isoproterenol-treated NHR myocytes—suggesting that pacing under near-physiological conditions minimizes response difference [65]. Notably, male/female differences in response to isoproterenol were evident in hypertrophic myocytes. Hypertrophic female myocytes did not exhibit the accentuated elevation in Ca2+ transient amplitude observed in male HHR myocytes, even though both female and male HHR had similarly augmented contractile responses. This suggests a Ca2+-independent component determining adrenergic response selectively in females. The mechanism is unclear, but may be related to differential cellular metabolism profile—with hypertrophic hearts drawing on sexselective energy reserves to effect an adrenergic-driven contractile response [5, 66]. Cardiomyocyte Ca2+ handling and responsiveness during ischemia and reperfusion is different in hypertrophy in females and males In this investigation, distinct mechanisms underlying vulnerability to ischemia/reperfusion in female- and male-origin cardiomyocytes are identified. These findings build on our prior observations involving isolated ex vivo hearts where we established that the more robust functional recovery of female hearts to ischemic insult was abrogated in hypertrophy [23]. In that study, HHR male and female hearts performed equally poorly, displayed similar necrotic damage (lactate dehydrogenase release) and the enhanced activation of the pro-survival reperfusion injury survival kinase (RISK; [23]) pathway seen in NHR female hearts was absent in HHR female hearts. In the two heart strains, coronary flow during ischemia/reperfusion in that study was also not different—eliminating vascular perfusion status as an underlying causative agent in sex- and strainrelated performance. Our new in vitro finding presented Bell et al. Biology of Sex Differences (2016) 7:32 in this investigation, that overall isolated cardiomyocyte viability loss through simulated ischemia/reperfusion was not different between sexes or strains, indicates that the measures of ex vivo heart damage reflect primarily cell rupture injury attributable to in situ tissue torsion (likely during contracture) and not cell-origin oncotic lesion. During ischemia, female NHR showed a surprising tolerance for elevated cardiomyocyte Ca2+ levels—suggesting that the interplay between Ca2+, Na+ and pH which is an important determinant of electromechanical status at end reperfusion operates differently in normal male and female myocytes. Related, but qualitatively different, observations have been previously reported. The sex differences in ionic homeostatic responses to simulated ischemia may depend on precise experimental conditions [24]. Sex-comparative data relating to hypertrophic cardiomyocytes have not previously been presented. An exploration of how these ionic flux responses may be differentially manipulated via pharmacologic means may provide a valuable therapeutic lead for new post-ischemia sex- and disease-specific intervention approaches. Cell shortening is typically reduced by approximately 50 % relative to basal in the first 5 min of simulated ischemia, though this reduction was attenuated in female HHR cardiomyocytes (Fig. 4a). This 50 % reduction is similar to that described previously [24, 67], though not as extensive as other studies describing near cessation of myocyte shortening [35, 36, 68–70]. As was observed in isolated HHR hearts [23], at the end of ischemia, male and female isolated cells showed similar levels of contractile recovery (whereas in NHR sex-specific performance, differences were apparent). Most dramatically, different underlying Ca2+ handling and response characteristic produced this similar net effect in HHR cell types. In the male HHR myocytes, a significant elevation of Ca2+ transient amplitude occurred with a major offsetting reduction in myofilament sensitivity to Ca2+. In the female HHR myocytes, this combination of Ca2+ adaptations was not apparent. In relation to adverse outcomes in early reperfusion, these findings may be extrapolated to suggest that in acute reperfusion, Ca2+ overload arrhythmogenic predisposition presents as a male myocyte vulnerability. In contrast, the blunted female myocyte Ca2+ transient response to β-adrenoceptor inotropic influence has the potential for compromised functional performance. Thus, ischemic recovery is similarly impaired in both males and females—but via different cellular mechanisms. Clinically, the importance of gender differences in arrhythmia substrate is increasingly recognized—and advancing the understanding of underlying cellular processes involved is essential [71]. The present study, of young adult rodents at prime estrogenic life stage, demonstrates the absence of a Page 12 of 15 female ‘protected’ state in the context of hypertrophy. The conventional view of female oestrogen cardioprotection may require refinement [15, 72]. Oestrogen receptors (ERα, oestrogen receptor β (ERβ), G-proteincoupled oestrogen receptor (GPER)) are expressed in male and female myocardium. When expression/activity is manipulated genetically or pharmacologically, dual sex effects may be observed [73]—yet oestrogen exposure is not always favourable [15]. Experimentally, where female hearts have shown more ischemic resilience than male hearts, this has been most evident when the context involved baseline elevated Ca2+ loading levels [20–22]. New information suggests that local myocardial androgen-to-oestrogen conversion may be an element in determining the sex specificity of responses to cardiac challenge [74–76], and in hypertrophy, local steroid levels may become more important in modulating function under stress conditions. More detailed studies of the specific molecular processes involved in regulating ionic homeostasis in male- and female-origin cardiomyocytes in defined disease hypertrophic states and acute stress settings are warranted. A limitation of the present study is the in vitro and ‘unloaded’ conditions under which myocyte recordings are undertaken. However, the advantage of the approach is the capacity to achieve a high level of control over cellular ambience to resolve key functional differences. Conclusions In summary, this experimental investigation demonstrates that the responses of hypertrophic male and female cardiomyocytes to β-adrenoceptor stimulation and ischemia/ reperfusion challenges are fundamentally different at the cellular level—and that hypertrophy in females confers a distinctive liability. Our findings indicate that therapeutic interventions targeted to the early reperfusion period may have differential sex-specific efficacy depending on the presence of an underlying comorbid hypertrophic state. Where clinical trials have previously failed to identify benefit in early reperfusion manipulation of cellular processes impacting on myofilament Ca2+ sensitivity, there may be a case for review in selected at-risk female subgroups. This study provides a basis to support relevant focused pre-clinical research to pursue this possibility. Additional files Additional file 1: Table S1. Basal sex differences in cardiomyocyte performance suppressed with hypertrophy. (sex p < 0.05, #strain p < 0.05; mean ± SEM, n = hearts or animals in brackets for each group). (PPTX 68 kb) Additional file 2: Table S2. Sex differences in cardiomyocyte performance following either β-adrenoceptor stimulation or stimulated ischemia. (Values expressed as % basal. *sex p < 0.05, # strain p < 0.05; mean ± SEM, n = cells in brackets for each group). (PPTX 62 kb) Bell et al. Biology of Sex Differences (2016) 7:32 Abbreviations ANOVA, analysis of variance; CaMKII, Ca2+/calmodulin-dependent kinase II; CWI, cardiac weight index; ERα, oestrogen receptor α; ERβ, oestrogen receptor β; GPER, G-protein-coupled oestrogen receptor; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HHR, hypertrophic heart rat; NHR, normal heart rat; RISK, reperfusion injury salvage kinase; SHR, spontaneously hypertensive rat Acknowledgements Career support was provided through the National Heart Foundation of Australia and the University of Melbourne R. Douglas Wright Faculty Trust (JRB). Research support was provided through Grant-in-Aid from the National Heart Foundation of Australia (LMDD, JRB and CLC) and the National Health and Medical Research Council of Australia (LMDD, SBH and JRB). Authors’ contributions JRB, CLC, MVP and LMD conceived and designed the experiments. JRB, CLC, TWH and MVP performed the experiments and analysed the data. SBH and LMD generated the HHR model. JRB, CLC and LMD wrote the manuscript. All authors contributed to the discussion, critical revision and approved the final manuscript. Authors’ information None Competing interests The authors declare that they have no competing interests. Author details 1 Department of Physiology, University of Melbourne, Melbourne, Victoria, Australia. 2Centro de Investigaciones Cardiovasculares, Centro Cientifico Tecnologico La Plata, Facultad de Ciencias Medicas, Universidad Nacional de La Plata, La Plata, Argentina. 3Cardiac Phenomics Laboratory, Department of Physiology, University of Melbourne, Melbourne, Victoria 3010, Australia. Received: 18 February 2016 Accepted: 22 June 2016 References 1. Lam CSP, Little MS. Sex and cardiovascular risk. Circ. 2012;126:913–5. 2. Go AS, Mozaffarian D, Roger VL, et al. Executive summary: heart disease & stroke statistics. Circ. 2014;129:399–410. 3. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med. 1990;322:1561–6. 4. Deo R, Vittinghoff E, Lin F, Tseng ZH, Hulley SB, Shlipak MG. 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https://openalex.org/W2904527631	https://repositum.tuwien.at/bitstream/20.500.12708/790/2/Filipovic%20Lado%20-%202018%20-%20Review--System-on-Chip%20SMO%20Gas%20Sensor%20Integration%20in...pdf	English	null	Review—System-on-Chip SMO Gas Sensor Integration in Advanced CMOS Technology	Journal of the Electrochemical Society	2,018	cc-by	21,468	Review—System-on-Chip SMO Gas Sensor Integration in Advanced CMOS Technology Lado Filipovic z and Ayoub Lahlalia Institute for Microelectronics, Technische Universit¨at Wien, E360, 1040 Vienna, Austria The growing demand for the integration of functionalities on a single device is peaking with the rise of IoT. We are near to having multiple sensors in portable and wearable technologies, made possible through integration of sensor fabrication with mature CMOS manufacturing. In this paper we address semiconductor metal oxide sensors, which have the potential to become a universal sensor since they can be used in many emerging applications. This review concentrates on the gas sensing capabilities of the sensor and summarizes achievements in modeling relevant materials and processes for these emerging devices. Recent advances in sensor fabrication and the modeling thereof are further discussed, followed by a description of the essential electro-thermal-mechanical analyses, employed to estimate the devices’ mechanical reliability. We further address advances made in understanding the sensing layer, which can be modeled similar to a transistor, where instead of a gate contact, the ionosorped gas ions create a surface potential, changing the ﬁlm’s conduction. Due to the intricate nature of the porous sensing ﬁlms and the reception-transduction mechanism, many added complexities must be addressed. The importance of a thorough understanding of the electro-thermal-mechanical problem and how it links to the operation of the sensing ﬁlm is thereby highlighted. p g y g g © The Author(s) 2018. Published by ECS. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 License (CC BY, http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse of the work in any medium, provided the original work is properly cited. [DOI: 10.1149/2.0731816jes] Manuscript submitted August 10, 2018; revised manuscript received December 5, 2018. Published December 15, 2018. This was Paper 1469 presented at the Seattle, Washington Meeting of the Society, May 13–17, 2018. While aggressive device scaling has taken the front stage in the semiconductor industry for many decades, there is currently an ever- increasing demand for functional integration in a single device. This means not only the integration of an increasing number of transistors along the path of Moore’s Law, but also the integration of multiple ap- plications on a single device, appropriately named More-than-Moore. The rise of the Internet of Things and the Internet of Everything are clear indicators of this trend. The ﬁrst attempt at in-package integra- tion dealt with connecting different dies with varying functionalities using bonding wires. Gas Sensing Mechanisms A large part of how we perceive the environment is shaped by the presence of various gases in our vicinity. As a natural sensor the hu- man nose is able to detect hundreds of different odors, but it is not able to detect all harmful gases and fails absolutely when there is a need to detect speciﬁc gas concentrations. The ability to electrically detect our environment and the air we breathe has been a topic of extensive research over many decades. A wide range of applications and indus- tries have a vested interest in gas sensor development including health and safety,3 automotive,4 environmental monitoring,5–7 and chemi- cal warfare detection,8,9 among others. The feasibility to detect toxic and harmful gases in our environment through wrist watches, smart phones, tablets, and wearables is of particular interest, triggering sub- stantial research.10–12 Furthermore, fabrication and process controls as well as laboratory analytics can be made more affordable with cheaper gas sensing equipment. Currently, a variety of gas sensing principles are being implemented in industry and research, e.g. semiconduc- tor, optical, thermal conductivity, infrared (IR), quartz microbalance, catalytic, dielectric, electrochemical, and electrolyte sensors.13–15 y p The use of silicon as a substrate material for added functional- ity, including analog and radio frequency (RF) circuits, sensors and actuators, or biochips, allows for the efﬁcient integration of micro- electro-mechanical systems (MEMS) and complementary metal oxide semiconductor (CMOS) structures into a truly monolithic device. This is highly challenging, since the typically high temperatures associated with sensor fabrication has a negative inﬂuence on CMOS front end of line (FEOL) devices and back end of line (BEOL) metallization. However, the challenge is deemed well worth the effort since the inte- gration of gas sensors with CMOS electronics is seen as a key enabler of smart gas sensors for mobile applications, allowing low power, low costs, and portability.1,2 Gas sensors can be classiﬁed as those whose sensing is based on a variation in electrical properties or variation in other properties.16 An excellent review of the different gas sensors is given by G. Korotcenkov in Ref. 14, while A. Dey recently reviewed semiconduc- tor metal oxide (SMO) gas sensors,15 discussing the materials used, their sensitivity, selectivity, and stability. Dey’s review thoroughly de- scribes the characterization of the sensing material itself, including its response time, detection limits, and temperature of operation, while concentrating primarily on ammonia detection. Review—System-on-Chip SMO Gas Sensor Integration in Advanced CMOS Technology However, this method can negatively impact performance and power dissipation, since long wires result in a high resistance/capacitance (RC) delays and an increased circuit resistance, limiting high frequency performance and reducing device lifetimes. The highest efﬁciency is reached when no bond wires are required and all functionalities are fabricated on a single substrate, deemed System-on-Chip (SoC). microheaters. Additionally, recent designs and models are presented and analyzed in this review. Before discussing the SMO sensor, the following subsection is meant to put the SMO sensor in context with other available gas sensor technologies. Its advantages over alternative solutions make it abundantly clear why these sensors are the subject of extensive research. zE-mail: ﬁlipovic@iue.tuwien.ac.at ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP B862 B862 Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) ) unless CC License in place (see abstract). ecsdl.org/site/terms_use ess. Redistribution subject to ECS terms of use (see ) unless CC License in place (see abstract). ecsdl.org/site/terms_use ms of use (see Semiconductor Metal Oxide Sensors Recently it was shown that the sensing is not only due to a surface redox reaction with adsorbed oxygen, but that even in the absence of oxygen, a thin accu- mulation layer forms around the surface, thereby changing the ﬁlm’s resistivity and introducing a sensor signal. In addition, many studies have shown that the introduction of a dopant metal can improve the sensitivity and selectivity of SMO sensors. A model which includes all the effects of an additional dopant metal does not currently exist, while such a model would be beneﬁcial in order to develop predictable models and a technology computer aided design (TCAD) environment for SMO sensor designs. The advantages and disadvantages of SMO alternatives are brieﬂy mentioned here in order to convey to the reader the advantages that SMOs present over alternatives and that further work on improv- ing the sensor’s microheater as well as selectivity is well worth the effort. Even though there are several challenges, including the afore- mentioned selectivity and ensuring mechanically stable microheater integration, SMO sensors still provide meaningful advantages toward the development of integrated smart sensors. The primary disadvantage of catalytic combustion is the lack of selectivity in the detection of a desired gas. Electrochemical sensors are not easily miniaturized to a portable size and suffer from poor stability, durability, and response time. Thermal conductivity sensors have very poor sensitivity and selectivity, while the power dissipation, portability, and cost trail behind those of an SMO sensor. Infrared (IR) absorption sensors have a very high selectivity and sensitivity, but are very difﬁcult to be made portable with complex maintenance and high fabrication costs. If recent advances in the integration of IR sensors with silicon technologies prove to be fruitful, these hurdles may be overcome, resulting in a near ideal gas sensing device.25 In the mean- time, the SMO sensor is one which has been embraced by industry and has been commercialized due to its high sensitivity, fast response CMOS integration.—The semiconductor metal oxide sensor is on its way to becoming a universal sensor, since it can be used for many emerging applications in sensor networks, medical applications, food quality monitoring, and wearable devices.40,41 At the same time, the thin SMO ﬁlm can detect a variety of gases, essential for mea- suring indoor and outdoor air pollution and toxicity in our environ- ment, aspects of primary concert to our global health and safety. Gas Sensing Mechanisms We review the integration of microheaters for SMO gas sensors. E = Excellent, G = Good, time, low maintenance, cheap fabrication costs, and portability.26–31 Some questions still remain regarding the SMO’s selectivity, stability, and durability, with room for improvement on several fronts. integration, fabrication, and understanding the operation of SMO gas sensors. The sensor structure is discussed, including its fabrication and electro-thermal-mechanical operation. Subsequently, the recent advances in furthering our understanding of the sensing mechanism and its modeling are given. The primary concerns for SMO sensors are: r The functioning principle of the gas sensor is based on heating the metal oxide sensing layer to temperatures between 250◦C and 500◦C in order to provide enough energy for the necessary surface reactions to take place. Reducing the temperature to below 100◦C, while having a good sensing response, would lead to a reduction in the power consumption and improve the sensor reliability, since the associated thermal stress would be signiﬁcantly reduced. One of the main research ﬁelds is, therefore, attempting to reduce the operating temperature of the sensing layer. Another signiﬁcant advantage of SMO sensors is their repro- ducibility and repeatability.24 The ability to repeatedly fabricate the same structure with predictable operating conditions is essential when developing commercial devices. Without this, the industry would have never moved toward commercialization, since in mass production, high levels of certainty must exist when not every individual device can be precisely tested prior to its delivery to customer. While many other potential devices have been researched over the years, including graphene and other two-dimensional materials, these technologies are simply not mature enough to reach the reproducibility of SMO de- vices; this is why SMOs still dominate the semiconductor gas sensor market. r The need for operation at high temperatures means that a mi- croheater is required. Due to the required high temperatures, thermal isolation from the surrounding devices becomes essential, compli- cating the design and fabrication processes. This is also the reason why stability and durability should be improved to ensure long device lifetimes. In addition, since the provided temperature inﬂuences the sensing, knowing the exact microheater behavior is essential. This is not trivial and characterizing different types of sensors is a research study in itself. Semiconductor Metal Oxide Sensors The detection in SMO sensors is based on changing electrical properties in the presence of a target gas. More speciﬁcally, the resis- tance of the ﬁlm changes due to the interaction of gas molecules at its surface. From the summary of different gas sensors in use today given in Table I, it is clear why SMOs are currently the most popu- lar choice, with its only ﬂaw being selectivity, which is currently an active research ﬁeld. In fact, similar to the piezoelectric sensor, the SMO’s selectivity is primarily achieved using a sensor array, which allows for a simultaneous detection at multiple optimizations, such as at different operating temperatures or using different dopants. The collected sensor data can then be post-processed using a variety of methods to introduce selectivity. r Another concern is with the SMO’s selectivity, which is cur- rently being addressed using sensor array structures.32–38 Sensors can be individually engineered to increase sensitivity toward a particular gas and when many sensors are combined, the collected data can be analyzed using post-processing techniques such as neural networks in order to better pinpoint which gas has adsorbed at the SMO surface.39 The required data analysis makes it even more attractive to inte- grate the sensor with digital CMOS circuits, since it allows for the post-processing to take place on the same chip, increasing speed and reducing losses associated with long interconnect lines. g g r A thorough understanding of all the processes taking place dur- ing the SMO sensor’s operation is not yet known. Recently it was shown that the sensing is not only due to a surface redox reaction with adsorbed oxygen, but that even in the absence of oxygen, a thin accu- mulation layer forms around the surface, thereby changing the ﬁlm’s resistivity and introducing a sensor signal. In addition, many studies have shown that the introduction of a dopant metal can improve the sensitivity and selectivity of SMO sensors. A model which includes all the effects of an additional dopant metal does not currently exist, while such a model would be beneﬁcial in order to develop predictable models and a technology computer aided design (TCAD) environment for SMO sensor designs. r A thorough understanding of all the processes taking place dur- ing the SMO sensor’s operation is not yet known. Gas Sensing Mechanisms The review of Korot- cenkov deals primarily with identifying the different ways in which gas detection can be implemented and the advantages and disadvan- tages of those options. These are very clearly laid out in Ref. 14, which was also summarized more recently in Ref. 15 and enhanced and updated in this review, shown in Table I. The table includes the original characterizations by Korotcenkov from 2007, the update by Dey from 2018, as well as additions and updates for piezoelectric and photoionization sensors summarized from Refs. 17–19 and 20–23, respectively. This review concerns itself primarily with the CMOS This review discusses recent achievements in the integration of semiconductor metal oxide (SMO) MEMS gas sensors within an ad- vanced CMOS technology, for which all fabrication steps, required for the sensor fabrication, are below 450◦C. The discussion is split into four main sections: The ﬁrst one introduces semiconductor metal oxide sensors and their composition, while the second looks at the de- sign of the microheater element, an essential component of the SMO gas sensor. The third section discusses the methods used to analyze the complex SMO structure, including the suspended membrane, while the ﬁnal discussion looks at the modeling and simulation capabilities which have been developed in order to better analyze the sensing ﬁlm itself. This type of in-depth analysis has enabled a signiﬁcant improve- ment in the design and power optimization of advanced sensors and Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B863 Table I. Summary of available gas sensing devices. We review the integration of microheaters for SMO gas sensors. E = Excellent, G = Good, F = Fair, and P = Poor. Parameter SMO Catalytic pellistor Piezo-electric Electro-chemical Thermal pellistor Photo-ionization Infrared adsorption Sensitivity E G E G P E E Accuracy G G E G G E E Selectivity F P F G P F E Response time E G E F G E F Stability G G G P G E G Durability G G F F G E E Power E E F G G P F Cost E E G G G F F Footprint E G G F G E P Table I. Summary of available gas sensing devices. We review the integration of microheaters for SMO gas sensors. E = Excellent, G = Good, F = Fair and P = Poor gas sensing devices. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP Semiconductor Metal Oxide Sensors A typical integrated resistive sensor circuit is shown in Figure 1. The complexity for its SoC integration is immediately ev- ident: It requires a microheater, a sensing element, and analog and digital circuitry, all on a single chip.42,43 The colored sections in Fig- ure 1 highlight elements, whose integration requires special attention: A typical integrated resistive sensor circuit is shown in Figure 1. The complexity for its SoC integration is immediately ev- ident: It requires a microheater, a sensing element, and analog and digital circuitry, all on a single chip.42,43 The colored sections in Fig- ure 1 highlight elements, whose integration requires special attention: r Green - The sensitive semiconductor metal oxide layer must be exposed to the ambient, thereby requiring to be deposited at the end of the CMOS sequence at low temperatures. q p r Red - The microheater is required to heat the SMO layer to high temperatures, conducive to gas sensing. In this review we concen- trate on the design and implementation of metallic microheaters in a suspended, full, or perforated membrane. The integration between an SMO sensor and CMOS is twofold: First, CMOS electronics are required to enable the control of the volt- age supplied to the heater to provide the increase in temperature, when operating in power-saving pulsed mode. In addition, the sensing sig- nal itself can be processed using an analog/digital CMOS circuit, as shown in Figure 1. The second integration deals with the fabrication of the sensor devices. Allowing for the devices to be processed us- ing a mature CMOS technology is essential to allow for highly cost- and power-efﬁcient fabrication. Park et al.1 recently introduced an interface system between a metal oxide sensor and CMOS electron- ics, which is intended to provide a switching scheme for pulse width modulation to control the temperature of the heater and measure the sensor output simultaneously. These types of designs led to the de- velopment of fully CMOS integrated portable sensor modules for IoT applications.43 There are two main fabrication techniques for the membrane gen- eration using front-side or back-side etching, which result in two membrane types, the suspended membrane or the closed membrane, Figure 2. Membrane structures for an SMO sensor with (a) left, a suspended membrane and right, a full or closed membrane. Semiconductor Metal Oxide Sensors The discoveries which have enabled the integration of thin SMO ﬁlms B864 Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) Figure 1. Typical schematic of a single SMO sensor with interface blocks. The sensor requires a heating element, a voltage follower, and an analog-to-digital converter (ADC). In order to analyze the obtained data, it is passed to a microcontroller, which is enabled with a read-only-memory (ROM), random access memory (RAM), and input/output (I/O) interfaces.42,43 Figure 1. Typical schematic of a single SMO sensor with interface blocks. The sensor requires a heating element, a voltage follower, and an analog-to-digital converter (ADC). In order to analyze the obtained data, it is passed to a microcontroller, which is enabled with a read-only-memory (ROM), random access memory (RAM), and input/output (I/O) interfaces.42,43 forward to implement within a CMOS fabrication sequence, with spray pyrolysis being the most cost-effective option and the one in wide use today.57 within a CMOS fabrication sequence have opened up a world of pos- sibilities for sensors integrated in electronic components and a broad integration of sensors in our daily lives, which is already packed with electronic components and devices. The CMOS integration has also made device production much more affordable than its alternatives. y The highest complexity in the fabrication of an SMO sensor is the inclusion of a microheater. The microheater requires thermal iso- lation from surrounding components, achieved with the formation of a suspended membrane, which uses air as a thermal insulator to the underlying silicon wafer, as shown in Figure 2. The formation of this membrane is the most challenging fabrication component, for which two main membrane types are currently being used: The closed membrane, which is etched from the back of the wafer, and the sus- pended membrane, which is etched from the front of the wafer.66,67 The two types are illustrated side by side in Figure 2a, where the top and side views of the two membrane types are shown. In Fig- ure 2b, the side view of the active area is depicted in more detail,68 showing the effective air isolation below the membrane, while Fig- ure 2c depicts the membrane materials, with a microheater sand- wiched between isolation layers (SiO2 or Si3N4). In addition, the SMO ﬁlm is shown, deposited on top of electrodes. However, it is also not uncommon to have electrodes deposited on top of the sensing ﬁlm. Semiconductor Metal Oxide Sensors In (b) the side view is shown, while in (c), the membrane area is zoomed in, showing the location of the membrane materials (SiO2/SiN), the microheater, the electrodes, and the active sensing ﬁlm. Sensor fabrication.—In Ref. 44, Lackner et al. show the impor- tance of sensor integration with the CMOS fabrication technique, resulting in very low production costs and low power consumption. The crucial step is the integration of a microheater and suspended membrane. The microheater must be made of materials, preferably metals, readily available in a CMOS fabrication environment. The suspended membrane, which comprises the microheater and isolating layers, such as oxides and nitrides, also needs to be made possible within a CMOS fabrication technology, if the cost of the ﬁnal device is to be minimized. Another critical processing step, in addition to the fabrication of a suspended membrane, is the deposition of a metal oxide layer to act as the sensing element. The deposition of the sensing ﬁlm it- self can be incorporated with the CMOS fabrication sequence in one of several ways. These include sol-gel processing,45–48 chemical va- por deposition,49–51 sputtering,52–56 spray pyrolysis,57–63 pulsed-laser deposition,64 and rheotaxial growth and vacuum oxidation.65 Sputter- ing, chemical vapor deposition, and spray pyrolysis are quite straight Figure 2. Membrane structures for an SMO sensor with (a) left, a suspended membrane and right, a full or closed membrane. In (b) the side view is shown, while in (c), the membrane area is zoomed in, showing the location of the membrane materials (SiO2/SiN), the microheater, the electrodes, and the active sensing ﬁlm. Figure 2. Membrane structures for an SMO sensor with (a) left, a suspended membrane and right, a full or closed membrane. In (b) the side view is shown, while in (c), the membrane area is zoomed in, showing the location of the membrane materials (SiO2/SiN), the microheater, the electrodes, and the active sensing ﬁlm. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 ownloaded on 2020-01-10 to IP Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B865 Figure 3. Etching techniques to release the MEMS membrane for SMO gas sensors. Front-side etching: (1) Start with a silicon wafer, (2) Deposit membrane layers, (3) Etch holes in the membrane to create the suspension beams, and (4) Apply wet chemical (or plasma) etching to create a hole below the membrane. Semiconductor Metal Oxide Sensors For front-side fabrication, there are two main methods to etch the hole, one of which is the use of highly selective wet chemical etchants such as potassium hydroxide (KOH),69 ethylenediamine py- rocatechol (EDP),67 or tetramethylammonium hydroxide (TMAH).70 These etchants require the use of a silicon nitride or silicon oxide ﬁlms as etch-stop layers.71 The wet chemical etching technique is very ex- pensive and low-cost alternatives are sought after. The alternative low-cost option is the use of selective plasma etch processes, such as SF6-based plasma chemistries, often used for silicon and silicon diox- ide etching.68,72 Although plasma etching is cheap and fully CMOS compatible, it is very difﬁcult to avoid the lateral etching which takes place during this process. This is primarily because lateral etching is desired in one direction, in order to release the membrane, but not in the other, as it increases the size of the resulting air pocket. This process results in a very wide well, where more of the membrane is suspended than necessary. One way to deal with this is to introduce vertical nitride or oxide blocking layers, which would be costly to implement, or using a combination of back side etching with a ﬁnal front-side etch of the membrane to create the suspension beams, as is represented visually in step 5 of the back side etch process given in Figure 3.68,72 More recently, STMicroelectronics has realized another method in generating the suspended membrane on a silicon wafer.75–77 First, an isolating silicon dioxide is deposited using thermal oxidation or chemical vapor deposition (CVD). A sacriﬁcial Polyimide HD8820 is subsequently spin-coated and selectively etched to form the sensor cavity, as shown in the right side of Figure 3 and in Figure 4. The mem- brane material is composed of a Tantalum-Aluminum (TaAl) micro- heater, sandwiched between two silicon nitride (Si3N4) layers. Si3N4 is deposited using low pressure chemical vapor deposition (LPCVD), while TaAl is patterned using physical vapor deposition (PVD) and subsequent plasma etching. The sensing material is deposited on top of the membrane, followed by the metal contacts.78 This novel tech- nique allows for a fully front-side fabrication without the need for corrosive wet chemical etchants, while being compatible with CMOS fabrication. The applied sensing layer is tin dioxide (SnO2), one of the most promising metal oxide materials for gas sensor applications. Semiconductor Metal Oxide Sensors Backside etching: (1) Start with a silicon wafer, (2) Deposit membrane layers and back side SiO2, (3) etch back side photoresist to open area below the sensor, and (4) Etch up to the membrane using wet chemical etching or DRIE, and (5) Optionally open the membrane from the top. Polyimide etching: (1) Start with themal oxide on silicon, (2) Etch a hole in the SiO2, (3) Deposit a layer of polyimide HD8820 whih ﬁls the hole, (4) Deposit membrane layers, (5) Etch holes in the membrane, and (6) Selective plasma etches away polyimide through the holes, leaving the silicon and membrane materials in tact. Figure 3. Etching techniques to release the MEMS membrane for SMO gas sensors. Front-side etching: (1) Start with a silicon wafer, (2) Deposit membrane layers, (3) Etch holes in the membrane to create the suspension beams, and (4) Apply wet chemical (or plasma) etching to create a hole below the membrane. Backside etching: (1) Start with a silicon wafer, (2) Deposit membrane layers and back side SiO2, (3) etch back side photoresist to open area below the sensor, and (4) Etch up to the membrane using wet chemical etching or DRIE, and (5) Optionally open the membrane from the top. Polyimide etching: (1) Start with themal oxide on silicon, (2) Etch a hole in the SiO2, (3) Deposit a layer of polyimide HD8820 whih ﬁls the hole, (4) Deposit membrane layers, (5) Etch holes in the membrane, and (6) Selective plasma etches away polyimide through the holes, leaving the silicon and membrane materials in tact. by thermal conduction and convection through the entire membrane, a principal heat loss component in these structures. In suspended membranes, the conduction only takes place through thin suspension beams.73 The back side etching can be performed using the same wet chemical etch techniques described for the front-side etching process or by using deep reactive ion etching (DRIE), which is cheaper, but more complex to implement.74 By adding an extra etching step form the front side, the closed membrane can be released to be identical to a suspended membrane. However, this requires non-trivial alignment between the front and back sides of the wafer. respectively, as depicted in the left and middle fabrication sequences in Figure 3. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see ) ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 aded on 2020-01-10 to IP Semiconductor Metal Oxide Sensors As mentioned previously, SnO2 can be deposited using a variety of g The air isolation below the active area can also be achieved by etching to the membrane from the back of the wafer, meaning that no suspension beams are required, but rather that a full membrane is used. This method, labeled as back side etching in Figure 3 (steps 1– 4), although cheaper, has a reduced power efﬁciency, since heat is lost Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B866 Figure 4. Two-dimensional cross-section cut through the material stack mak- ing up the microheater.77 The primary materials are shown, including the sacri- ﬁcial Polyimide, which, when removed, forms the air cavity and the suspended membrane, ensuring thermal isolation between the membrane microheater and the underlying silicon wafer. given by given by Sensitivity = Rair −Rgas Rair · 100%, [1] [1] where Rair represents the baseline resistance after stabilization in am- bient air and Rgas is the resistance of the sensitive layer after exposure to a target gas mixed in air. The response to the presence of CO shown in Figure 5a has been measured at several temperatures for a spray py- rolysis deposited ﬁlm with a thickness of 50nm. The authors note that the addition of a 2nm evaporated gold (Au) ﬁlm results in a signiﬁcant improvement in the sensor response. For a 50nm thin ﬁlm, varying the temperature from 350◦C to 400◦C did not greatly inﬂuence the response. Figure 4. Two-dimensional cross-section cut through the material stack mak- ing up the microheater.77 The primary materials are shown, including the sacri- ﬁcial Polyimide, which, when removed, forms the air cavity and the suspended membrane, ensuring thermal isolation between the membrane microheater and the underlying silicon wafer. p The CO response shown in Figure 5b has been measured at 300◦C for a spray deposited ﬁlm, which also includes impurities in the form of platinum (Pt) nanoparticles.87 The presence of Pt impurities results in a signiﬁcant increase in the sensing performance, but only when a very small amount of Pt (0.2wt%) is present. When the Pt concentration increased to 2wt%, the sensing response is actually reduced. While no accepted physical explanation exists for this phenomenon, the presence of Pt increases the sensitivity due to the dissociation of oxygen on platinum. Semiconductor Metal Oxide Sensors Then, the activated oxygen species reach the SnO2, where they ﬁnally react with CO.87 The low signal when 2wt% Pt is used may lead to “localized” CO consumption without electron transfer, resulting in no changes in the SnO2 ﬁlm’s resistivity.87 More discussion on this topic is given in the ﬁnal section, dealing with surface chemisorption of noble metal doped SMO ﬁlms. The symbols in the ﬁgure represent measured data while the solid lines are best-ﬁt power-law lines, discussed in more detail in Ref. 88. techniques: CVD, sputtering, pulsed-laser deposition, sol-gel process, and spray pyrolysis. The choice of sensing material is discussed in more detail in the following section. Choice of sensing ﬁlm.—The miniaturization of transistors and other electronic devices has proven to be essential in advancing our technological capabilities. However, the chemical sensor ﬁeld, until recently, lagged behind the overall progress of CMOS devices. Dis- coveries in the application of metal oxide semiconductors have nar- rowed this somewhat by enabling sensor miniaturization and integra- tion with other electronics. In the early 1950s Brattain and Bardeen79 demonstrated for the ﬁrst time that several semiconducting materials display a sensitivity toward the presence of gas molecules, especially when heated to high temperatures. The conductivity of these materials changed when the chemical composition of the ambient gas changes. Following this discovery, in the early 1960s, the ﬁrst gas sensing de- vice based on a thin zinc oxide (ZnO) ﬁlm was proposed, operating at 485◦C.80 By 1967, Shaver showed a new method to improve the material’s sensitivity by adding small amounts of noble metal dopants such as platinum, rhodium, iridium, gold, and palladium.81 p The inﬂuence of metal additives to improve selectivity was re- cently further addressed and tested by Tangirala et al. in Ref. 89. The inﬂuence of copper (Cu), platinum (Pt), and palladium (Pd) doped SnO2 on CO sensing was studied. The authors proposed incorporat- ing metal dopants with chemical and impregnation methods by using urea and ammonia as precipitating agents. They found that the highest sensitivity was achieved when doping was incorporated using chemi- cal methods with urea precipitator, while Cu:SnO2 provided enhanced sensitivity, when compared to Pt or Pd,89 shown in Figure 5c. Semiconductor Metal Oxide Sensors The im- provement achieved using a urea precipitator was attributed to this method providing uniform and homogeneous nanoparticles during synthesis, while the high response of Cu:SnO2 is due to the excellent association and dissociation of oxygen (O) in the presence of CO at the sensor operating temperature to form CuO.89 p g p Researchers have since intensiﬁed the search for new gas sens- ing materials and the combinations of dopants which can improve their sensitivity and selectivity. Many SMOs have been extensively studied for their gas sensing properties, including indium oxide (In2O3), indium-tin oxide (ITO), cadmium oxide (CdO), zinc tin ox- ide (ZnSnO4), lead oxide (PbO), and many more. Among the most promising, and those now beginning to enter commercialization, are tin oxide (SnO2), zinc oxide (ZnO), and tungsten trioxide (WO3). These three SMOs fulﬁll most, if not all, of the requirements for a good gas sensing performance, which is their sensitivity to a broad spectrum of potentially harmful gases, ease of deposition, and low cost of fabrication.13 The ﬁrst SMO based gas sensing device was patented by Taguchi and it was dedicated to safety monitoring using a porous tin oxide (SnO2) ﬁlm with a palladium doping.82,83 Since then, a plethora of research has been centered around ﬁnding the perfect metal oxide material for speciﬁc gas sensing capabilities. Re- cently, it has become clear that SnO2 is likely the best metal ox- ide for gas sensors due to its ability to detect almost all relevant gases.13,84 ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP Microheater Design In (b) the M¨adler et al.87 discussed the inﬂuence o platinum (Pt) doping on a spray deposited SnO2 ﬁlm. In (c) V. K. K Tangirala et al.89 discussed the inﬂuence of various dopants, doping methods, and precipitatin agents in the sensitivity of an SnO2 ﬁlm toward CO. The temperatures used during the measurements correspond to the optimal temperature of operation for th detection of the target gas. Ra and Rg are the ﬁlm resistances in air and in the presence of a target gas, respectively. (a) (b) (b) (a) (b) ( ) (c) ( ) (c) (c) Figure 5. SnO2 thin ﬁlm sensor response during exposure to a varying concentration of carbon monoxide (CO) gas. In (a) K¨ock et al.44,86 showed the inﬂuence of temperature and evaporated gold particles on the sensor response on a 50nm thin spray deposited ﬁlm. In (b) the M¨adler et al.87 discussed the inﬂuence of platinum (Pt) doping on a spray deposited SnO2 ﬁlm. In (c) V. K. K Tangirala et al.89 discussed the inﬂuence of various dopants, doping methods, and precipitating agents in the sensitivity of an SnO2 ﬁlm toward CO. The temperatures used during the measurements correspond to the optimal temperature of operation for the detection of the target gas. Ra and Rg are the ﬁlm resistances in air and in the presence of a target gas, respectively. low fabrication costs, low CTE, low Poisson’s ratio, and most im- portantly, high compatibility with MEMS and CMOS fabrication techniques.66 low fabrication costs, low CTE, low Poisson’s ratio, and most im- portantly, high compatibility with MEMS and CMOS fabrication techniques.66 In the early years of micro-hotplate development, the frequently used materials were those readily available in a CMOS fabrication facility, including polysilicon, aluminum, and gold.98,99 Eventually it was found that these materials are not ideal since they suffer from electromigration defects and have poor contact properties. Microheater Design As previously mentioned, the microheater is one of the key com- ponents of the SMO gas sensor since the sensing ﬁlm is only activated at elevated temperatures. The choice of microheater and membrane materials and microheater geometry is essential in enabling a uniform temperature distribution across the active sensor region and ensuring a minimal power dissipation, since the heater is also the sensor’s most power-hungry component. The heater must be able to provide a pre- dictable temperature, so that the power/temperature relationship can be appropriately characterized. Otherwise, if the temperature deliv- ered is not the one expected, the operation of the sensing element will be incorrect. Microheater material.—Different materials have been used for the microheater, including silicon carbide (SiC),90 polysilicon,91,92 molybdenum,93,94 platinum,95 and tungsten.96 The membrane, which surrounds the microheater, is usually comprised of some combination of silicon dioxide (SiO2) and silicon nitride (Si3N4).70,78,97 The mem- brane is important in providing a platform on which the microheater and sensing ﬁlm are suspended. Recent interest in SiC based micro- heaters stray from the typical SiO2/Si3N4 stack, but their fabrication is very complex and thereby also cost intensive.90 The SiC microheaters are also not compatible with CMOS fabrication and therefore will not be covered here. g The gas sensing capability of SnO2 is well known and, over the last few years, it has become the most commonly used SMO material for gas sensing, resulting in its commercialization.26–31 SnO2 is also the material which was shown to be the easiest to integrate into the CMOS silicon technology.13,44,74,85 Figure 5a shows the response of an SnO2 thin ﬁlm to the presence of carbon monoxide (CO) in the en- vironment, compiled from recent publications from A. K¨ock et al.44,86 Here, and in subsequent ﬁgures, the sensitivity is represented as the ratio, in percent, of the resistance reduction when in the presence of a target gas, compared to the resistance in air or inert ambient, ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 ownloaded on 2020-01-10 to IP Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B867 (a) (c) (b) Figure 5. SnO2 thin ﬁlm sensor response during exposure to a varying concentration of carbon monoxide (CO) gas. In (a) K¨ock et al.44,86 showed the inﬂuenc of temperature and evaporated gold particles on the sensor response on a 50nm thin spray deposited ﬁlm. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP ec address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP ) unless CC License in place (see abstract). ecsdl.org/site/terms_use n subject to ECS terms of use (see Microheater Design However, increased intricacies in the geometries and attempts to reduce the pitch mean an increased complexity in the fabrication technique and poten- tial reliability concerns. Having many corners and sharp turns for the current could result in current crowding and increase the likelihood of early electromigration failure, cracking, and localized deformations. Circular structures and rounded corners are therefore generally pre- ferred as an alternative to the typical rectangular meander shape from Figure 7. predictable temperature uniformly across the entire active sensor area is essential to provide conﬁdence in the sensor’s response. However, increased intricacies in the geometries and attempts to reduce the pitch mean an increased complexity in the fabrication technique and poten- tial reliability concerns. Having many corners and sharp turns for the current could result in current crowding and increase the likelihood of early electromigration failure, cracking, and localized deformations. Circular structures and rounded corners are therefore generally pre- ferred as an alternative to the typical rectangular meander shape from Figure 7. Power efﬁciency.—An extensive amount of work has recently been devoted to novel microheater designs to improve the temperature uni- formity, power consumption, and thermal isolation, while easing the fabrication requirements. The relationship between the input power and microheater temperature is near linear, allowing for the use of a microheater efﬁciency parameter in terms of ◦C/mW. However, this is insufﬁcient when trying to characterize microheaters across a broad range of sizes, since the heater area plays a signiﬁcant role in the power dissipation. Therefore, in this review we treat power efﬁciency as the temperature increase when 1mW of power is applied to a microheater with equivalent area of 1mm2, in terms of mm2·K/mW. The efﬁciency for recently published microheaters since 2000 is summarized in Table II. In this review we concentrate primarily on CMOS-integrable designs. The summary is sorted by year of publication, ranging from 2000 until 2018.73,74,78,91,100,106–121 From Table II we further extract a general trend that larger mi- croheaters have a higher value for the power efﬁciency. This is most evident in the works of Siegele et al.118 and Ali et al.74 In these works, the authors fabricated two microheaters each using the identical fab- rication process, but with different surface areas. This resulted in the larger heater requiring more power, but in it having a better power efﬁciency. Microheater Design Platinum is frequently used as a heating element today due to its ability to deal with high current densities and it being chemically inert at high temperatures.66 The main drawback of platinum is its cost and it having a positive temperature coefﬁcient of resistance (TCR), which magniﬁes hotspot effects, leading to concerns over the long-term re- liability of the microheater and potential response drift.100 Tungsten was also suggested as a potential material,74 and it seems ideal since it is effectively resistant to electromigration, but its tendency to form an oxide at temperatures above 300◦C makes it problematic for use as a heating element. Currently, research into nickel and nickel alloys for micro-heaters is intensiﬁed67,101–103 due to their low coefﬁcient of thermal expansion (CTE), resistance to humidity, and high Young’s modulus. Tantalum-Aluminum (TaAl) is another promising compos- ite material, recently suggested in Ref. 97, the advantage of which is its ability to maintain mechanical strength at high temperature and the negative TCR of about −100ppm/◦C. Microheater geometry.—Many attempts have been made over the years to optimize the geometry of the heater in order to achieve tem- perature uniformity in the active membrane region. Several designs involve the placement of a highly thermally conductive element (sil- icon, polysilicon, or metal) below or above the microheater in order to distribute the heat more uniformly.1 However, this method results in additional lithography steps and increased fabrication costs. An additional and widely used approach is the efﬁcient modulation of the microheater geometry. The geometries can be broadly classiﬁed as rectangular, square, circular, or irregular, further subdivided into honeycomb, drive wheel, elliptical, etc. shown in Figure 6 and char- acterized by several research groups.76,104,105 The meander design, shown in Figure 7, is most commonly used in combination with rect- angular or circular geometries. The line widths and the separation between lines has a signiﬁcant inﬂuence on the efﬁciency of the tem- perature distribution. Minimizing the separation, or pitch, can lead to an improved power uniformity and lower power dissipation. The microheater geometry plays an integral role in deﬁning the sensor performance. The quality of the microheater to provide a A good microheater material is characterized with having a low thermal conductivity, high melting point, high electrical resistivity, Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B868 Figure 6. Microheater Design Microheater geometries characterized and modeled by different research groups.76,104,105 These include several shapes: (a) Meander, (b) S-meander, (c) Curved, (d) S-curved, (e) Double spiral, (f) Drive wheel, (g) Elliptical, (h) Circular, (i) Plane plate, (j) Fin shape, (k) Honeycomb, and (l) Irregular. Figure 6. Microheater geometries characterized and modeled by different research groups.76,104,105 These include several shapes: (a) Meander, (b) S-meander, (c) Curved, (d) S-curved, (e) Double spiral, (f) Drive wheel, (g) Elliptical, (h) Circular, (i) Plane plate, (j) Fin shape, (k) Honeycomb, and (l) Irregular. With regard to Table II, it should be noted that in Ref. 119 the values used were only mentioned as typical values for power consumption of micro-hotplates using an estimated efﬁciency of 15◦C/mW, which was then applied to their structure’s geometry to ﬁnd the power dis- sipation. It is not clear what was precisely measured or simulated to obtain this number. In some of the studies mentioned, the active area covers a very small section of the microheater; this is not uncommon since temperature uniformity is almost impossible to achieve up to the edge of the microheater. However, in Ali et al.74 the active area covers only 7% and 0.6% of the membrane area for the 26mW and 11mW heaters, respectively. This can help improve the power efﬁciency num- bers since the rest of the heater can have very poor uniformity or be thermally isolated, while only the small active area is exposed, ex- plaining their very good power efﬁciency. At ﬁrst sight it may seem that a better efﬁciency evaluation should be dependent on the required active area. However, our aim is to evaluate the microheater design itself and not the size of the sensor it is heating. Also, introducing this metric would then force the introduction of other factors includ- ing temperature uniformity, fabrication complexity, microheater and membrane thicknesses, among many others. Our goal in this review was to summarize the efﬁciency of the complete microheater design and heater area purely concerning the power-temperature relationship, irrespective of the active area or performance factors. It is clear that having an efﬁciency metric, which takes all components into account, would be desirable to have in the future. predictable temperature uniformly across the entire active sensor area is essential to provide conﬁdence in the sensor’s response. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP ) unless CC License in place (see abstract) ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see Microheater Design A possible explanation for this is the fact that heaters with a larger surface area have a lower ratio of circumference to surface. Since the presented heaters have a closed membrane, much of the heat is lost on the sides, or along the circumference of the microheater. This can explain the trend of improved power efﬁciency with increasing size. In Ali et al.74 the larger microheater has a circumference which is approximately 46% wider than that of the smaller design, resulting in an efﬁciency improvement of about 32%. Similarly, the results from Siegele et al.118 suggest an increase in the side length of a square heater by 30% results in an improvement of the power efﬁciency by about 23%. There are clearly additional effects which need to be con- sidered to properly evaluate a microheater, but these works validate the general hypothesis that fewer larger microheaters are preferred to many smaller ones. In the context of a sensor array, shown in Figure 8, it would be much more efﬁcient if the sensors did not each have an Figure 7. Meander microheater design with a square pattern. The second line is used for temperature measurement. Figure 7. Meander microheater design with a square pattern. The second line is used for temperature measurement. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B869 Table II. Characteristics and efﬁciency of recently-published microheaters. The power efﬁciency is calculated as the temperature increase above room temperature, in Kelvin, over a 1mm2 area when 1mW is applied to the microheater (mm2 · K/mW). Area x1000 (um2) Heater material Microheater type Temperature (◦C) Power (mW) Efﬁciency (mm2 · K/mW) Ref. Microheater Design A design which takes this into consideration is discussed in the next section. Recent microheater designs.—The recent designs achieved by Lahlalia et al. in Ref. 78 have an area of 0.9mm × 0.6mm, with a structural membrane stack formed with layers of 500nm thick silicon dioxide (SiO2), 300nm thick silicon nitride (Si3N4), and an additional 500nm thick SiO2. Platinum was used for the microheater and elec- trodes for its stability, linearity, and resistance to oxidation at a broad range of operating temperatures. Aluminum-copper (AlCu) is chosen for the microheater pads for its high electrical conductivity and low thermal conductivity. For electrical insulation between microheater and sensor contact metallization, a 300nm thick silicon dioxide layer is deposited on top of the microheater. The two recently suggested novel geometries, which address key concerns for gas sensor devel- opers are the microheater array design and the dual hotplate design, shown in Figure 9. A short description of the designs will be given here, while an in-depth analysis is provided in Ref. 78. The principal goals of the designs are to provide a power-efﬁcient sensor array and to enable a uniform temperature distribution across the active sensor Microheater array.—The microheater array design is shown in Figure 9a and combines small resistances in an array instead of a conventional single-layer microheater. This allows for the localized heating of the sensing layer to different temperateness at different locations, thereby allowing for a natural integration with a sensor array using a single heat source and a reduced power consumption. The small resistances also provide an ultra-fast thermal response time allowing the microheater, which can operate in ultra-short pulse mode, to further reduce the average power consumption to a few hundred μW.1 Since the sensitivity of the SMO ﬁlms toward target gases is opti- mal only at a single temperature, as depicted in Figure 10, the array can be used to simultaneously detect reactions at multiple tempera- tures. Microheater Design 22.5 Polysilicon S-Shaped 420 64.5 0.14∗ 106 90 Polysilicon MOSFET 300 100 0.25∗ 107 10 Polysilicon Meander 300 27.5 0.10 91 201.6 Platinum Meander 300 50 1.13 108 562.5 Platinum Meander 300 75 2.10 108 1.6 Platinum Meander 400 9 0.07 109 10 Platinum Meander 600 33 0.18 110 57.6 Polysilicon Meander 400 26 0.84 111 149.8 Tungsten Circular 500 100 0.72∗ 112 70.8 Polysilicon Circular 300 50 0.40 113 58.1 Platinum Circular 350 190 0.10 114 246 Tungsten Circular 500 26 4.55+ 74 70.8 Tungsten Circular 500 11 3.09+ 74 4000 Dilver P1 Meander 200 130 5.54 115 10 Polysilicon Meander 400 10 0.38 116 14.6 Platinum Meander 400 18 0.31 100 48.4 Platinum Meander 348 31.3 0.51∗ 117 10 Polysilicon Plate 400 24 0.16 118 4.9 Polysilicon Plate 470 18 0.12 118 4.9 Polysilicon Plate 495 17.5 0.13 118 283 Platinum paste Circular 600 35 4.69‡ 119 2.5 Platinum Meander 400 11.8 0.08 73 283 Silicon Circular 660 83 2.18 120 10 Tungsten Meander 480 20.1 0.23∗ 121 25.3 Platinum Circular 300 6.8 1.04∗ 78 ∗Simulation-based studies; ‡Mentioned in Ref. 119 as typical values, assuming an efﬁciency of 15◦C/mW; +The active area covers only 7% and 0.6% of the membrane area for the 26mW and 11mW heaters, respectively. Table II. Characteristics and efﬁciency of recently-published microheaters. The power efﬁciency is calculated as the temperature increase above room temperature, in Kelvin, over a 1mm2 area when 1mW is applied to the microheater (mm2 · K/mW). ﬁciency of recently-published microheaters. The power efﬁciency is calculated as the temperature increase above ver a 1mm2 area when 1mW is applied to the microheater (mm2 · K/mW). Simulation based studies; ‡Mentioned in Ref. 119 as typical values, assuming an efﬁciency of 15◦C/mW; +The active area covers only 7% and 0.6% of the membrane area for the 26mW and 11mW heaters, respectively. ; ‡Mentioned in Ref. 119 as typical values, assuming an efﬁciency of 15◦C/mW; +The active area covers only 7% and 0.6% of the membrane area for the 26mW and 11mW heaters, respectively. area without increased fabrication complexity, otherwise introduced by a heat spreading plate. By concentrating on these two properties, the designs describe the general trends of signiﬁcant interest to sen- sor manufacturers: miniaturization, power efﬁciency, and improved selectivity. individual microheater, but if one large microheater could be engi- neered to serve multiple sensing ﬁlms. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see Microheater Design Here, we see the sensitivity of doped and undoped SnO2 thin ﬁlms in detecting methane (CH4), H2, CO, propane (C3H8), isobutene (i-C4H10), ethanol (C2H5OH), and nitric oxide (NO) from several pub- lished works on SnO2 sensors.122–124 The collected measurements can subsequently be processed and treated using non-parametric analysis such as principal component analysis, discriminant functions, or neu- ral networks to distinguish between each gas, improving the overall selectivity of the SMO sensor.125,126 Recently, an additional improve- ment on this design was suggested by introducing two sensing layers, deposited within the same membrane structure.78 The design allows for further potential in adjusting the microheater dimensions and po- sition in order to incorporate even more sensing layers, effectively addressing the problem of selectivity in SMO gas sensors. With this design, a reduction in current crowding by about 20% was noted, when compared to conventional designs. Figure 8. Sensor unit showing a sensor array with interface electronics blocks. A sensor array allows for selectivity improvements, but requires multiple sen- sors to read the gates in the air at the same time. Figure 8. Sensor unit showing a sensor array with interface electronics blocks. A sensor array allows for selectivity improvements, but requires multiple sen- sors to read the gates in the air at the same time. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 ownloaded on 2020-01-10 to IP B870 Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) f y Figure 9. Novel microheater designs, including (a) microheater array and (b) dual hotplate geometries. Figure 10. Sensitivity of doped and undoped SnO2 toward different gases as a function of operating temperature. (a) The response from an undoped SnO2 ﬁ to several gases while varying temperature from 50◦C to 450◦C (b) The response of an electron beam evaporated SnO2 ﬁlm to a 300ppm concentration of seve Figure 9. Novel microheater designs, including (a) microheater array and (b) dual hotplate geometries. Figure 9. Novel microheater designs, including (a) microheater array and (b) dual hotplate geometries. Figure 10. Sensitivity of doped and undoped SnO2 toward different gases as a function of operating temperature. (a) The response from an undoped SnO2 ﬁlm to several gases while varying temperature from 50◦C to 450◦C. (b) The response of an electron beam evaporated SnO2 ﬁlm to a 300ppm concentration of several gases in an environment with 65% humidity. Microheater Design The silicon etch rate is dependent on the crystallographic orientation and, under the noted etch conditions, the rates for directions <100>, <110>, <111>, and <311> were found to be R100 = 13.3nm/s, R110 = 24.2nm/s, R111 = 0.1nm/s, and R311 = 23.9nm/s, respectively. Although the KOH structure displays a very clean geometry without undesirable lateral etching (Figure 12), the process can be very cor- rosive to the surrounding devices. For this reason, further analyses of the plasma etching on the same geometry have been carried out. This process is less corrosive and more compatible with CMOS fabrication, but suffers from profound lateral etching, as shown in Figure 12. Dual-hotplate.—The dual-hotplate design is shown in Figure 9b and is a combination of a single circular microheater, suggested by Elmi et al.,127 together with two passive micro-hotplates, suggested by Lahlalia et al.78 The hotplates are used for improved thermal unifor- mity in the active region. Even a small variation in the temperature over the sensing element can lead to baseline drift, changing the baseline resistivity of the SMO sensor, thereby requiring frequent calibrations. The fact that the additional hotplates are electrically passive ensures that the power consumption is kept at a minimum, which is also a critical factor to guarantee stability of the sensor baseline. A new membrane shape has been designed to accompany the dual- hotplate structure, formed by four curved micro-bridges, in order to provide improved insulation against heat losses to the substrate, shown in Figure 11b. In Figure 11a the three-armed membrane shape, used with the microheater array, is shown. The differences in the mechan- ical stability of the two membrane types, estimated by modeling, are discussed in the following section. Of note is the lack of sharp corners, but the use of rounding instead. This is in order to reduce the effects of current crowding, which could lead to void or hillock formation and eventual cracking. p g g Simulations of plasma etching have been carried out using the physics model described by the group at the University of California in Refs. 132 and 133, implemented within the ViennaTS tool.131 The key idea is to use a stochastic approach to the particles, which are present in the plasma chamber and which have an inﬂuence on the etching process. The particles can either be neutral, representing a chemical etch component, or ionic, representing a physical etch component. Microheater Design (c) The response of a sol-gel deposited pure SnO2 ﬁlm and (d) 1wt% Pt:SnO2 ﬁlm to the presence of CO, NO, and C3H8 at various temperatures.122–124 Figure 10. Sensitivity of doped and undoped SnO2 toward different gases as a function of operating temperature. (a) The response from an undoped SnO2 ﬁlm to several gases while varying temperature from 50◦C to 450◦C. (b) The response of an electron beam evaporated SnO2 ﬁlm to a 300ppm concentration of several gases in an environment with 65% humidity. (c) The response of a sol-gel deposited pure SnO2 ﬁlm and (d) 1wt% Pt:SnO2 ﬁlm to the presence of CO, NO, and C3H8 at various temperatures.122–124 ) unless CC License in place (see abstract ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 wnloaded on 2020-01-10 to IP ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 d on 2020-01-10 to IP Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B871 Figure 11. Structure of the SMO gas sensor membrane, used with the (a) microheater array and (b) dual-hotplate designs. can be generated by etching from the back of the wafer using deep reactive ion etching (DRIE) techniques or from the top, through open- ings in the membrane using wet chemical etching or plasma etching. When etching from the back, a closed membrane is generated, while etching through holes in the top results in a suspended membrane with suspension beams connecting the active region to the rest of the structure.66 The closed membrane thicknesses are in the range between 1μm and 2μm, but the required back-side etching techniques make the process more expensive and not fully CMOS-compatible.97,129 For this reason, we concentrate our studies on the suspended-membrane sensor. Figure 11. Structure of the SMO gas sensor membrane, used with the (a) microheater array and (b) dual-hotplate designs. A thorough analysis has been performed on a suspended mem- brane sensor with a typical geometry and an active area of 100μm × 100μm using both wet chemical potassium hydroxide (KOH) and SF6 plasma models.72 The simulation for KOH etching was achieved using the model described in Ref. 130 and the ViennaTS tool.131 Using a 150 minute etch with a KOH concentration of 30% and a temperature of 70◦C the suspended membrane was generated with a 100μm hole. Microheater Design The chemical etch component is the primary contributor to the lateral etching underneath the eventual suspended membrane. However, since the lateral etching proceeds in all directions, the overall size required by the sensor increases when this method is applied. Using SF6 plasma chemistry with a ﬂuorine ﬂux of 1×1019cm−2s−1 and disregarding any ion involvement, a 300 second etch was sufﬁcient to expose the membrane to the level shown in Figure 12. The ion involvement was not simulated because it only adds to the vertical etching, while the goal of a membrane release etch is to ensure a maximum lateral etching underneath the membrane. This is achieved using a fully isotropic etch, meaning removing the vertical inﬂuence of the ions. Modeling and Analysis of SMO Sensor Structures In order to have a complete picture of the fabrication, reliability, and operation of SMO gas sensors, measurements alone do not suf- ﬁce. Many modeling techniques and simulation tools are essential in order to provide an in-depth analysis of the interplay between differ- ent materials in this complex structure. In this section the essential modeling approaches used to gain a deeper understanding of SMO gas sensors are described. These include models for the fabrication of the CMOS-integrable devices, electro-thermal modeling using ﬁnite element methods (FEM) and compact models, as well as mechanical simulations using FEM. With regard to the microheater operation, the focus is on its power dissipation, thermo-mechanical properties, and mechanical stability,128 while the SnO2 metal oxide is discussed in terms of its conductive response in the presence of a target gas in the environment87 in the next section. In follow-up analysis, it was found that the lateral etching had no adverse effects on the stress distribution in the active sensor region.72,88 The modeled post-processing stress is a combination of the residual stresses in the layers which make up the entire membrane. The con- trol of the residual stress in multilayered structures is crucial for its stability. The typical values for the as-deposited stress in the Si3N4 SMO sensor fabrication techniques.—The complexity in the fab- rication of SMO gas sensor devices is heightened due to the need for a thermally-isolated membrane. As mentioned earlier, this membrane Figure 12. Side view of a suspended membrane structure for an SMO sensor, where the difference in proﬁle generated using plasma etching and wet chemical etching is pronounced. d membrane structure for an SMO sensor, where the difference in proﬁle generated using plasma etching and wet chemica Figure 12. Side view of a suspended membrane structure for an SMO sensor, where the difference in proﬁle generated using plasma etching and wet chemical etching is pronounced. Figure 12. Side view of a suspended membrane structure for an SMO sensor, where the difference in proﬁle generate etching is pronounced. Figure 12. Side view of a suspended membrane structure for an SMO sensor, where the difference in proﬁle generated using plasma etching and wet chemical etching is pronounced. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 nloaded on 2020-01-10 to IP ) unless CC License in place (see abstract ecsdl.org/site/terms_use address. Modeling and Analysis of SMO Sensor Structures Heat loss mechanisms in the SMO gas sensor and surroundings, where Th is the microheater temperature, required for sensor operation and Ta is the ambient temperature. followed by memory and computationally intensive simulations. The ambient conditions in the model are set to room temperature (20◦C) and the heat transfer coefﬁcient with the air is set to a value derived from a temperature-dependent function based on the ﬂuid motion and conduction through air.97 The value is calculated using a Nusselt number, assuming laminar ﬂow.137 The main sources of heat loss, depicted in Figure 13 are conduction and convection through the air and conduction through the membrane. There is a slight loss to radiation in the air, but this is relatively minor when compared to other losses. In Ref. 134 the heat conduction for a typical 100μm×100μm sensor structure was calculated, which required a total of 32.5mW to heat to 400◦C. Of the total dissipated 32.5mW, 18.9mW was lost to the air conduction above the membrane and 1.3mW to the air conduction below the membrane. A total of 12.2mW was lost to the conduction through the membrane beams, while a minimal 0.16mW was lost to radiation. Therefore, we can safely conclude that the bulk of the heat lost in a suspended membrane is through the membrane and air conduction through the top exposure to the air. For a closed membrane, most of the heat loss will be conduction through the solid material, along the sides of the active area and through the silicon wafer. Electro-thermal analysis.—Understanding the electro-thermal behavior of the sensor is essential to understanding its long-term reli- ability, sensitivity, and selectivity. In a recent work97 several means of characterizing the power-temperature behavior of a TaAl microheater was examined. These included the use of resistance temperature detec- tors (RTDs) of platinum and chromium silicon (CrSi) and comparing the characterizations to ﬁnite element simulations and an analytical model. Modeling the electro-thermal behavior of the sensor and mi- croheater in particular is essential to understanding the heat losses, shown in Figure 13, and thereby to minimizing the power dissipa- tion in the designed sensors. Modeling and Analysis of SMO Sensor Structures Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP B872 Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) Figure 13. Heat loss mechanisms in the SMO gas sensor and surroundings, where Th is the microheater temperature, required for sensor operation and Ta is the ambient temperature. and SiO2 layers are approximately 1GPa and −320MPa, respectively. These values were applied in the simulations in Ref. 72 and then the structure was allowed to relax. The average resulting stress in both structures was found to be on the order of 300MPa,72 which is outside of the acceptable range of residual stress, which should remain below 100MPa. The simulation nevertheless shows how the membrane deals with an applied external stress, which is most commonly the thermal stress during operation, since the membrane temperature regularly changes from several hundreds of Celsius to room temperature within short time spans.134 p Another aspect of the sensor fabrication which has been investi- gated is the deposition of the sensing SMO ﬁlm itself. This layer can be deposited in a variety of ways, including chemical vapor deposi- tion, sputtering, pulsed-layer deposition, sol-gel process, rheotaxial growth and vacuum oxidation, and spray pyrolysis.72,88 Sputtering and spray pyrolysis are methods which are quite straight forward and cost effective to implement within the CMOS sequence. Using both etching methods, the resulting geometry was analyzed with a topog- raphy simulator ViennaTS131 in Ref. 135. Spray pyrolysis showed a more isotropic coverage around corners and edges; however, the spray pyrolysis deposition requires elevated temperatures (400◦C), meaning that a thermal stress can develop in the ﬁlm due to the subsequent cooling to room temperature. This is not necessarily a disadvantage, since it results in having a ﬁlm which is relatively stress-free at elevated temperatures, considering that the SMO ﬁlm must be heated to temperatures in the range of 250◦C to 500◦C in order to activate sensing.88 The two main post-processing stress com- ponents analyzed are the intrinsic stress, which forms during the ﬁlm growth, and the thermo-mechanical stress, which results due to the difference in the deposition temperature and the subsequent cooling to room temperature.88,136 The thermo-mechanical stress is a concern when elevated temperatures are used for the deposition process due to the differences in the CTE between the depositing material and the substrate. Figure 13. Modeling and Analysis of SMO Sensor Structures Electrical equivalent Voltage (V) Permittivity (F/m) Electric resistivity ( · m) Electric resistance () Current (A) Electric conductivity (S/m) Charge (A · s) Electric capacitance (F) Temperature (K) Speciﬁc heat (J/Kg · K) Thermal resistivity (K · m/W) Thermal resistance (K/W) Heat ﬂow (W) Thermal conductivity (W/K · m) Heat (W.s) Thermal capacitance (J/K) Figure 15. Total displacement (mm) of the membrane stack when heated from room temperature (20◦C) to 300◦C. selectively test for several gases (Figure 10) during a single current pulse. This is due to the effective expansion in the membrane width due to the lateral etching component during plasma etching. The displace- ment in the novel membrane structures for the microheater array and dual-hotplate designs from Ref. 78, described earlier, are shown in Figure 15. There, the displacement is induced due to the thermal stress generated after heating the structure from 20◦C to 300◦C. We note a displacement of about 3μm for the 4-beam design and about 0.8μm for the 3-beam design. This means that the designs both im- prove upon the generic membrane structure from Figure 12 and that the 3-beam membrane displaces much less, indicating that it has a reduced likelihood of crack formation on the surface of the sensing element. In order to eliminate the need for FEM simulations and the associ- ated meshing and computational requirements, a model has been de- veloped, which can be used to represent the temperature ﬁeld and cal- culate the electro-thermal behavior of the sensor.78 Although FEM is a very powerful tool and can accurately predict many electro-thermal- mechanical phenomena, it requires a mesh to represent the entire sen- sor structure as well as the surrounding air. When dealing with very thin layers in a large structure, the aspect ratios required are large and the number of mesh elements can quickly increase to levels not easily manageable on a standard desktop computer. In addition, methods such as FEM or ﬁnite volumes, generate the complete set of equa- tions at each explicit node and for every temperature, meaning a large number of equations must be solved. As an alternative, an analytical model is frequently used, which emulates an integrated circuit (IC) us- ing thermal elements, represented by their electrical equivalents. The entire structure is effectively broken down into small segments, each represented with a parallel thermal resistor-capacitor-inductor circuit. Sensing Mechanism of the SMO Film A complete understanding of the sensing mechanism of the SMO sensor is not yet available, but signiﬁcant progress has been made recently in understanding conductivity and surface charge effects in one of the most frequently used SMOs, SnO2. The change in resis- tance of the sensing layer, when exposed to a target gas, is the factor which determines its sensitivity. Because the resistivity changes in the presence of a reducing or oxidizing gas, understanding the change in the resistivity, or conductivity, is the key to understanding the SMO’s sensing mechanism. As is the case for chemical sensors in general, the sensing effect is based on reception and transduction. The reception is that of an analyte gas with an SMO layer, through a surface chemical reaction, and transduction is of the changes at the SMO layer surface, Mechanical reliability analysis.—One of the major concerns re- garding the lifetime of SMO gas sensors is the long-term stability of the suspended membrane. The high temperatures, combined with a variety of materials with different CTEs, can lead to the buildup of ex- cessive stress in the membrane and eventual cracking or delamination failures.88 The stress in the membrane is a combination of the residual stresses in the layers which make up the complete membrane stack. This stress builds up due to two factors: intrinsic stress during depo- sition and thermal stress due to the post-deposition cooling to room temperature.41,88 The deposition of metal and semiconductor layers most often follows the Volmer-Weber growth mode, which involves the generation of islands, which then grow and impinge on each other, forming grains and grain boundaries.136 This type of stress buildup has been studied in the membrane layers as well as in the SMO ﬁlm itself,138 showing that spray pyrolysis deposited ﬁlms suffer from a higher stress at room temperature than sputtered ﬁlms. However, dur- ing operation at elevated temperatures, the sputtered ﬁlms experience increased thermal stresses. Figure 16. Temperature as a function of applied power to the microheater in a design presented in Ref. 97. The ﬁgure shows the power of the analytical model to replicate the realistic electro-thermal behavior of a SMO sensor.97 ) unless CC License in place (see abstract ecsdl org/site/terms use ms of use (see Using FEM, the effects of the as-deposited stress on the deforma- tion in the membrane layer have been studied. Modeling and Analysis of SMO Sensor Structures The analysis is usually performed in a ﬁnite element environment, which requires meshing the full geometry With this in mind, the microheater array and dual-hotplate designs, presented in the previous section, were optimized and signiﬁcantly improved, resulting in a total power dissipation of only 9.31mW and 8mW, respectively, when operating at 350◦C. In Figure 14, the thermal distribution for the two designs is shown. Here, we also note that the temperature uniformity across the active region is very good, with variations below 10◦C or below 3%. In the microheater array design in Figure 14b we also see two active regions with two different target temperatures operating simultaneously. This design allows us to heat the sensor to 270◦C and 350◦C at the same time, meaning that we can d (b) i h t d i Th i h t ll f t Figure 14. Simulation of the thermal distribution (◦C) in a (a) dual-hotplate design and (b) a microheater array design. The microheater array allows for concurrent operation at multiple temperatures. Figure 14. Simulation of the thermal distribution (◦C) in a (a) dual-hotplate design and (b) a microheater array design. The microheater array allows for concurrent operation at multiple temperatures Figure 14. Simulation of the thermal distribution (◦C) in a (a) dual-hotplate design and (b) a microheater array design. Th operation at multiple temperatures. ) unless CC License in place (see abstract ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 ownloaded on 2020-01-10 to IP Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B873 Figure 15. Total displacement (mm) of the membrane stack when heated from room temperature (20◦C) to 300◦C. Table III. Parameters for a thermal model equivalent of an electrical circuit model. Thermal parameter Electrical equivalent Temperature (K) Voltage (V) Speciﬁc heat (J/Kg · K) Permittivity (F/m) Thermal resistivity (K · m/W) Electric resistivity ( · m) Thermal resistance (K/W) Electric resistance () Heat ﬂow (W) Current (A) Thermal conductivity (W/K · m) Electric conductivity (S/m) Heat (W.s) Charge (A · s) Thermal capacitance (J/K) Electric capacitance (F) selectively test for several gases (Figure 10) during a single current pulse. Modeling and Analysis of SMO Sensor Structures In Table III the relationship between the heater’s thermal properties and an electrical component equivalent is shown. Using this strategy, the complete sensor geometry is discretized and an IC, which very accurately calculates the heat loss by convection, is generated. The power of this method is made obvious in Ref. 97, where the results are compared with measurements and an FEM simulation, shown in Figure 16. The method was able to accurately calculate the power- temperature relationship even for complex microheater geometries. ) unless CC License in place (see abstract) ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP Sensing Mechanism of the SMO Film Which sections of the surface are able to adsorb charge depends on the type of sensing ma- terial. A porous SMO ﬁlm is most commonly used, due to its granular structure, which increases its effective surface to volume ratio. A high surface to volume ratio exacerbates surface effects in the ﬁlm and is therefore desired for sensing devices. SMO sensor is not unlike a transistor; however, instead of directly ap- plying a potential at the gate, a potential is applied indirectly through the accumulation of charges at the surface. This charge stems from the ionosorption of gases at the SMO surface. Which sections of the surface are able to adsorb charge depends on the type of sensing ma- terial. A porous SMO ﬁlm is most commonly used, due to its granular structure, which increases its effective surface to volume ratio. A high surface to volume ratio exacerbates surface effects in the ﬁlm and is therefore desired for sensing devices. −→ jn = qnμn ⃗E + q Dn∇n, [4] −→ jn = qpμp ⃗E −q Dp∇p, [5] [5] where −→ jn and −→ jp are the current densities for n-type and p-type semi- conductors, respectively; R is the recombination rate; μn and μp are the mobilities of electrons and holes, respectively; Dn and Dp are the diffusion constants for electrons and holes, respectively; q is the elementary charge of an electron, and ⃗E is the applied electric ﬁeld. The n and p values are the electrons and hole concentrations in an n-type and p-type semiconductor, respectively. When a potential V is applied across the ﬁlm, the electric ﬁeld ⃗E is derived from the solution to the Poisson equation, with appropriate boundary conditions, which relate the derivative of the electrical potential to the surface charge density ρ and the permittivity ϵ0ϵr of the material with Porous SMO ﬁlms.—The typical SMO gas sensors in use today con- sist of a porous ﬁlm deposited on an insulating substrate. The porous ﬁlm is comprised of many grains, all of which have their own effective external surface, which can be exposed to a target gas, thereby increas- ing the surface to volume ratio. The conductivity involves conduction through grain-grain, grain-bulk, and grain-electrode interfaces, as de- scribed in some detail in Ref. 144. Sensing Mechanism of the SMO Film The limiting conducting factor is the grain-grain interface, since this has the highest resistance due to the limited charge carrier concentration and reduced mobility at the surfaces of the grains. Since the mobility of the charge carriers remains unchanged during gas molecule adsorption, the resistance de- pends purely on the charge concentration.139 The charge concentration can be manipulated through the sharing of charges with an adsorbed molecule, which simplistically describes the sensing mechanism. −∇· (ϵ0ϵr∇V (⃗x)) = ρ. [6] [6] The boundary conditions used are Dirichlet at the ohmic contacts, where a particular value for the potential is set, and Neumann condi- tions elsewhere. The electric ﬁeld is calculated by the negative value of the applied potential gradient: Modeling the conductivity at the grain-grain interface assumes that the Schottky approximation is valid, meaning that all donors are fully ionized. In this case the electron concentration at the surface is described by the Boltzmann distribution ⃗E (⃗x) = −∇V (⃗x) [7] [7] In an n-type ﬁlm, such as is the case with SnO2, only Equations 2 and 4 are considered using electrons as majority carriers, while in a p-type ﬁlm, such as CuO, the majority carriers are holes and they would have to be considered by solving 3 and 5. The discussion in the remainder of this sections deals with n-type materials only and more speciﬁcally with SnO2. How the equation would change when p-type materials are used can be extrapolated from this discussion. ns = NDexp −qVs kBT , [9] [9] where ND is the donor density, qVs is the energy of the surface band bending, which is depicted in Figure 19, kB is the Boltzmann constant, and T is the temperature. The resistance of the sensing layer is thereby proportional to the surface band bending qVs. When modeling the sensor, the surface charges can be used to calculate a surface potential Vs, which is solved for using the Schottky relation Furthermore, it is generally assumed that the depletion layer thick- ness is in comparable dimension to the mean free path of the charge carriers, allowing to eliminate the diffusion component from 4 and 5, thereby removing q Dn∇n and greatly simplifying the problem. Sensing Mechanism of the SMO Film Surprisingly, it has been found that the thermal stress distribution in the active area induced during thermal cycling is unaffected by the method of the mem- brane formation, whether using KOH or plasma etching.72 However, the maximum displacement, at the center of the membrane (with a 100μm×100μm active region), was found to be 8μm for the plasma- etched membrane, compared to 5μm for the KOH-etched membrane. Figure 16. Temperature as a function of applied power to the microheater in a design presented in Ref. 97. The ﬁgure shows the power of the analytical model to replicate the realistic electro-thermal behavior of a SMO sensor.97 ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B874 Figure 17. Temperature dependence on the electron concentration (n) and mobility (μn) of SnO2. The conductivity is a combination of both n and μn by σ = q · n · μn. which inﬂuence the electronic conductive properties of the sensing ﬁlm and charge transport therein.139 which inﬂuence the electronic conductive properties of the sensing ﬁlm and charge transport therein.139 SMO conductivity.—SnO2 is a wide bandgap n-type semiconduc- tor, which means its donor states are related to oxygen vacancies and that electrons are its majority carriers during conduction. The calcu- lated bandgap between the valence and conduction bands is 3.6eV,140 which was also measured and conﬁrmed experimentally.141 Drift-diffusion.—The electrical conduction of SMO ﬁlms is mod- eled using drift-diffusion equations.142 The drift diffusion equations are commonly used when modeling conduction in a semiconductor and are deﬁned by the diffusion and drift components, controlled by the number of charge carriers, which can be electrons or holes, the mobility of the charge carriers, and the applied electric ﬁeld. The drift diffusion equations are summarized by Figure 17. Temperature dependence on the electron concentration (n) and mobility (μn) of SnO2. The conductivity is a combination of both n and μn by σ = q · n · μn. ∇· −→ jn = R, [2] [2] ∇· −→ jp = −R, [3] [3] [3] SMO sensor is not unlike a transistor; however, instead of directly ap- plying a potential at the gate, a potential is applied indirectly through the accumulation of charges at the surface. This charge stems from the ionosorption of gases at the SMO surface. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP Sensing Mechanism of the SMO Film Therefore, from 4 we extract the conductivity σ = −→ jn / ⃗E, which can now be deﬁned as a combination of both the concentration n and the mobility μn with Vs = − qN 2 ef f 2ϵ0ϵr ND , [10] [10] σ = q · n · μn. [8] [8] where Nef f is the sum of the electrons which gain enough energy to reach the surface together with the external electrons donated from adsorbed or ionosorped gas ions.145 The resistance of the sensing layer is therefore proportional to the surface band bending according to The electron concentration n and the electron mobility μn can vary signiﬁcantly with temperature, even in a fully inert environment;143 therefore, also the conductivity is signiﬁcantly inﬂuenced by temper- ature. The inﬂuence of temperature on the electron mobility and con- centration is depicted in Figure 17, where the resulting temperature- dependent conductivity is also shown. The conductive behavior of the R ∝exp −qVs kBT . [11] [11] ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 on 2020-01-10 to IP Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B875 Figure 20. It is theorized that, after depleting all the oxygen at the SMO surface, CO gas molecules can directly interact with surface atoms, thereby donating atoms to the grain and forming an accumulation layer.40 Figure 18. Surface charge density [A · s/m2] in a synthetic air environment, with 80% N2 and 20% O2. This shows the effects of the surface ionosorption with O−and O2−ions. Figure 20. It is theorized that, after depleting all the oxygen at the SMO surface, CO gas molecules can directly interact with surface atoms, thereby donating atoms to the grain and forming an accumulation layer.40 species in SnO2 sensors using IR spectroscopy and summarized the steps in the transformation of an atmospheric oxygen (O2,gas) to a lattice oxygen (OO) as Figure 18. Surface charge density [A · s/m2] in a synthetic air environment, with 80% N2 and 20% O2. This shows the effects of the surface ionosorption with O−and O2−ions. Figure 18. Surface charge density [A · s/m2] in a synthetic air environment with 80% N2 and 20% O2. This shows the effects of the surface ionosorption with O−and O2−ions. Figure 20. Sensing Mechanism of the SMO Film It is theorized that, after depleting all the oxygen at the SMO surface, CO gas molecules can directly interact with surface atoms, thereby donating atoms to the grain and forming an accumulation layer.40 Figure 18. Surface charge density [A · s/m2] in a synthetic air environment, with 80% N2 and 20% O2. This shows the effects of the surface ionosorption with O−and O2−ions. species in SnO2 sensors using IR spectroscopy and summarized the steps in the transformation of an atmospheric oxygen (O2,gas) to a lattice oxygen (OO) as Since the sensor signal describes the change in resistance, the sen- sitivity can also be described by a difference in the surface band bending. 1 2 O2,gas + V 2+ O + 2e−⇀ ↽OO [12] [12] by including all intermediate steps and species, in the following set of reactions: by including all intermediate steps and species, in the following set of reactions: In summary, the ionosorption of gas molecules attracts electrons from the SnO2 bulk, resulting in band-bending at the interface between the material and the surrounding gas. The amount of band bending is proportional to the effective concentration of localized surface elec- trons Nef f .142 In Figure 18, the surface charge density is plotted versus time at various temperatures in a synthetic air environment, where O− and O2−ions are adsorbed at the surface. It is evident that the sur- face charge density saturates in accordance with the total number of ionosorption sites available. Also of note is the inﬂuence of tempera- ture; increasing the temperature from 250◦C to 350◦C allows for the oxygen ions to reach saturation more quickly. O2,gas ⇀ ↽O2,ads [13] [13] O2,ads + e−⇀ ↽O− 2,ads [14] [14] O2,ads + e−⇀ ↽O− 2,ads O2,ads + e−⇀ ↽O− 2,ads [15] O− 2,ads + e−⇀ ↽O2− 2,ads O− 2,ads + e−⇀ ↽O2− 2,ads [15] O2− 2,ads ⇀ ↽2O− ads [16] [16] Surface chemisorption.—The sensing of a desired gas, such as CO, occurs after a CO molecules reacts with a previously ionosorped oxygen atom, releasing it from the surface, as depicted in Figure 19. The chemical reactions taking place at the surface during the oxygen adsorption mechanism has been researched extensively. Sensing Mechanism of the SMO Film Molecular species and atomic species have been proposed to interact with the SnO2 surface.144,146 Degler139,147 recently studied the active oxygen O− ads + e−⇀ ↽O2− ads ⇀ ↽OO [17] [17] The adsorption of carbon monoxide (CO) molecules then proceeds according to: COgas + O2− 2,ads + (2 −α) · e−⇀ ↽(CO3)α− [18] [18] Figure 19. Gas sensing and conduction mechanism for a porous metal oxide, where the oxygen and reducing gas can penetrate to interact with each grain. In (a) the oxygen ion is adsorbed on the grain surface, forming a depletion region around the grain. In (b) after the introduction of a reacting gas such as CO, which reacts with some of the oxygen ions, the depletion region thickness is reduced. [19] COgas + OO ⇀ ↽(CO2)O [19] Based on the above description, sensing reducing gases, such as CO, would only be detectible in the presence of oxygen; however, it was recently found that, even without O2, the SMO ﬁlm continues to show a sensing response, while no oxidation product (CO2) was found in the exhaust.148 This led to the expansion of the above description to include a subsequent step, which is the formation of a donor species (e.g. CO+) which adsorbs and directly injects electrons into the con- duction band.148 This is shown graphically in Figure 20, where the injection of electrons leads to the formation of a narrow accumulation layer. An explanation for this behavior is the adsorption of CO on a tin (Sn) site, whereby charge is transferred directly from the CO molecule to the solid:149,150 COgas ⇀ ↽COads [20] [20] Figure 19. Gas sensing and conduction mechanism for a porous metal oxide, where the oxygen and reducing gas can penetrate to interact with each grain. In (a) the oxygen ion is adsorbed on the grain surface, forming a depletion region around the grain. In (b) after the introduction of a reacting gas such as CO, which reacts with some of the oxygen ions, the depletion region thickness is reduced. In summary, there are several cases of gas reception on SnO2 ﬁlms and they each behave somewhat differently, but nevertheless are based on the interplay of the surface reduction and re-oxidation by atmospheric oxygen. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) N2 ambient) the surface en- ergy bands are ﬂat and the number of charges on the surface corresponds to the number of charges in the bulk. p g 2. In the presence of oxygen gas, the SMO ﬁlm’s surface oxygen vacancies are ﬁlled and a charge is trapped at the formed lattice oxygen species, thereby ensuring that the donor concentration at the surface is lower than that in the bulk and creating a depletion layer. S = 1 + (kreact/kdes) · pCO κ · pH2O 1/(α + γ) , [25] y 3. If only a reacting gas (CO) is present, with no atmospheric oxy- gen, a surface oxygen vacancy reacts with CO, thereby reducing the surface. Therefore, the donor concentration at the surface is increased, when compared to the bulk, and a thin accumulation layer is formed, without the release of oxidation by-products. [25] where κ = 1 + kH2O,ads · [SnSn]2/kdes and γ is used to account for the effects of humidity. A deeper investigation for the modeling of power law exponent n = 1/(α + γ) has been performed by Hua et al.152–154 and is described in the next section. y y p 4. When both O2 and CO are found in the atmosphere, the surface oxygen will be formed by adsorption to the oxygen vacancy site. This will subsequently be removed during oxidation of CO to form CO2 and the surface will be continuously re-oxidized. In high concentrations of atmospheric oxygen, this will lead to a reduction in the depletion layer. In low oxygen concentration, or in the presence of interfering gases (e.g. H2O), the surface oxygen vacancy concentration is higher than that in bulk and an accumulation layer is formed. However, after the oxygen is depleted, there is an observed switch in conduction mechanisms from depletion-layer controlled to accumulation-layer controlled, discussed in the previous section and illustrated in Figure 19 and Figure 20. This change is signiﬁcant for transduction, since conductance is no longer proportional to the surface concentration of free charge carriers, but rather the average over the accumulation layer. The reception of gas molecules (e.g. CO) is also different, since they no longer only react with the adsorbed oxygen, but also with SnO2.40 A fully worked out model for this in- teraction is not yet available, but the following surface reactions are suggested in Ref. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) 40: While the adsorption of CO through the intermediate adsorption of oxygen is discussed here, several studies also deal with gas detection through the intermediate adsorption of water (H2O) instead. For an in-depth analysis of the use of hydroxide as an intermediate species, further reading in Refs. 139,151 is recommended. COgas + SD,CO ⇀ ↽CO+ D + e−, [26] [26] Surface reaction model for CO on SnO2.—The reactions described above are the closest researchers have come to understanding the sur- face interactions leading to gas detection in SMO ﬁlms. This model is quite intricate and involves many steps with many reaction constants, as summarized in Figure 21.147 With this model, the full simulation of a complex structure is very difﬁcult and cumbersome, with several ﬁtting parameters necessary, including the adsorption and desorption rates of all reactions. When attempting to simulate a sensor, whether it be a porous ﬁlm40 or a nanowire,142 several estimations are generally made. Primarily, the intermediate reactions are left out and the ad- sorption of oxygen, water vapor, and subsequently carbon monoxide is treated using three reactions40 H2Ogas + SD,H2O ⇀ ↽H2O+ D + e−, [27] [27] The reaction suggests the direct adsorption of reactive species on the surface and the addition of an electron, which contributes to the formation of an accumulation layer. Experimentally it was shown that an approximate amount of downward band bending, qVS from Figure 20, results in a change in the ﬁlm’s resistance by40 The reaction suggests the direct adsorption of reactive species on the surface and the addition of an electron, which contributes to the formation of an accumulation layer. Experimentally it was shown that an approximate amount of downward band bending, qVS from Figure 20, results in a change in the ﬁlm’s resistance by40 R ∝exp −qVs 2kBT , [28] [28] which has an extra 1 2 in the exponent, compared to 11. Since it is expected that the donor species (e.g. CO+ D) determines the sensing effect, the electron concentration will be proportional to the concen- tration of the adsorbing gas molecule (e.g. CO). The conductance is proportional to the average electron concentration in the accumulation layer, which is proportional to the square root of the electron surface concentration. This square root dependence of the conductance on the partial pressure of CO can explain the extra 1 2 in the exponent in 28. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B876 Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) Figure 21. Proposed surface chemistry model for the reception of small re- ducing gases on a pristine SnO2 surface.147 oxygen species, and e−represents an electron in the conduction band. It becomes evident that only the surface oxidation/re-oxidation (situ- ation 4. above) is treated in this model, as a comprehensive model for the situations 2. and 3. are still a work in progress. The mass action law stemming from the reactions described by 21, 22, and 23 can then be applied to generate a rate equation for the oxygen surface coverage [O−α S ] in the steady state d O−α S dt = kads · p 1 2o2 · [S] · nα S −kdes · O−α S −kH2O,ads · pH2O · [SnSn]2 · O−α S d O−α S dt = kads · p 1 2o2 · [S] · nα S −kdes · O−α S −kH2O,ads · pH2O · [SnSn]2 · O−α S Figure 21. Proposed surface chemistry model for the reception of small re- ducing gases on a pristine SnO2 surface.147 + kH2O,des · [O H]2 · e · nα S −kreact · O−α S · pCO, [24] [24] reactions at elevated temperatures for simple molecules, which do not produce species through intermediate surface reactions:139,151 reactions at elevated temperatures for simple molecules, which do not produce species through intermediate surface reactions:139,151 where the square brackets [ ] denote surface coverage, kads/des are the reaction constants for oxygen adsorption/desorption, kH2O,ads/des are the reaction constants for water vapor adsorption/desorption, kreact is the reaction constant for Equation 23, pO2/CO are the partial pressures of O2/CO, and nS is the concentration of electrons at the surface. Since the concentration of tin atoms at the surface is very large com- pared to those of the hydroxyl groups and with surface coverage θ = [O−α S ]/[S], the sensor signal S = RCO/Rair can be modeled with a power law approximation 1. With no reacting gas present (e.g. N2 ambient) the surface en- ergy bands are ﬂat and the number of charges on the surface corresponds to the number of charges in the bulk. 1. With no reacting gas present (e.g. Sensing Mechanism of the SMO Film The following situations are suggested to describe all possible In summary, there are several cases of gas reception on SnO2 ﬁlms and they each behave somewhat differently, but nevertheless are based on the interplay of the surface reduction and re-oxidation by atmospheric oxygen. The following situations are suggested to describe all possible ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) This phenomenon has yet to be fully described and modelled, with discussions still ongoing on what exactly causes the power law re- sponse experimentally observed in SMO sensors and how to properly 1 2 O2,gas + S + α · e−⇀ ↽O−α S [21] [21] H2Ogas + 2 · SnSn + O−α S ⇀ ↽2 · (SnO H) + α · e−+ S [22] COgas + O−α S →CO2,gas + α · e−+ S, [23] COgas + O−α S →CO2,gas + α · e−+ S, [23] where S is a surface adsorption site, α = 1 for strongly ionized oxygen, α = 2 for doubly ionized oxygen, O−α S is a chemisorbed where S is a surface adsorption site, α = 1 for strongly ionized oxygen, α = 2 for doubly ionized oxygen, O−α S is a chemisorbed Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B877 this is still a missing piece toward the development of a comprehensive model of the SMO sensing mechanism. model the response to the sensing, as given by n = 1/(α + γ) in 25, which is discussed in the next section. model the response to the sensing, as given by n = 1/(α + γ) in 25, which is discussed in the next section. An additional aspect, which should be included when performing experiments on a SMO sensor, is the ability to accurately measure the temperature provided by the microheater in the active region. Tem- perature plays a signiﬁcant role in deﬁning the sensor signal, both the reception and transduction components; however, as it was dis- cussed in an earlier section, the temperature uniformity and accurate measurements of the temperature are not easily obtained. In Ref. 97, the authors used several methods in order to attempt to extract the temperature, while to date no error-free method is known to the au- thors. In addition, humidity does not only inﬂuence the way in which the sensing proceeds, but it also changes the power dissipation of the SMO microheater. The change in power dissipation also changes the thermal resistive behavior of the microheater itself, effectively reduc- ing the provided temperature for the active sensor area, ultimately inﬂuencing the sensing performance and introducing another layer of uncertainty. When developing SMO sensing behavior models based on experimental results, the accuracy of the experimental setup and proper extraction is of upmost importance. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) Nt is deﬁned as the number of donors of the charge density ND, found in the depletion region. Solving 9, 10, and 11 and substituting in the transducer function from 29 the authors determine the transducer function as The only relevant transducer function, treated by the authors, is the double-Schottky barrier model across neighboring grains, visualized in Figure 19. Nt is deﬁned as the number of donors of the charge density ND, found in the depletion region. Solving 9, 10, and 11 and substituting in the transducer function from 29 the authors determine the transducer function as Inﬂuence of noble metal additives.—As previously discussed, the introduction of noble metal additives has been used to increase the sensitivity and selectivity of SMO gas sensors. A perfect example is the use of Pt in SnO2 sensors for CO detection, shown in Figure 10d. Recently, researchers have attempted to address the inﬂuence of plat- inum additives in the reception-transduction behavior of the SnO2 sensor using operando spectroscopy.156 The authors found that nano- sized clusters of Pt are formed on the sensing ﬁlm’s surface. These act as primary reaction sites for CO oxidation, thereby signiﬁcantly increasing the sensing effect, when compared to pristine SnO2 ﬁlms. The amount of platinum also determines whether CO oxidation or CO sensing will dominate, therefore simply having more of an additive does not automatically mean an improved sensor response, even if the additive is more reactive. This is clearly demonstrated in Ref. 87 and in Figure 10b, where the introduction of 0.2wt% Pt resulted in an improved performance, while increasing this to a 2wt% reduced the sensing response to below pure SnO2 levels. A reproduction of these experiments in Ref. 139 in a humid environment showed that the re- sponse of the 2wt% loaded ﬁlm does not fall below that of the undoped ﬁlm, but is nevertheless less sensitive than the 0.2wt% alternative. d R dNt = Nt (L D ND)2 = m L D ND , [30] [30] where L D is the Debye length and m is deﬁned to refer to the reduced surface charge density using where L D is the Debye length and m is deﬁned to refer to the reduced surface charge density using m = Nt L D ND . [31] [31] The second part of the function given in 29 is the reduction, which involves several components. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) One way to ensure a proper link between the characterized and simulated temperatures is to avoid the complex microheater and membrane structure while performing the measurements, but instead using a heat chamber. This is most commonly done and it ensures the same conditions for the simulation and characterization. However, understanding how the sensor behaves when the temperature distribution across the SMO ﬁlm is not uniform would be an important study in itself. In addition, including the in- ﬂuence of humidity on the change in the microheater temperature, and not only on the surface reactions, would go a long way in the formation of a fully-encompassed SMO simulator. Only through such a simulator, which covers multiple aspects of an SMO device, will a leap in the development and scaling of these devices be made possible. Power-law response.—Recently, Hua et al.152–154 investigated means to physically model the power-law response of SMO gas sen- sors. The exponent in the power-law response is one of the primary problems in fully understanding the sensing mechanism of MOS gas sensors and in providing an accurate physical model. The exponent was shown to be speciﬁc to the particular gas and temperature.152–154 However, other results also show the exponent varying with prepara- tion conditions of the sensing ﬁlm and the relative humidity.155 The model proposed by Hua et al. is summarized here, but for a more detailed discussion, the reader should refer to.152–154 The study has been performed by observing the power response of SnO2 to exposure to CO in the presence of dry air. The transducer model is built on two conducing mechanisms, the Schottky barrier model and grain model, while the receptor functions were calculated using the law of mass action for oxygen with different concentrations of reducing gas. The primary novelty in the work is the suggestion that the exponent n can be fully broken down into two components, one dealing with the transducer function and one the receptor function. The receptor function Rce deals with the adsorption of oxygen and subsequent reaction with CO or H2, while the transducer function Ts deals with the inﬂuence of the added charges on band bending and resistance: d R d P = d R dNt × dNt d P [29] [29] The only relevant transducer function, treated by the authors, is the double-Schottky barrier model across neighboring grains, visualized in Figure 19. ) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.131.44.51 Downloaded on 2020-01-10 to IP Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) Essentially, the authors solve this in the same way as previously discussed, using the mass action law. For the different oxygen species O−, O− 2 , and O2−the derived equation for the exponent was n = 1 2 1 − 1 m2 + 1 , [32] n = 1 − 1 m2 + 1, and [33] n = 1 4 1 − 1 2m2 + 1 , [34] n = 1 2 1 − 1 m2 + 1 , [32] n = 1 2 1 − 1 m2 + 1 , [32] n = 1 − 1 m2 + 1, and [33] [32] n = 1 − 1 m2 + 1, and [33] [33] The ways in which a noble metal atom can inﬂuence the sens- ing ﬁlm is described in some detail in several recent works156,157 and are partially summarized in Figure 22. The two primary mechanisms are chemical and electrical sensitization. The chemical sensitization mechanism is related to the spill-over of gases, which is primar- ily attributed to metallic clusters which adsorb oxygen or reducing gases.158 The adsorbed gas molecules are subsequently activated and transferred to the SnO2 surface, increasing the reactivity of the sens- ing ﬁlm, thereby improving the sensitivity, as depicted in Figure 22a. The electrical sensitization mechanism is based on the Fermi level alignment of the SMO material and noble metal phase, due to the fact that the metal phase has a different work function. The contact be- tween the two materials leads to an alignment of the Fermi levels and thereby surface band bending. This band bending is now controlled by the contact between the two materials and not by the presence of an ionosorbed species, depicted in Figure 22b. n = 1 4 1 − 1 2m2 + 1 , [34] [34] respectively. The Equations 32, 33, and 34 can be described by a single equation n = α 2β 1 − 1 βm2 + 1 , [35] [35] where α = 1 or α = 2 for dissociative or non-dissociative adsorption, respectively, and β is the charge state of oxygen adsorbates on the surface. A power law exponent is furthermore calculated for the ad- sorption of reducing gases, and the adsorption of oxidizing gases. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) In the absence of a chemical and electrical interaction between the metal phase and SMO ﬁlm, a competitive reaction on the noble metal phase takes place, decreasing the concentration of the target gas, thereby hindering the sensing effect, shown in the top surface reaction in Figure 22b.139 However, an oxidation reaction at the interface of the two solids could enhance the gas reception and thereby the sensing behavior.157 The noble metal atoms or ions can also be incorporated in the SMO’s lattice, changing the chemical and electrical behavior of the ﬁlm and thereby the reception and transduction effects. In Figure 22c the various locations of noble metal atom incorporation in the SMO ﬁlm and their effects are shown, as adapted from Ref. 139. If the valence state of the metal ion is different from that of the replaced cation, additional acceptor or donor states can be introduced.157 Par- ticularly doping with Pt leads to a lowered resistance of the sensing ﬁlm and an enhanced CO oxidation due to the increased number of oxygen vacancies generated.157,159 steps were analyzed using process simulation tools. For mechanical simulations, in order to understand the stress buildup and distribution in the complex structure, ﬁnite element analysis have been carried out. Electro-thermal characterization has been performed using FEM and an analytical model has been developed as a means to represent the thermal behavior of the sensor in an equivalent electrical circuit form. Using these simulations, we can perform a complete electro-thermal- mechanical simulation to better understand the long-term mechanical reliability of the complex membrane structure of the SMO sensor. Re- cent designs have been described, which show the improved behavior of suspended membranes with rounded corners, due to the reduced current accumulation effects, which could lead to high stresses and eventual failure. Finally, recent models which describe the sensing mechanism of SMO ﬁlm through the reception-transduction behavior have been pre- sented. Additional calculations have been performed in order to char- acterize the effects of gas ionosorption at the surface of SMO thin ﬁlms. The adsorbed charges generate an effective surface potential, which forms an electric ﬁeld, inﬂuencing the behavior of the charge carriers in the SMO and thereby also its conductance. While the con- ductivity of the SMO ﬁlm can be described using drift diffusion equa- tions with reasonable accuracy, the exact chemisorption steps are still not fully known. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) The calculated values seem to agree with the experiments shown; however, the full picture is also here not given. The subsequent adsorption of gas molecules to form a thin accumulation layer is not addressed and where α = 1 or α = 2 for dissociative or non-dissociative adsorption, respectively, and β is the charge state of oxygen adsorbates on the surface. A power law exponent is furthermore calculated for the ad- sorption of reducing gases, and the adsorption of oxidizing gases. The calculated values seem to agree with the experiments shown; however, the full picture is also here not given. The subsequent adsorption of gas molecules to form a thin accumulation layer is not addressed and Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) B878 Figure 22. Cross section of a grain within a SMO ﬁlm, together with a noble metal doping. (a) The red metal atom can serve to spill over an oxygen molecule, slitting it into two atoms and allowing it to adsorb to the surface. (b) Fermi level control mechanism based on the alignment of the Fermi levels of the SMO ﬁlm and the noble metal. Also, a competing catalytic reaction with the metal atom is shown. (c) Atomic sites are shown, with the metal atom acting as a reactive site and possibly as an acceptor/donor. The images are adapted from Ref. 139. Figure 22. Cross section of a grain within a SMO ﬁlm, together with a noble metal doping. (a) The red metal atom can serve to spill over an oxygen molecule, slitting it into two atoms and allowing it to adsorb to the surface. (b) Fermi level control mechanism based on the alignment of the Fermi levels of the SMO ﬁlm and the noble metal. Also, a competing catalytic reaction with the metal atom is shown. (c) Atomic sites are shown, with the metal atom acting as a reactive site and possibly as an acceptor/donor. The images are adapted from Ref. 139. Journal of The Electrochemical Society, 165 (16) B862-B879 (2018) The most recent studies on the surface chemisorption of CO on SnO2 have been described in this review. While the general opinion was that the sensing mechanism involves the adsorption of oxygen at the surface, which creates a depletion layer, followed by a reaction of that oxygen with CO gas molecules to form CO2, thereby reducing the depletion layer once more. Although this is accurate in most cases, there are still events which are less clear, including the evi- dence of a sensing behavior even when little or no oxygen is present in the ambient or in an inert/CO ambient. The most recent understanding of these phenomena have been summarized in this review along with a discussion on the inﬂuence of noble metal additives to the SMO ﬁlm. Primarily, the additives can improve the sensitivity, and sometimes selectivity, of the ﬁlm; however, increasing the amount of additive metal does not immediate mean an increase in the sensitivity. There is a more complex relationship, which still requires further research to completely understand. Conclusions There has been signiﬁcant progress recently in the design, model- ing, and understanding of SMO gas sensors. These types of sensors are currently the cheapest and most portable option available for the detec- tion of harmful environmental pollutants. The ability to integrate the device with CMOS circuitry and to fabricate it using a mature CMOS foundry are primary reasons for its success in industry and commer- cialization. However, in order to make a truly integrated smart gas sensor using an SMO ﬁlm, there are several aspects which still need to be improved upon. Those include an improvement in the reliability of the different layers used as well as a complete understanding of the sensing mechanism itself, which is still lacking. Advanced modeling and simulation tools have been used to obtain a deeper understanding of the fabrication, reliability, and operation of semiconductor metal oxide gas sensors. The advanced analysis and characterization have allowed us to recently develop optimized sensor designs which reduce the power consumption, improve temperature uniformity, and minimize stress accumulation. Of primary importance is reducing the power dissipation of gas sensor arrays to levels com- patible with portable technologies, while ensuring proper temperature uniformity across the active sensor area. In this review, recent de- signs were compared for their power efﬁciency, showing that few large microheaters are preferred to many small ones. Recent designs introduce the concept of a microheater array, which takes advantage of this knowledge to ensure improved power dissipation while still allowing for multiple sensors in order to introduce selectivity to the SMO device. In addition, researchers are always searching for new materials, which have improved microheater properties; however, this is difﬁcult as it is still desired to have these readily available in a CMOS fabrication facility, due to the associated cost reduction. ORCID Lado Filipovic https://orcid.org/0000-0003-1687-5058 Ayoub Lahlalia https://orcid.org/0000-0002-4680-0498 References 1. J.-H. Park et al., IEEE Sens. J., 17(3), 784 (2017). 2. D. R¨uffer et al., Sensors, 18(4), 1052 (2018). 3. H. Li et al., IEEE Sens. J., 14(10), 3391 (2014). 4. R. Moos et al., Sens. Actuators B, Chem, 83(1–3), 181 (2002). 5. G. Martinelli et al., Sens. Actuators B, Chem, 55(2–3), 99 (1999). ec address. 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https://openalex.org/W2153380371	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/C8E670BB1F0E2185F0EDDFB4B8C5AB8E/S0047279414000828a.pdf/div-class-title-social-investment-after-neoliberalism-policy-paradigms-and-political-platforms-div.pdf	English	null	Social Investment after Neoliberalism: Policy Paradigms and Political Platforms	Journal of social policy	2,014	cc-by	9,508	Jnl Soc. Pol. (2015), 44, 2, 297–318 C⃝Deeming, C. & Smyth, P. 2014. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Jnl Soc. Pol. (2015), 44, 2, 297–318 C⃝Deeming, C. & Smyth, P. 2014. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:10.1017/S0047279414000828 Abstract The concept of the ‘social investment state’ refocuses attention on the productive function of social policy eclipsed for some time by the emphasis on its social protection or compensation roles. Here we distinguish between different social investment strategies, the Nordic ‘heavy’ and the Liberal ‘light’, with particular reference to the inclusive growth approach adopted in Australia. In 2007, social democrats in Australia returned to government with a clear mandate to reject the labour market deregulation and other neoliberal policies of its predecessor, and to tackle entrenched social and economic disadvantage in Australian society. For the last ﬁve years, social investment and inclusive growth has been at the centre of the Australian social policy agenda. Against this background, the article examines and critically assesses the (re)turn to ‘social investment’ thinking in Australia during Labor’s term in ofﬁce (2007–13). Analysis focuses not just on what was actually achieved, but also on the constraining role of prevailing economic and political circumstances and on the processes that were used to drive social investment reform. In many ways, the article goes some way to exposing ongoing tensions surrounding the distinctiveness of ‘social investment’ strategies pursued by leftist parties within the (neo)liberal state. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Social Investment after Neoliberalism: Policy Paradigms and Political Platforms CHRISTOPHER DEEMING∗and PAUL SMYTH∗∗ ∗School of Geographical Sciences, University of Bristol, UK email: chris.deeming@bristol.ac.uk ∗∗School of Social and Political Sciences, University of Melbourne, Australia email: p.smyth@unimelb.edu.au Introduction In this article, we critically examine the concept of ‘social investment’ and the political strategies pursued by social democratic parties in power. Here we distinguish between investment platforms, the Nordic ‘heavy’ and the Liberal ‘light’, with particular reference to the inclusive growth strategy adopted by the Australian Labor Party during its term in ofﬁce (2007–13). We critically examine the reasons for Labor adopting a ‘social investment’ approach. In so doing, the article provides a critical analysis of the content and coherence of Labor’s social investment strategy in terms of policy change (cf. Hall, 1993). Analysis focuses not just on what was actually achieved, but also on the constraining role of prevailing economic and political circumstances and on the processes that were used to drive social investment reform. More broadly, we are interested in situating the Australian case within the growing literature on ‘social investment’, org/10.1017/S0047279414000828 Published online by Cambridge University Press christopher deeming and paul smyth 298 and we contrast the Australian ‘light’ approach with the ‘heavy’ investment strategies pursued elsewhere in Northern Europe. In many ways, the article goes some way to exposing ongoing tensions surrounding the distinctiveness of ‘social investment’ strategies pursued by leftist parties within the (neo)liberal state. The concept of a ‘social investment state’ has a deep resonance with an Australian social policy history strongly characterised by productivist values and giving each other a ‘Fair Go’ (opportunity). We illustrate this brieﬂy from the classic moments in the evolution of the ‘Australian Way’ before reﬂecting on the (re)turn to ‘social investment’ policies in the early part of the twenty-ﬁrst century. We show how this productivist revival helped break the austerity constraints on Australian social policy created in the neoliberal period. At the same time, we ﬁnd that the contemporary concept of ‘the social investment state’ is only a partial expression of this productivist tradition and remains politically fragile because of deep uncertainties around the future of domestic economic policy. We also ﬁnd that, because of its rather unique history and policy pathway of achieving redistributive efforts using the instruments of wage regulation (rather than public expenditure in traditional areas of welfare), Australia has a particular challenge of reviving the productive dimension of social policy in a way that is not detrimental to its protective or compensatory functions. Introduction Needless-to-say, some of the longer-term outcomes and returns on investments in areas such as early childhood development, mental health, education and social mobility, for example, cannot be accommodated in this review. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Social investment: emerging concept and policy context Social investment: emerging concept and policy context ‘Socialinvestment’arguablyrepresentstheverylatestjustiﬁcationforsocialpolicy to guide the development of the economy and society in the twenty-ﬁrst century. Theearlyoriginsofthesocialinvestmentperspectivearetraceabletothefounding of the Swedish social-democratic welfare state in the 1930s, and the arguments of Swedish social democrats who viewed social policy as an investment rather than a cost (Tilton, 1993). Since the late 1990s, new ideas and strategies concerning the role of social policy for societal development have been formulated, and, internationally, policy agendas now point towards a similar policy logic based around notions of ‘social investment’ (Midgley, 1999). The notion of ‘social investment’ makes reference to policies that aim to help disadvantaged citizens to succeed in education and the labour market. As Jenson (2012) argues, ‘social investment’ can be distinguished both from the more traditional ‘Keynesian’ social policy approach based on income and job protection and from the ‘neoliberal’onebasedonderegulationandthecommodiﬁcationofhumanlabour. Nevertheless, social policy scholars do not share a singular theoretical core on a par with macro demand-side management or supply-side economic liberalism, nor could we say that the ‘social investment’ perspective has fully emerged as the https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press social investment after neoliberalism 299 dominant policy paradigm (see, for example, Hemerijck, 2012). Despite some of the conceptual vagueness, two core features may be observed: investment in human capital and the objective of full labour market participation. Indeed, the notion that the social investment state seeks to rebuild the welfare state around work has become iconic in the European context but, as we shall see, this has been a long-standing tradition in the Australian context. Conceptually, scholars attempt to separate out ‘social investment’ spending from social spending on ‘old social risks’ relating to pensions, healthcare and unemployment beneﬁts (but this can be problematic as we shall see). More broadly, scholarship continues to observe distinctions between different ‘worlds’ (Morel et al., 2012) and ‘varieties’ of social investment regime (Bonoli, 2009), with further distinctions between different investment strategies. These may be described as ‘strong’ or ‘weak’ for example (Bonoli, 2012), or (as we prefer) ‘heavy’ and ‘light’, and our notion of ‘Social Policy as Productive Factor’ may go beyond that originally envisaged in Europe in the late 1990s (European Commission, 2000). https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Social investment: emerging concept and policy context The experience of the Nordic countries suggests that it may be perfectly possible to combine strong social protection (i.e., ‘old’ social spending) with heavy investment in human capital (i.e., ‘new’ social spending, particularly in education and early childhood policies) in order to secure greater levels of equality and foster the human capital of future generations (Kvist 2014). According to Esping-Andersen et al. (2002) social policy should actively mobilise the productive potential of citizens in order to mitigate the ‘new’ social risks of the post-industrial knowledge-based economy (adversely affecting low-skilled workers, women, young adults and children). New family- and child-centred investment strategies, for example, can break patterns of social inheritance and exclusion. Social protection, however, is of paramount importance even in the most productivist of welfare states, as Esping-Andersen (2001) argues. The human capital-based investment strategy will inevitably leave some citizens behind, and, thus, it remains an imperative to have a secure welfare safety net of minimum income support underpinning the social investment strategy. Social democrats in market liberal economies such as Labour (between 1997 and 2010) in the UK, by contrast, have pursued lighter social and human capital- based investment strategies alongside the pro-market employment orientation of welfare provision. Here ‘passive’ welfare systems, which had created a ‘moral hazard’, were transformed by ‘active’ social policies.1 The role of the state was to facilitate ‘activation’, with new training programmes to improve skills, and new forms of welfare ‘conditionality’ to tackle the wider cultural problem of ‘worklessness’ in society. Tax and beneﬁt reforms, and incentives, helped to ‘make work pay’. This policy platform was inspired by ‘Third Way’ social democratic thinking advocated by Anthony Giddens (1998), and later embraced by the European Commission and the OECD (Hemerijck, 2012; Jenson, 2010). In Figure 1, we see the different investment strategies which show the Nordic https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press 300 christopher deeming and paul smyth Social investment expenditure Compensatory expenditure − + + Traditional compensatory welfare systems Southern and Continental Europe Dual investment liability: protection and promotion Nordic countries − Hidden welfare state USA Productive human capital investment and low protection UK Figure 1. Four worlds of social investment Source: Adapted from Morel et al. (2012: 358). dual investment approach (i.e., protection and promotion), and the UK’s human capital investment approach combined with low social protection. The USA is taken as an example of a weakly developed social investment state. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Social investment: emerging concept and policy context The policy context in Australia, however, is very different to that found in Europe. Here we ﬁnd the emergence and foundations of Australia’s ‘social investment state’ at the end of the nineteenth century and the beginning of the twentieth century (this has been well documented elsewhere, Perkins et al., 2005; Smyth, 2008). Its novelty lay in the emphasis on the role of social policy as economic investment. Thus, social investment played a key role in the economy, with extensive government investment in capital infrastructure, utilities and state enterprises. Industrial regulation provided a formative social investment pathway and the orientation towards a ‘welfare society’, rather than a ‘welfare state’. Minimum wage laws were introduced in South Australia in 1894 and then, in 1907, the ‘Harvester Judgment’ gave courts in Australia the power to determine acceptable working conditions including (minimum) living wages for families. Thus, a social economy with a fair wage was the order of the day. Clearly, an inclusive growth strategy has always been at the heart of the ‘Australian Way’. Even the so-called ‘wage earners’ welfare state’ only made sense – and in fact only ever worked – when integrated within a successful growth strategy designed to deliver more and better-paid manufacturing jobs in the industrial period. Across the 1980s and 1990s, social policy had progressively reduced its role to socialprotectionorcompensation,vacatingthesphereofeconomicproductionto the newly revived free market economics. Welfare slowly shrank to the dimension of an income ‘safety net’, and thus seemingly faced a future of permanent austerity. However, with the election of the Rudd Labor government in 2007, we observe a return to the characteristically Australian productivist tradition of active state investment. The ‘social investment’ agenda emerged ﬁrst as an economic policy agenda aimed at lifting the performance of the nation’s ‘human https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Pre social investment after neoliberalism 301 capital’ (especially disadvantaged groups who were measurably underachieving). This was overlaid by a ‘social inclusion’ agenda belatedly adopted from Europe. As we shall show below, social policies were soon being politically reframed in the languages of ‘investment’ and ‘inclusion’, although the narratives lacked depth and the policies any rigorous framework of the means for and goals of social investment (Saunders, 2013). In his critique of ‘social investment’, Nolan (2013) distinguishes the ‘paradigm’ from the ‘platform’. We shall show that social investment is already embedded as a platform in Australia. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Social investment: emerging concept and policy context But we also explore what the Australian experience can tell us about it as a paradigm. Here we have three main points of departure. The ﬁrst arises from the observation that the agenda requires a more integrated approach to economic and social policy than that which was obtained for much of the postwar period (Smyth and Buchanan, 2013). Especially after the triumph of neoliberal economics in the 1980s and 1990s, social policy found itself subordinated to the market with little room to construct a positive role for state intervention. Thus, when economic policy actually shifted to allow space for a social investment agenda which would tackle the negative effects of poverty and inequality, even while promoting economic growth, many in social policy remained suspicious of an ongoing subordination of the social to the economic (Nolan, 2013). But the challenge to a better integration of economic and social policy runs more deeply through the discipline. Thus, Beblav´y et al. (2013) describe the difﬁculties in integrating a study of education with social protection in the OECD, when the ﬁrst is positioned as being about ‘opportunities’ and the second about ‘outcomes’. Education of course is the vanguard of the social investment approach. The narrow preoccupation of the social policy tradition with the tax and transfer system has also stiﬂed dialogue with development policy where the inclusive growth approach offers a more complete integration of the economic and the social than that so far afforded by the social investment framework. Midgley (1999), for example, writes of social policy’s inability to take full account of the role of social policy in growth and structural change. To progress ‘social investment’, the new role of social policy cannot just be about protection but about building the productive capacities of citizens – principally, but not exclusively, their education and health. This leads to our second challenge. While the positive social policy environment in Australia has partly reﬂected an economy that has avoided recession for over two decades, today’s global economic environment highlights that a social investment platform by itself is not enough if the economy does not deliver sufﬁcient employment opportunities. Here social policy cannot be indifferent to the different implications for its role of either a persistent neoliberal ‘growth ﬁrst’ strategy or a shift to the alternative of ‘inclusive growth’ currently being developed by the OECD and Bretton Woods institutions. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Social investment: emerging concept and policy context In the org/10.1017/S0047279414000828 Published online by Cambridge University Press 302 christopher deeming and paul smyth inclusive growth strategy, government seeks to inﬂuence the pattern of economic development to ensure opportunities for all citizens (OECD, 2014). The third challenge – and one very much a focus of this paper – is already well canvassed in the literature: that is the tendency for the productivist values of social investment to be promoted at the expense of, rather than as a complement to, the value of social protection. Here the Australian case is of particular interest because we show how the recent renewal of the characteristically productivist welfare tradition has indeed been accompanied by greater constraints around aspects of its income support system. Australia is an interesting site on which to explore these issues. The neoliberal period brought radical not incremental change to the traditional Australian welfare state model. In the mid-1990s, the Australian welfare regime was well recognised in the international literature as a fourth – ‘radically redistributive’ – world, an exception to Esping-Andersen’s threefold typology. Australia became routinely identiﬁed as the ‘wage earners’ welfare state’ (see Deeming, 2013a, for a critical review of this literature). By the close of the twentieth century, this model had been largely conﬁned to history and the new social investment state was being designed around the needs of ‘hardworking’ Australian families. Australia’s (re)turn to social investment Australia s (re)turn to social investment If the oil crises of the 1970s helped to usher in the neoliberal chapter in Australian politics, then the global ﬁnancial crisis helped to instigate a return to social investment (Saunders and Deeming, 2011). The Rudd Labor government, which had been elected in 2007, reverted to ‘Keynesian’ social investment policies in order to stimulate the economy in the face of recession (showing the interchange of economic ideas, see Hall, 1989, on different variants of Keynesianism). ‘Nation Building’, rather than neoliberal austerity measures, point to the productive interpretation and value of social interventions as ‘social policy’ under Labor. Capital spending on national infrastructure projects and new investment in road, rail and port infrastructure, for example, may be outside the more narrow conﬁnes of the list of policy proposals contained in the Social Investment Package produced by the European Commission (2013), but we might well question the logic of this framework. Such questioning of the term and of what might reasonably be included or excluded under a ‘social investment strategy’ is a recurring theme in this article. Labor politicians now distanced themselves from neoliberal ideology, arguing instead for social democratic values and principles of social investment and inclusion to help make Australia a fairer society. As we shall see, however, there were strong continuities but also critical differences from the ‘neoliberal’ agenda, particularly with the role of the ‘enabling’ or ‘active’ investment state taking greater responsibility for workforce participation and for skills, training social investment after neoliberalism 303 and employment strategies, as articulated by social democrats in Europe (Esping- Andersen et al., 2002). Keynesian principles of social investment in human capital and infrastructure were also revived under Labor. The traditional social demo- cratic concern with social justice also remained, but the relationship between the state and recipients of welfare was now recast in a new contract of ‘rights and responsibilities’, framed by new thinking about inclusive economic growth (SmythandBuchanan,2013).TheLaborgovernmentnowﬁrmlybelievedinsocial inclusion through paid work. Policymakers agreed that all working-aged adults whocouldworkshouldbeengagedinthelabourforce,thusincreasingthecentral- ity of paid work to the securing and production of family well-being in Australia. In the lead-up to the 2007 election, Labor had announced its support for a ‘social inclusion’ policy. The approach rested on the assumption that a purely income-based approach to disadvantage does little to address the underlying causes of social disadvantage. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Australia’s (re)turn to social investment Social inclusion initiatives focused on social and economic participation as a means of overcoming social disadvantage. The social inclusion agenda had two guiding principles: ﬁrstly, it must tackle the social exclusion of individuals and communities; and, secondly, it must invest in the human capital of citizens, especially the most disadvantaged. The goal for Labor was to reduce state spending on unproductive areas of social welfare, such as social security beneﬁts, by increasing labour market participation and inclusion. Access to paid work was not only seen as the best welfare guarantee, but higher rates of labour market participation also increases the tax base and thus helps to sustain other welfare state functions, such as healthcare and pensions for older citizens in retirement. The Labor government regarded work as the best route out of poverty for unemployed Australians, although it was now content to pursue the (diminished) goal of full employability, as opposed to full employment. Australia has one of the lowest unemployment rates among the advanced economies (Figure 2), and the number of people out of work has risen following the economic downturn in 2008. Nevertheless, the legitimate role of the active welfare state was to mobilise the full productive potential of Australian citizens. New ‘activation’ and job training programmes helped to reskill workers and increase their job readiness. Long-term unemployment can deskill in the knowledge economy. Training unemployed citizens in the skills required by employers was therefore part of the supply-side approach adopted by the Labor government to overcome shortfalls in the economy. Compared to European countries, however, the level of state investment in human capital policies and active labour market programmes (ALMPs) in Australia remained relatively low by comparison, and their effectiveness to generate successful employment outcomes in the Australian context is questionable (Belchamber, 2013). Table 1 shows total annual expenditureonactivelabourmarketpoliciesinAustraliaat0.3percentofGDPfor org/10.1017/S0047279414000828 Published online by Cambridge University Press 304 christopher deeming and paul smyth TABLE 1. Australia’s (re)turn to social investment Public expenditure on active labour market policies in the advanced economies (% of GDP) 2004 2005 2006 2007 2008 2009 2010 2011 Denmark 1.8 1.6 1.6 1.3 1.4 1.7 2.1 2.3 Sweden 1.1 1.2 1.2 1.0 0.9 0.9 1.1 1.1 Finland 1.0 0.9 0.9 0.9 0.8 0.9 1.1 1.0 France 1.0 0.9 0.9 0.9 0.9 1.0 1.1 0.9 Germany 1.1 1.0 0.9 0.7 0.8 1.0 0.9 0.8 Norway 0.8 0.7 0.6 0.6 − − − − UK 0.5 0.4 0.3 0.3 0.3 0.4 − − Australia 0.4 0.4 0.3 0.3 0.3 0.3 0.3 0.3 New Zealand 0.4 0.4 0.4 0.3 0.4 0.4 0.3 0.3 United States 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs- data-en). Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs- data-en). Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs- data-en). 0 1 2 3 4 5 6 7 8 9 OECD Australia Figure 2. Unemployment rates (all adults) in the advanced economies, 2000–13 Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en). Figure 2. Unemployment rates (all adults) in the advanced economies, 2000–13 Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en Figure 2. Unemployment rates (all adults) in the advanced economies, 2000–13 Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en). the years 2007–11. Denmark had the highest expenditure on ALMPs at 2.3 per cent of GDP in 2011. The relatively low level of investment in ALMPs may surprise, given Labor’s ﬁrm commitment to social investment and inclusion. However, if social investment in ‘human capital policies’ is more broadly conceived to include education (including vocational education and training in schools and universities), we ﬁnd Australia has above average spending and performs well on educational and training outcomes (Burke, 2013). There is widespread https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press social investment after neoliberalism 305 0.00 0.10 0.20 0.30 0.40 0.50 0.60 Figure 3. The strength of the link between individual and parental earnings in the advanced economies Note: The height of each bar measures the extent to which sons’ earnings levels reﬂect those of their fathers. The higher the value, the greater is the persistence of earnings across generations, thus the lower is the intergenerational earnings mobility. Source: OECD (2010: 181–98). Figure 3. Australia’s (re)turn to social investment The strength of the link between individual and parental earnings in the advanced economies Note: The height of each bar measures the extent to which sons’ earnings levels reﬂect those of their fathers. The higher the value, the greater is the persistence of earnings across generations, thus the lower is the intergenerational earnings mobility. Source: OECD (2010: 181–98). consensusthatadvancedwelfarestateshavetomaintainahighlyskilledandhighly productive workforce in order to maintain living standards and competitive advantage in the global economy. The Labor government embraced the latest social investment principles that prioritise high levels of employment for both men and women. New family- and child-centred social policies were introduced that effectively ‘hollowed out’ the old ‘male bread-winner/female home-maker’ (or ‘wage earner’) model of welfare in favour of the new dual-earner model of family-centred welfare production (Deeming, 2013a). In terms of policy redirection, this welfare effort looked to privilege the active stages of the life course, but here Labor was also attempting to promote social mobility. For Labor’s policy of social inclusion also committed the government to tackling social exclusion at birth. Comparatively, Australia performs well on most indicators of intergenerational social mobility, which is concerned with the relationship between the socio-economic status of parents and the status their children will attain as adults. Figure 3, for example, shows the strength of the link between the individual and parental earnings. In Australia, this link is relatively low compared to the UK, where it is relatively high. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press 306 6 christopher deeming and paul smyth Labor focused much of its efforts on strengthening the system of welfare for ‘hardworking families’; this term, which has positive connotations, was extensively used by Rudd and Gillard during the 2007 federal election. Successive Labor budgets (2008–12) were mildly progressive, intent on improving the living standards for this group. The government provided tax relief for low-income households, the Low Income Tax Offset, and child-care tax rebates to the tune of $47 billion, such as the Child Care Rebate and Family Tax Beneﬁt, to help provide for their needs. The Labor administration also introduced the national Paid Parental Leave scheme in 2011. Thus, a new model of employment relations is in the making whereby both men and women share both working and family time. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Australia’s (re)turn to social investment However, the Australian ‘social investment’ discourse was clearly not just about protecting low-income working families from the perils of the free market. Labor had embraced the latest social investment principles with the new family- and child-centred policies, child-care rebates and tax breaks that now saw more afﬂuent‘middle-class’familiesdrawnintothe‘ﬁscal’welfaresystem,thuscreating a ‘dual’ welfare system in Australia. Signiﬁcantly, this ‘social investment state’ reform agenda in Australia emerged from economic not social policy – perhaps explaining why social policy research has beensoslow torecognisethemajorwelfarereformtrenditembodies. Also it originated under the Liberal–National coalition government led by John Howard (1996–2007), underscoring its bipartisan appeal. The catalyst was the call fora‘thirdwaveofnationalreform’bytheVictorianstategovernment(Australian Department of the Premier and Cabinet, 2005). It described the success of the ﬁrst two waves in reinvigorating a national economy after signiﬁcant decline in the 1980s. Thus, the ﬁrst wave opened up the economy in that decade principally through tariff reductions and ﬁnancial deregulation, while the second focused on microeconomic reform and was driven through the National Competition Policy framework established in 1995. While emphasising the ongoing value of these reforms, the report insisted that participation and productivity had now emerged as the critical drivers of future prosperity. ‘The most effective way’, it said, to boost productivity and participation is to develop our human capital. Improving health, learning and work outcomes is how we build a healthy, skilled and motivated society, and a high income economy that is among the world’s best’ (Australian Department of the Premier and Cabinet, 2005: 8). The Council of Australian Governments’ (COAG) Reform Council was established in 2006 and a national reform agenda commenced in 2008. Chairman John Brumby (2013) summarised the work after ﬁve years in a way which shows the coalescence of economic and social goals. ‘There were two main elements’, he said, ‘further regulation reform, and investment in human capital . . . (it) was all about improving the lives of Australians – their health and well-being, their skill levels and education, their participation in work and in society more broadly . . . It would be good for individuals, good for families, but also good for an economy whose greatest future strength would lie in the resilience, creativity and capacities of its people’. The key social policy areas related to the early years, schooling, the employment participation of youth and at-risk groups; better health services and systems; and closing the gap on indigenous disadvantage. Australia’s (re)turn to social investment The very notion of ‘hardworking Australian families’, the preferred expression of Prime Minister Gillard in her 2010 Address to the Nation, is not just a rhetorical device aimed at a political constituency – it also articulates a range of normative assumptions about the way in which life should be lived in Australia in the twenty-ﬁrst century. Work is seen as the best form of welfare for Australian families, not only because work provides greater ﬁnancial rewards, but also because it promotes individual and social well-being. Therefore, regulatory work-based welfare measures that had been introduced in the 1990s were strengthened in order to help move welfare claimants into paid employment. Thus, increased beneﬁt conditionality was perfectly justiﬁable in order to tackle what policymakers saw as the growing social problem of ‘welfare dependency’ in Australia and the culture of ‘worklessness’ that had evolved out of the passive system of social security transfers. With the emphasis on individual responsibility, reciprocity and obligation, there are strong continuities here with neoliberal workfare-orientated policies, as beneﬁt claimants, particularly parents with young children, now found new conditions and forms of enforcement attached to social security payments: being available for work and actively seeking employment and being able to demonstrate this, for example, were minimum codes of conduct. Harsh punitive sanctions help to guard against non-compliance (Blaxland, 2013). Labor’s Fair Work Act reversed the unpopular labour market polices of the 1990s and improved job security for workers. In particular, Labor restored to Australian workers the legal right to appeal against harsh or unfair dismissals from their place of work – a right that had been rescinded by the Workplace Relations Amendment Act 2005, known as WorkChoices. Once again, collective bargaining was encouraged and wages were to reﬂect relative living standards and the needs of workers (as well as the potential impact of changes on the labour market and unemployment levels). Low-paid workers clearly beneﬁted. Despite the falling value of minimum wages during the 1980s and 1990s, minimum wage protection levels in Australia increased during the 2000s, offering low-paid workers better protection from the vagaries of the market. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press social investment after neoliberalism 307 Targeted tax cuts for working families and the Fair Work initiative, which reinstated the notion of appropriate minimum wage rates on social justice grounds, clearly marked a return to more familiar territory for Australian social policy. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Australia’s (re)turn to social investment It is a policy proﬁle which matches broadly that associated with the Social Investment Package in Europe – relating to the early years, youth, activation and health (European Commission, 2013). https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press 08 christopher deeming and paul smyth 308 While often referred to as the ‘human capital’ agenda, the breadth of the COAG reforms points to the inﬂuence on Australian policymakers of Sen’s work on capabilities (Sen, 2005), and the capabilities approach has also underpinned the Federal Treasury’s economic well-being framework. This approach, said COAG’s ‘Third Wave’ report, ‘considers not only incomes, but also health and education outcomes . . . A human capital approach therefore supports not only economic outcomes, but also the public interest more broadly’ (Australian Department of the Premier and Cabinet, 2005). The COAG Reform Council’s ﬁve-year progress review found that the implementation and outcomes had been a mixed success. While endorsing the overall policy agenda, it noted that there was some loss of reform momentum as a result of the global ﬁnancial crises, the changing political composition of member governments and the general challenges created to national reform agendas by Australia’s federal system of government. Further, many signiﬁcant areas of reform relating to human capital have subsequently occurred outside of the COAG framework. Whether or not COAG continues as the vehicle of national reform in Australia remains to be seen. However the policy directions which it has set have provided Australia with the base for that transition from a welfare to a social investment state integral to the inclusive growth approach (Smyth and Buchanan, 2013). The Labor administration also secured major investment in ‘old’ social welfare protection against social risks during its term in ofﬁce, securing real improvementstothestandardoflivingforpensionersandpeoplewithadisability, although the extent to which all forms of ‘social spending’ may be differentiated between ‘social investment’ spending (looking for ‘returns’) and ‘non-social investment’ spending is conceptually problematic, and currently contested as previously discussed (Nolan, 2013). Superannuation policy, for instance, in Australia is arguably social investment in ﬁnancial capital accumulation (i.e., part of the ‘piggy-bank’ function of the welfare state alongside the ‘Robin Hood’ protective welfare function that redistributes resources within society). https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press social investment after neoliberalism 309 Figure 4. Total public social expenditure in the advanced economies, 2000–13 (as a percentage of gross domestic product) Source: OECD Social Expenditure Statistics (SOCX data, DOI: 10.1787/socx-data-en). Figure 4. Total public social expenditure in the advanced economies, 2000–13 (as a percentag of gross domestic product) Source: OECD Social Expenditure Statistics (SOCX data, DOI: 10.1787/socx-data-en). Australia’s (re)turn to social investment The minimum-income protection ﬂoor for pensioners was raised to 28 per cent of average weekly male earnings, following the Harmer Review (2009) of the pensions system, and the Superannuation Guarantee rate was set to increase from 9 per cent in 2012 to 12 per cent by 2018, following the Cooper Review (2010), beneﬁting 8.4 million Australians. The Australian health service was also strengthened under Labor, another key social democratic reform, and Labor introduced DisabilityCare Australia to support people with signiﬁcant and permanent disability. In many ways, the Labor government was an ambitious welfare-state builder but, in other respects, the Australian system of welfare was arguably attempting to catch up with social welfare systems found in many of the other developed economies. Importantly, Figure 4 does show a notable rise in the level of public expenditure as Labor strengthened health, education, pensions and disability services. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press social investment after neoliberalism 309 https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Social investment and equality Social investment and equality In this section, we move to consider in more detail the social security policy framework in Australia and some of the distributional impacts and consequences of Labor’s social investment and inclusion strategy. We are particularlyinterestedinsocialprotectionandout-of-workbeneﬁts;andthelong- running trends in poverty and inequality, thus placing Australian trends within a comparative context. We also consider gender inequalities and Indigenous peoples’ disadvantage. Proponents of social investment maintain that a secure welfare safety net of minimum income support is an imperative but, with the focus on productive investment and activation in the Australian context, it is far from self-evident that adequate social protection has been secured during the latest round of social democratic reforms. Most importantly, unemployment risks themselves are not evenly distributed across society, and risk is increasingly concentrated at the bottom of the socio-economic class structure. Low-skilled workers, for example, face a different and altogether higher risk of unemployment than skilled workers and middle-class professionals. There is good evidence now emerging that suggests investment in human capital may have been accompanied by greater constraints imposed on Australia’s system of income support. So, while minimum wage schemes and working tax credits helped to ensure that work paid, wage replacement rates fell in real terms by comparison with average earnings. In 2005, for example, unemployment org/10.1017/S0047279414000828 Published online by Cambridge University Press 310 christopher deeming and paul smyth 310 christopher deeming and paul smyth 0 10 20 30 40 50 60 70 80 90 100 Figure 5. Unemployment beneﬁt replacement rates for adults in the advanced economies (% of average wage levels in 2011, plus 2007 for Australia) Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en). Figure 5. Unemployment beneﬁt replacement rates for adults in the advanced economies (% of average wage levels in 2011, plus 2007 for Australia) Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en). beneﬁt replacement rates for an adult stood at 33 per cent of average wages compared to just 23 per cent in 2011 – a 10 per cent fall against average earnings. Beneﬁt levels in Australia continue to be amongst the lowest in the Western world. Thus, Australians appear to accept the risk of a relatively low standard of living and poverty if they are unable to work or become unemployed. Beneﬁt replacement rates, paid in the initial phase of unemployment, are low compared to other advanced economies. For an unemployed adult, beneﬁt replacement rates amount to about one-ﬁfth of average wages; only unemployed adults in the UK receive less (Figure 5). Wage replacement rates for single parents and couples with children in Australia are better, but they remain relatively low in comparison to the other OECD nations (Figures 6 and 7). In many ways, Labor’s approach to social investment and inclusion remained distinctly neoliberal or ‘light’, characteristic of the ‘Work First’ approach (any job is better than inactivity). Labor continued to develop the workfare-orientated strategy that relies on setting very strong work incentives, largely at the expense of income support allowances – already low by international standards. Beneﬁt conditionality increased under Labor and spending on ALMPs, again relatively low by international standards, remained unchanged over the 2007–13 period. Investment in the social security system for unemployed workers and vulnerable citizens living on out-of-work beneﬁts was not accorded a priority. social investment after neoliberalism 311 0 10 20 30 40 50 60 70 80 90 100 Figure 6. Unemployment beneﬁt replacement rates for a single parent with two children in the advanced economies (% of average wage levels in 2011, plus 2007 for Australia) Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en). Figure 6. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press 310 christopher deeming and paul smyth Unemployment beneﬁt replacement rates for a single parent with two children in the advanced economies (% of average wage levels in 2011, plus 2007 for Australia) Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en). 0 10 20 30 40 50 60 70 80 90 100 Figure 7. Unemployment beneﬁt replacement rates for a couple with two children in the advanced economies (% of average wage levels in 2011, plus 2007 for Australia) Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en). Figure 7. Unemployment beneﬁt replacement rates for a couple with two children in the advanced economies (% of average wage levels in 2011, plus 2007 for Australia) Source: OECD Employment and Labour Markets Statistics (DOI: 10.1787/lfs-data-en). https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press 312 christopher deeming and paul smyth 312 christopher deeming and paul smyth 0 5 10 15 20 25 30 35 40 45 50 Figure 8. Income inequality in the advanced economies, mid-1980s to the late 2000s (Gini coefﬁcients) Note: Arrows indicate the change and direction of income inequality. Source: OECD Income Distribution and Poverty Database (IDD in OECD.Stat). Figure 8. Income inequality in the advanced economies, mid-1980s to the late 2000s (Gini coefﬁcients) Note: Arrows indicate the change and direction of income inequality. Source: OECD Income Distribution and Poverty Database (IDD in OECD.Stat). Liberal welfare states have long assumed that individuals prefer not to work if minimum wages are undermined by decent social security beneﬁts (i.e., ‘moral hazard’); in the Australasian context this has particular resonance as work incentives have long taken priority over any meaningful standard of adequacy in social policy (Deeming, 2013a). During Labor’s term in ofﬁce, social policy scholars(e.g.,SoldaticandPini,2012)continuedtocallintoquestiontheadequacy of social security beneﬁts for those not in paid work. Labor, however, was essentially content to preserve Australia’s system of redistribution and arguably accepted the political limits and constraints imposed by the low-tax approach to social democracy (Deeming, 2013b; Wilson, 2013). In Australia, the burden of tax on waged labour is low by international standards, only Switzerland and New Zealand appear to have lower tax rates than Australia. Indeed, a major independent review of taxation policy in 2009 (Henry Review, 2009) argued that the country’s tax and transfer system, which has long favoured market freedom and individual opportunity, had served Australia well, and the Labor government accepted this verdict. These reforms go to the heart of the question of whether growth in the Australian economy has been inclusive. A strong economy and low levels of unemployment have lifted many but not all boats; a small minority of the Australian population has remained jobless. Most of the gains from economic growth were highly concentrated at the top end of the income distribution, and as a result we ﬁnd relative poverty and inequality has risen in recent times. Figure 8, for example, shows widening inequality since the mid-1980s, increasing by 1.6 percentage points on the Gini index.2 social investment after neoliberalism 313 0 5 10 15 20 25 30 Figure 9. Poverty rates in the advanced economies, mid-1990s to the late 2000s (60% of median income) Note: Arrows indicate the change and direction of income poverty. Source: OECD Income Distribution Database (IDD in OECD.Stat). Figure 9. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press 312 christopher deeming and paul smyth Poverty rates in the advanced economies, mid-1990s to the late 2000s (60% of median income) Note: Arrows indicate the change and direction of income poverty. Source: OECD Income Distribution Database (IDD in OECD.Stat). PovertyinAustraliahasalsoincreasedsincethemid-1990s,byonepercentage point. Over a ﬁfth (22 per cent) of Australians now live with disposable household income below the 60 per cent poverty threshold. As shown in Figure 9, this puts Australia above most of the other advanced economies on this measure of hardship. And while some of the latest evidence on social exclusion in Australia points to a decline in levels of exclusion overall as a result of Labor’s policies, nevertheless prospects for some of Australia’s most excluded citizens did not improve over the 2007–13 period (Saunders, 2013). So, while the size of the group in this position appears to have shrunk, its distance from the mainstream has undoubtedly grown. ProgressontacklingIndigenouspeoples’disadvantageremainedslowdespite a range of policy initiatives such as ‘Closing the Gap’, designed to create a more inclusive Australia. The unemployment rate for people of Aboriginal and Torres StraitIslanderorigininAustralia,at20percent,isoverthreetimesthenationalav- erage.IndigenousAustralianstendtoliveinareasofsocio-economicdisadvantage and experience a gap in life expectancy at birth estimated to be 11.3 years for males and9.5yearsforfemales,comparedtototalmalesandfemales.Lessthanone-third ofyoungIndigenousAustralians(agedbetweenseventeenandeighteenyears)and onlyabouthalfofyoungpeoplefromlowsocio-economicbackgroundsobtainthe school leaving qualiﬁcation (Whiteford, 2013). Youth unemployment continues to be a concern and gender inequalities stubbornly persist in Australian society. Australia is a long way from gender pay parity – a key measure of fairness – despite thepolicyof‘equalpayforequalwork’.From2010,workingparentsweregiventhe org/10.1017/S0047279414000828 Published online by Cambridge University Press 314 christopher deeming and paul smyth righttorequestﬂexibleworkarrangementsandworkingtimes.Statepolicyclearly matters but state-level provision is shaped in important ways at the organisational level and within the workplace. Women are much more ‘ﬂexible’ workers than men. In Australia, 70 per cent of part-time employment is female (so in practice Australia now has mainly one (male) and a half (female) ‘bread-winner’ families). Part-time work and ﬁxed-term contracts help to explain the inferior labour market position of women, who are disproportionately responsible for care work (Craig and Mullan, 2011). The re-engineering of welfare policy in the name of social investment by the Labor government arguably struggled to tackle some of the deeply entrenched gender, cultural and ethnic inequalities. Drawing together the continuities and breaks in ‘social investment’ policy, as implemented by the Australian Labor Party, we ﬁnd improvements in living standards for most Australians, but continued economic growth has not been inclusive for all. 312 christopher deeming and paul smyth Persistent poverty, multiple and entrenched social disadvantage and growing social inequality continue to trouble Australian society (Leigh, 2013). In the European context, social policy scholars warn that the shifting emphasis of social policy towards social investment in human capital policies and labour market integration may well come at the expense of social protection and inclusion for all. Certainly there is growing evidence for this in Europe (Cantillon and Van Lancker, 2013; Van Kersbergen and Hemerijck, 2012), and this must be the concern within the Australian context. Social security against the adverse effects of the labour market is still of value to society and the citizens who are protected by social policy. Thus, employment ‘ﬂexibility’ and traditional social protection, in the form of a secure safety net that provides workers with an adequate standard of living in the event of unemployment is a precondition for an effective ‘heavy’ social investment strategy that promotes equality and protects against poverty in the event of unemployment. Early on in its term, the Labor government appears to have acknowledged that it faced heavy constraints and inevitable limits were placed on its strategy of ‘social investment’. Thus, the government posed the relevant policy question: ‘Does Australia’s level of social expenditure underwrite the social investments necessary for an inclusive society?’ The answer, it would seem, turns out to be decisive: Even after allowing for difﬁculties in harmonising different accounting systems, the conclusion is inevitable that Australia stands near the bottom of the list of relative social expenditures (Australian Government, 2009: 81, emphasis added). Here Labor seems to have accepted that the necessary platform for social investment in Australia was ‘light’ at best; essentially concluding that Australia’s overall level of social expenditure was probably unlikely to be sufﬁcient to underwrite the level of ‘heavy’ social investment needed for a more inclusive https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press social investment after neoliberalism 315 society. Even after allowing for difﬁculties in harmonising international data, as the government suggests, it is clear Australia’s social investment strategy looks different from many other comparable European countries in a number of important respects. Investment in ALMPs remains low, as do social expenditure levels more generally, although health and education have beneﬁted from additional investment. Out-of-work beneﬁts, already low by international standards, lost value against earnings. 312 christopher deeming and paul smyth In this context, Labor’s legacy appears to have been the recognition that greater investment was needed, but the accompanying revenue-raising strategy was out of reach. Hence, the low tax road towards ‘social investment’. https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press Conclusions In many ways, the Australian experience reﬂects the general move away from the neoliberal model of economy and society, that dominated the late twentieth century,andthedesiresofAustraliansocialdemocrats,whoreturnedtothe‘social investment’ paradigm that seeks inclusive growth (this time for all Australians) and once again regards social policy as economic investment. In a number of important respects, however, the Australian approach (and that of other liberal market economies) differs from the social investment strategies being pursed in some European contexts (Morel et al., 2012). In the liberal state of Australia, as we have shown, there has been a decisive move to strengthen the social policy framework for ‘hardworking families’ – ‘the battlers’ against the ‘dole bludgers’ – as rhetorical incarnations of economic struggle (or the ‘strivers’ verses the ‘skivers’ in the British context).3 In the battle over the political middle ground, more afﬂuent middle-class families have been brought into the social security system. Dual-earner families on low-to-middle incomes now receive ‘welfare-in-work’. Under Labor, social investment was orientated towards meeting their needs and protecting their living standards – although greater efforts are required to tackle gender inequalities in the labour market and caring roles in the home. Nevertheless, the range of initiatives implemented by Labor can be seen as forging a new de facto ‘contract’ whereby the state has now absorbed greater responsibility for complementing the ‘market family wage’, at least for working families. From this perspective, there has been a complete transformation of the Australian welfare state; the dual-earner family- centred mode of household welfare production is fast replacing the vacuum left by the ‘male bread-winner’ model. Here we might also wish to draw attention to the continued role of politics in the policy process, determining social policy outcomes. The old ‘male breadwinner/female home-maker’ family model of welfare grew out of the democratic class struggle and, in turn, it is a new politics that has shaped, if not constrained, the path of ‘social investment’ in Australia (Deeming, 2013b). https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Press https://doi.org/10.1017/S0047279414000828 Published online by Cambridge University Pre 6 christopher deeming and paul smyth 316 Labor’s deﬁning achievement has been to recreate a state of investment for ‘working families’, and, in the social democratic tradition, one that has beneﬁted working families at the lower end of the income distribution, which also protects their living standards in retirement, and all Australians in the event of poor health and/or disability. Conclusions In other ways, Labor’s ‘light’ approach to social investment strengthened the policy of ‘workfarism’ it had inherited from the Howard-led Liberal–National coalition government (1996–2007), taking a more punitive stance towards social assistance recipients in an effort to move non- working citizens off welfare beneﬁts and into paid employment. A more fully engaged ‘heavy’ social investment scenario that offers better insurance against the risk of unemployment is costly and has never carried much appeal with the Australian electorate. Under the Labor government, social protection lost ground to rising living standards in the working population, and social inequality has in many respects become more visible. The article therefore exposes some of the tensions inherent in the pursuit of ‘social investment’ and inclusion strategies by leftist parties within the ‘neoliberal state’ (Plant, 2012). Social democrats in Australia face particular challenges where political institutions facilitate the pursuit of private ends, rather than deﬁned political goals like ‘social investment’ for a more just society. At the same time, we also ﬁnd the Nordic welfare states in the midst of transformation (Kananen, 2014). All eyes are now on the Nordic countries to see if ‘heavy investment’ which has produced ‘high efﬁciency’ in the capitalist market economy with ‘high equity’ in the distribution of resources and life chances in society can be maintained in the face of global forces and pressures from the new international economic order. Notes 1 The notion that unemployment beneﬁts are ‘passive’ can be problematic. Out-of-work beneﬁts help to support livelihoods and aid workers in their search for new employment. 1 The notion that unemployment beneﬁts are ‘passive’ can be problematic. Out-of-work beneﬁts help to support livelihoods and aid workers in their search for new employment. 2 The Gini index measures the extent to which the income distribution deviates from a perfectly equal distribution: 0 represents perfect equality and 100 implies perfect inequality. 2 The Gini index measures the extent to which the income distribution deviates from a perfectly equal distribution: 0 represents perfect equality and 100 implies perfect inequality. 3 Regrettably, such misleading rhetoric being used by politicians seriously risks undermining trust and social welfare services still further. 3 Regrettably, such misleading rhetoric being used by politicians seriously risks undermining trust and social welfare services still further. Acknowledgements This article was presented at the conference: ‘Assessing the Social Investment Strategy’, University of Lausanne, Switzerland, 10th–11th April 2014. 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https://openalex.org/W2108284976	https://europepmc.org/articles/pmc2990334?pdf=render	English	null	RNAi Knockdown of a Salivary Transcript Leading to Lethality in the Pea Aphid,<i>Acyrthosiphon pisum</i>	Journal of insect science	2,006	cc-by	3,137	RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum Navdeep S. Mutti1,2, Yoonseong Park1, John C. 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https://openalex.org/W3154590113	https://www.nature.com/articles/s41416-021-01278-0.pdf	English	null	Comparative performance of lung cancer risk models to define lung screening eligibility in the United Kingdom	British journal of cancer	2,021	cc-by	9,405	1International Agency for Research on Cancer, Lyon, France; 2The Institute of Cancer Research, London, UK; 3University of Cambridge, Cambridge, UK; 4Cancer Epidemiology Unit, Nufﬁeld Department of Population Health, University of Oxford, Oxford, UK; 5University of Manchester, Manchester, UK; 6Leeds Teaching Hospitals, Leeds, UK; 7Nottingham University Hospitals and University of Nottingham, Nottingham, UK and 8Division of Cancer Epidemiology and Genetics, Department of Health and Human Services, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Correspondence: Hilary A. Robbins (robbinsh@iarc.fr) BACKGROUND In the USA, the US Preventive Services Task Force (USPSTF) guidelines use categorical criteria to determine who is eligible for screening. Eligibility by the 2013 guideline required age 55–80 years, at least 30 pack-years smoked, and for former smokers, no more than 15 years since quitting.5 The 2020 draft guideline expands eligibility by lowering the age-to-start from 55 to 50 years, and lowering the pack-year threshold from 30 to 20 pack- years.15 However, secondary analyses of the NLST demonstrated that lung screening may be more efﬁcient and cost-effective when eligibility is based on individual lung cancer risk, estimated using a continuous risk prediction model.16–19 Lung screening in the UK was implemented using individual risk-based eligibility from the beginning, and the NHS England protocol speciﬁes that indivi- duals aged 55–74 years can be screened if their lung cancer risk exceeds 1.51% by the PLCOm2012 model (6-year risk) or 2.5% by the Liverpool Lung Project version 2 (LLPv2) model (5-year risk).14 Lung cancer is the leading cause of cancer death worldwide.1,2 Two large, randomised trials have now demonstrated that screening by low-dose computed tomography (LDCT) can reduce mortality from lung cancer among people with a heavy smoking history. Lung cancer mortality was reduced by 20% over 5 years in the USA National Lung Screening Trial (NLST) with 3 annual LDCT screens3 and by 24% (men) and 33% (women) over 10 years in the Dutch-Belgian NELSON trial with 4 LDCT screens over 5.5 years.4 g y The USA issued a national recommendation for lung screening in 2014.5 In the United Kingdom, there have been several successful pilot studies, including the Manchester Lung Health Checks6,7 and the Liverpool Healthy Lung Programme.8 Compared with the implementation of lung screening in the USA, the UK has often been more successful in terms of overall uptake and engagement of populations with low socioeconomic status (SES),6,9,10 and lung cancer detection rates have often exceeded those in the NLST.3,6,11,12 Building on this success, the National Health Service (NHS) England is implementing a £70 million programme of “Targeted Lung Health Checks” in 10 areas with high lung cancer mortality.13,14 p g j y The choice of which risk model to use for screening eligibility is important. Poor model discrimination or calibration can reduce the efﬁciency and cost-effectiveness of screening and even lead to net harm if models select individuals who are unlikely to beneﬁt from screening. British Journal of Cancer (2021) 124:2026–2034; https://doi.org/10.1038/s41416-021-01278-0 British Journal of Cancer (2021) 124:2026–2034; https://doi.org/10.1038/s41416-021-01278-0 www.nature.com/bjc ARTICLE Epidemiology Comparative performance of lung cancer risk model lung screening eligibility in the United Kingdom Hilary A. Robbins 1, Karine Alcala 1, Anthony J. Swerdlow2, Minouk J. Schoemaker2, Nick Wareham3, Ruth C Philip A. J. Crosbie5, Matthew Callister6, David R. Baldwin7, Rebecca Landy8 and Mattias Johansson 1 BACKGROUND: The National Health Service England (NHS) classiﬁes individuals as eligible for lung cancer scre prediction models, PLCOm2012 and Liverpool Lung Project-v2 (LLPv2). However, no study has compared the p cancer risk models in the UK. METHODS: We analysed current and former smokers aged 40–80 years in the UK Biobank (N = 217,199), EPIC-U Generations Study (N = 25,777). We quantiﬁed model calibration (ratio of expected to observed cases, E/O) a discrimination (AUC). RESULTS: Risk discrimination in UK Biobank was best for the Lung Cancer Death Risk Assessment Tool (LCDRA CI = 0.81–0.84), followed by the LCRAT (AUC = 0.81, 95% CI = 0.79–0.82) and the Bach model (AUC = 0.80, 95 Results were similar in EPIC-UK and the Generations Study. All models overestimated risk in all cohorts, with ranging from 1.20 for LLPv3 (95% CI = 1.14–1.27) to 2.16 for LLPv2 (95% CI = 2.05–2.28). Overestimation incre socioeconomic status. In the combined cohorts, USPSTF 2013 criteria classiﬁed 50.7% of future cases as scree LCDRAT and LCRAT identiﬁed 60.9%, followed by PLCOm2012 (58.3%), Bach (58.0%), LLPv3 (56.6%), and LLPv CONCLUSION: In UK cohorts, the ability of risk prediction models to classify future lung cancer cases as eligib best for LCDRAT/LCRAT, very good for PLCOm2012, and lowest for LLPv2. Our results highlight the importan prediction tools in speciﬁc countries. British Journal of Cancer (2021) 124:2026–2034; https://doi.org/10.1038/s41416-021-01278-0 Published by Springer Nature on behalf of Cancer Research UK Received: 22 July 2020 Revised: 4 January 2021 Accepted: 13 January 2021 Published online: 12 April 2021 Comparative performance of lung cancer risk models to deﬁne lung screening eligibility in the United Kingdom Hilary A. Robbins 1, Karine Alcala 1, Anthony J. Swerdlow2, Minouk J. Schoemaker2, Nick Wareham3, Ruth C. Travis4, Philip A. J. Crosbie5, Matthew Callister6, David R. Baldwin7, Rebecca Landy8 and Mattias Johansson 1 BACKGROUND: The National Health Service England (NHS) classiﬁes individuals as eligible for lung cancer screening using two risk prediction models, PLCOm2012 and Liverpool Lung Project-v2 (LLPv2). However, no study has compared the performance of lung cancer risk models in the UK. METHODS: We analysed current and former smokers aged 40–80 years in the UK Biobank (N = 217,199), EPIC-UK (N = 30,813), and Generations Study (N = 25,777). We quantiﬁed model calibration (ratio of expected to observed cases, E/O) and discrimination (AUC). ( ) RESULTS: Risk discrimination in UK Biobank was best for the Lung Cancer Death Risk Assessment Tool (LCDRAT, AUC = 0.82, 95% CI = 0.81–0.84), followed by the LCRAT (AUC = 0.81, 95% CI = 0.79–0.82) and the Bach model (AUC = 0.80, 95% CI = 0.79–0.81). Results were similar in EPIC-UK and the Generations Study. All models overestimated risk in all cohorts, with E/O in UK Biobank ranging from 1.20 for LLPv3 (95% CI = 1.14–1.27) to 2.16 for LLPv2 (95% CI = 2.05–2.28). Overestimation increased with area-level socioeconomic status. In the combined cohorts, USPSTF 2013 criteria classiﬁed 50.7% of future cases as screening eligible. The LCDRAT and LCRAT identiﬁed 60.9%, followed by PLCOm2012 (58.3%), Bach (58.0%), LLPv3 (56.6%), and LLPv2 (53.7%). CONCLUSION: In UK cohorts, the ability of risk prediction models to classify future lung cancer cases as eligible for screening was best for LCDRAT/LCRAT, very good for PLCOm2012, and lowest for LLPv2. Our results highlight the importance of validating prediction tools in speciﬁc countries. METHODS We analysed longitudinal data from the UK Biobank, European Prospective Investigation into Cancer and Nutrition (EPIC)-UK, and Generations Study cohorts. The UK Biobank is a prospective cohort study of 500,000 people aged 40–72 years at recruitment (2006–2010).25 EPIC-UK recruited participants aged 45–74 years in Cambridge and aged ≥20 years in Oxford during 1993–2000.26 EPIC-Cambridge used population-based recruitment of patients of general practitioners, while EPIC-Oxford was comprised of both population-based recruitment and a subset targeted at “health conscious” individuals. Finally, the Generations Study recruited 112,000 women aged ≥16 years during 2003–2011, of whom about one-third had a mother, daughter, or sister also participat- ing in the study.27 In all cohorts, cancer and death ascertainment relied on registry linkages at minimum, sometimes with additional active follow-up.25–27 g p g Calibration estimates for all risk models were >1 in all cohorts, indicating that the models predicted more lung cancer cases (or for LCDRAT, lung cancer deaths) than were observed over the time period speciﬁed by the model (Fig. 1). The extent of over- estimation of risks (and therefore poorest calibration) was the highest in the Generations Study and the lowest in UK Biobank for each model. Across the risk models, in UK Biobank, LLPv3 was best calibrated (E/O = 1.20, 95% CI = 1.14–1.27), followed by PLCOm2012 (E/O = 1.30, 95% CI 1.23–1.36), the Bach model (E/O = 1.39, 95% CI 1.31–1.47), LCRAT (E/O = 1.44, 95% CI 1.36–1.52), and LCDRAT (E/O = 1.52, 95% CI 1.41–1.64). Over- estimation was the highest for LLPv2 (E/O = 2.16, 95% CI 2.05–2.28). The order in which models were ranked in EPIC-UK and the Generations Study was similar to UK Biobank, but E/O statistics were higher. From all participants in these cohorts, we restricted to those known to be current or former smokers who were aged 40–80 years at enrolment, including 217,199 in UK Biobank, 30,813 in EPIC-UK, and 25,777 in the Generations Study (total N = 273,789). Never smokers and participants with unknown smoking status were excluded. After these restrictions, substantial amounts of missing data were present for some variables in some cohorts, such as 31% missing smoking intensity (cigarettes per day) in UK Biobank. Missing data were handled using various approaches within the framework of multiple imputation (see Supplement). Among participants who were alive and free of lung cancer at the end of follow-up (i.e. METHODS in whom future lung cancer status would be unknown), follow-up time was at least 6 years for all participants in UK Biobank and EPIC-UK and for 88% in the Generations Study. Differences in discrimination estimates (AUCs) across models were modest. In UK Biobank, discrimination was the highest for LCDRAT (AUC = 0.82, 95% CI 0.81–0.84) and the lowest for LLPv2 (AUC = 0.77, 95% CI 0.76–0.78) (Fig. 2). However, this ordering differed in EPIC-UK, ranging from AUC = 0.84 for the Bach model (95% CI 0.81–0.87) to 0.81 for PLCOm2012 (95% CI 0.77–0.85). Discrimination in the Generations Study ranged from AUC = 0.84 for LCDRAT (95% CI 0.77–0.90) to 0.78 for PLCOm2012 (95% CI 0.71–0.85). To further investigate model overestimation, we calculated E/O estimates stratiﬁed by demographic and smoking characteristics in UK Biobank (Table 2). Analogous estimates for discrimination (stratiﬁed AUCs) are presented in Supplementary Table 2. For all models, there was a strong positive relationship between model overestimation and SES, which was measured by the area-level Townsend deprivation index. For example, for LCDRAT, E/O statistics across SES quartiles were 2.03 (highest SES), 1.95, 1.61, and 1.26 (lowest SES). Patterns for other characteristics differed across models, though frequent patterns included more over- estimation in men than in women, in former smokers than in current smokers, and at the extremes of age (40s and 70s). When stratifying by quintiles of predicted risk (Supplementary Fig. 1), PLCOm2012 substantially underestimated risk in the lowest-risk quintile while modestly overestimating risk in the upper categories. Overestimation tended to be higher at higher risks for LLPv2, LLPv3, and Bach, while it was higher at lower risks for LCDRAT and LCRAT. y We evaluated 8 lung cancer risk models. These included the PLCOm2012 and LLPv2 models, which are proposed for use in selecting screening participants in the NHS protocol.11,14,17,20 We also evaluated the Bach model,28 the LCDRAT,18 the Lung Cancer Risk Assessment Tool (LCRAT),18 the original LLP model,20 the LLPv3,29 and the Hoggart model.30 Each of these models is either a USA-based model that performs well in USA data (Bach, LCDRAT, LCRAT, PLCOm2012)21 or a European model whose performance in European data is unknown (LLP, LLPv2, LLPv3, Hoggart). Risk thresholds above which screening can be offered have been proposed for LCRAT and LCDRAT,19,31 and LLPv3,29 in addition to PLCOm2012 and LLPv2. RESULTS h Among the 217,199 current or former smokers aged 40–80 years in UK Biobank, 1265 lung cancer cases were diagnosed within 5 years of enrolment, and 700 lung cancer deaths occurred in this period (Table 1). In EPIC-UK, 156 lung cancers and 100 lung cancer deaths occurred over 5 years among 30,813 participants, and in the Generations Study, 53 lung cancers and 26 lung cancer deaths occurred over 5 years among 25,777 participants. Distributions of demographic and smoking variables differed across cohorts. BACKGROUND include; for example, the LLP/LLPv2/LLP model version 3 (LLPv3) models include only one measure of smoking (duration), whereas the Lung Cancer Death Risk Assessment Tool (LCDRAT) includes smoking duration, pack-years, quit-years, and intensity.18,20 Most models, including PLCOm2012 and LCDRAT, were developed using USA data, whereas the LLP/LLPv2/LLPv3 models were developed in the UK.17,18,20 Although both PLCOm2012 and LLPv2 have been implemented successfully in screening studies, the absence of outcome data on individuals who were not eligible (and thus not screened) has precluded evaluation of whether either of these is the optimal model.6,11 Several models have been evaluated in population cohort studies in the USA,21,22 but non- USA evaluations are scarce,23,24 and none include data from UK cohorts. We calculated calibration as the ratio of expected to observed (E/O) lung cancer cases or deaths, overall and in subgroups. We quantiﬁed discrimination using the area under the receiver- operating curve (AUC) statistic. The 95% conﬁdence intervals (CIs) for calibration and discrimination statistics account for within and between imputation variance.21 Here we performed a comparative evaluation of lung cancer risk models to deﬁne lung screening eligibility in the UK. We analysed 3 cohort studies to quantify the calibration and discrimination of risk models and then compared their ability to classify future lung cancer cases into a group deﬁned as eligible for screening. BACKGROUND Risk models differ in the variables that they Published by Springer Nature on behalf of Cancer Research UK © World Health Organization 2021, corrected publication 2021 Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. 2027 LLPv2 (5-year), and LLP (5-year) were calculated directly.17,20 We present results for two models in Supplementary Table 1 and do not include them in discussions below, due to redundancy with LLPv2/LLPv3 (LLP) and very high overestimation of risk (Hoggart). We present results for LLPv2 in the main manuscript, even though it may be eventually replaced by LLPv3, because LLPv2 is listed in the NHS England protocol. LLPv2 (5-year), and LLP (5-year) were calculated directly.17,20 We present results for two models in Supplementary Table 1 and do not include them in discussions below, due to redundancy with LLPv2/LLPv3 (LLP) and very high overestimation of risk (Hoggart). We present results for LLPv2 in the main manuscript, even though it may be eventually replaced by LLPv3, because LLPv2 is listed in the NHS England protocol. include; for example, the LLP/LLPv2/LLP model version 3 (LLPv3) models include only one measure of smoking (duration), whereas the Lung Cancer Death Risk Assessment Tool (LCDRAT) includes smoking duration, pack-years, quit-years, and intensity.18,20 Most models, including PLCOm2012 and LCDRAT, were developed using USA data, whereas the LLP/LLPv2/LLPv3 models were developed in the UK.17,18,20 Although both PLCOm2012 and LLPv2 have been implemented successfully in screening studies, the absence of outcome data on individuals who were not eligible (and thus not screened) has precluded evaluation of whether either of these is the optimal model.6,11 Several models have been evaluated in population cohort studies in the USA,21,22 but non- USA evaluations are scarce,23,24 and none include data from UK cohorts. METHODS Risk estimates for the LCRAT (5-year time horizon), LCDRAT (5-year), and Hoggart (1-year) models were generated using the lcmodels package in R.32 Estimates for the Bach model used code adapted from lcmodels to reduce the time horizon to 5 years. Estimates for PLCOm2012 (6-year time horizon), LLPv3 (5-year), Table 3 considers the hypothetical impact of using each risk model to determine who is screening eligible in the combined Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. 2028 Table 1. Characteristics of current and former smokers in UK Biobank, EPIC-UK, and the Generations Study. METHODS COPD chronic obstructive pulmonary disease, NA not applicable. aEligibility by the US Preventive Services Task Force (USPSTF) 2013 criteria requires age 55–80 years, at least 30 pack-years, and no more than 15 quit-years. Eligibility by the draft USPSTF 2020 criteria requires age 50–80 years, at least 20 pack-years, and no more than 15 quit-years. HA Robbins et al. 202 Table 1 shows data prior to imputation of missing data. UK educational categories were mapped to USA categories as described in the Supplement. Body mass index categories were deﬁned as follows: <18.5 underweight, 18.5–24.9 normal weight, 25–29.9 overweight, and ≥30 obese. “Asbestos exposure” reﬂects self- reported occupational asbestos exposure. COPD chronic obstructive pulmonary disease, NA not applicable. aEligibility by the US Preventive Services Task Force (USPSTF) 2013 criteria requires age 55–80 years, at least 30 pack-years, and no more than 15 quit-years. Eligibility by the draft USPSTF 2020 criteria requires age 50–80 years, at least 20 pack-years, and no more than 15 quit-years. Overestimation Underestimation 1 2 3 Bach LCDRAT LCRAT LLPv2 LLPv3 PLCOm2012 Risk models Ratio of expected to observed number of cases (log scale) Cohorts UKB EPIC GS Calibration Fig. 1 Calibration of lung cancer risk models in the UK Biobank, EPIC-UK, and Generations Study cohorts, as measured by the ratio of expected to observed cases. UKB UK Biobank, GS Generations Study. Estimates for UK Biobank also appear in Table 2 and Supplementary Table 3. Fig. 1 Calibration of lung cancer risk models in the UK Biobank, EPIC-UK, and Generations Study cohorts, as measured by the ratio of expected to observed cases. UKB UK Biobank, GS Generations Study. Estimates for UK Biobank also appear in Table 2 and Supplementary Table 3. population of UK Biobank, EPIC-UK, and the Generations Study. In the combined population, after imputing missing data, 15.0% of individuals were eligible for screening by USPSTF 2013 criteria (age 55–80 years, at least 30 pack-years, no more than 15 quit-years). The thresholds that would screen the same number of individuals using risk-based eligibility were 0.8% for LCDRAT (5-year lung cancer death risk), 1.4% for LCRAT (5-year lung cancer risk), 1.5% for PLCOm2012 (6-year risk), 1.6% for Bach (5-year risk), 1.3% for LLPv3 (5-year risk), and 2.3% for LLPv2 (5- year risk). cancer deaths (N = 415) over 5 years would be screening eligible, among which some fraction could be prevented by earlier detection. METHODS Characteristic UK Biobank EPIC-UK Generations Study All participants 217,199 30,813 25,777 Lung cancer cases Within 1 year of enrolment 194 23 5 Within 2 years of enrolment 420 58 16 Within 5 years of enrolment 1265 156 53 Within 6 years of enrolment 1461 196 68 Lung cancer deaths Within 5 years of enrolment 700 100 26 Eligible by USPSTF 2013 criteriaa 26,644 (12.3%) 2484 (8.1%) 1110 (4.3%) Eligible by USPSTF 2020 criteriaa 45,065 (20.7%) 5179 (16.8%) 2994 (11.6%) Lung cancer cases within 5 years of enrolment Among USPSTF 2013 eligible individualsa 616 (48.7%) 59 (37.8%) 17 (32.1%) Among USPSTF 2020 eligible individualsa 805 (63.6%) 75 (48.1%) 24 (45.3%) Age at recruitment, years 40–44 19,881 (9.2%) 3508 (11.4%) 3815 (14.8%) 45–49 24,954 (11.5%) 5782 (18.8%) 4124 (16.0%) 50–54 30,633 (14.1%) 5277 (17.1%) 4773 (18.5%) 55–59 39,632 (18.2%) 4302 (14.0%) 5417 (21.0%) 60–64 56,626 (26.1%) 3813 (12.4%) 4481 (17.4%) 65–69 44,389 (20.4%) 3877 (12.6%) 2067 (8.0%) 70–74 1084 (0.5%) 3268 (10.6%) 799 (3.1%) 75–80 0 (0.0%) 986 (3.2%) 301 (1.2%) Sex Male 108,043 (49.7%) 13,191 (42.8%) 0 (0.0%) Female 109,156 (50.3%) 17,622 (57.2%) 25,777 (100.0%) Smoking status Current 50,151 (23.1%) 6721 (21.8%) 4116 (16.0%) Former, 0–4 quit years 15,094 (6.9%) 2227 (7.2%) 2057 (8.0%) Former, 5–9 quit years 14,431 (6.6%) 2583 (8.4%) 1972 (7.7%) Former, 10–14 quit years 12,513 (5.8%) 3374 (10.9%) 2259 (8.8%) Former, 15–19 quit years 12,753 (5.9%) 3590 (11.7%) 2772 (10.8%) Former, ≥20 quit years 54,610 (25.1%) 10,025 (32.5%) 11,579 (44.9%) Former, Unknown 57,647 (26.5%) 2293 (7.4%) 1022 (4.0%) Cigarettes smoked per day ≤10 43,870 (20.2%) 9845 (32.0%) 11,223 (43.5%) 11–19 31,488 (14.5%) 8249 (26.8%) 7278 (28.2%) 20–29 55,168 (25.4%) 5427 (17.6%) 2905 (11.3%) 30–39 11,468 (5.3%) 711 (2.3%) 408 (1.6%) ≥40 8619 (4.0%) 435 (1.4%) 125 (0.5%) Unknown 66,586 (30.7%) 6146 (19.9%) 3838 (14.9%) Educational level Less than secondary 44,950 (20.7%) 6248 (20.3%) 6 (0.0%) Secondary degree 58,098 (26.7%) 12,031 (39.0%) 10,947 (42.5%) Some post-secondary training 33,688 (15.5%) NA NA Some university 16,126 (7.4%) NA 7996 (31.0%) University graduate 60,363 (27.8%) 6369 (20.7%) 6223 (24.1%) Unknown 3974 (1.8%) 6165 (20.0%) 605 (2.3%) Body mass index Underweight 1100 (0.5%) 356 (1.2%) 167 (0.6%) Normal weight 64,430 (29.7%) 14,776 (48.0%) 12,220 (47.4%) Overweight 93,806 (43.2%) 11,927 (38.7%) 8658 (33.6%) Obese 56,651 (26.1%) 3754 (12.2%) 4233 (16.4%) 028 Table 1. Characteristics of current and former smokers in UK Biobank, EPIC-UK, and the Generations Study. Table 1. METHODS continued Characteristic UK Biobank EPIC-UK Generations Study Personal history of cancer 10,651 (4.9%) 1123 (3.6%) 1722 (6.7%) First-degree family history of lung cancer 18,039 (8.3%) Missing Missing COPD or emphysema 4046 (1.9%) Missing Missing Prior pneumonia 1346 (0.6%) Missing Missing Asbestos exposure Missing 2167 (7.0%) Missing Table 1 shows data prior to imputation of missing data. UK educational categories were mapped to USA categories as described in the Supplement. Body mass index categories were deﬁned as follows: <18.5 underweight, 18.5–24.9 normal weight, 25–29.9 overweight, and ≥30 obese. “Asbestos exposure” reﬂects self- reported occupational asbestos exposure. COPD chronic obstructive pulmonary disease, NA not applicable. aEligibility by the US Preventive Services Task Force (USPSTF) 2013 criteria requires age 55–80 years, at least 30 pack-years, and no more than 15 quit-years. Eligibility by the draft USPSTF 2020 criteria requires age 50–80 years, at least 20 pack-years, and no more than 15 quit-years. Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. 2029 Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. population of UK Biobank, EPIC-UK, and the Generations Study. In the combined population after imputing missing data 15 0% of cancer deaths (N = 415) over 5 years would be screening eligible, among which some fraction could be prevented by earlier Overestimation Underestimation 1 2 3 Bach LCDRAT LCRAT LLPv2 LLPv3 PLCOm2012 Risk models Ratio of expected to observed number of cases (log scale) Cohorts UKB EPIC GS Calibration Fig. 1 Calibration of lung cancer risk models in the UK Biobank, EPIC-UK, and Generations Study cohorts, as measured by the ratio of expected to observed cases. UKB UK Biobank, GS Generations Study. Estimates for UK Biobank also appear in Table 2 and Supplementary Table 3. Table 1. continued Characteristic UK Biobank EPIC-UK Generations Study Personal history of cancer 10,651 (4.9%) 1123 (3.6%) 1722 (6.7%) First-degree family history of lung cancer 18,039 (8.3%) Missing Missing COPD or emphysema 4046 (1.9%) Missing Missing Prior pneumonia 1346 (0.6%) Missing Missing Asbestos exposure Missing 2167 (7.0%) Missing Table 1 shows data prior to imputation of missing data. UK educational categories were mapped to USA categories as described in the Supplement. Body mass index categories were deﬁned as follows: <18.5 underweight, 18.5–24.9 normal weight, 25–29.9 overweight, and ≥30 obese. “Asbestos exposure” reﬂects self- reported occupational asbestos exposure. DISCUSSION L Applying USPSTF 2020 criteria (age 50–80 years, at least 20 pack-years, no more than 15 quit-years) yielded lower risk thresholds for screening and higher percentages of cases classiﬁed as screening eligible, as expected (Table 3). USPSTF 2020 criteria identiﬁed 26.1% of participants and 66.4% of future lung cancer cases as screening eligible. If risk models also screened the 26.1% highest-risk participants, LCDRAT would identify 77.0% of cases at a threshold of 0.4% 5-year risk (N = 1135) followed by Bach (76.7%, N = 1131 at 0.8% 5-year risk), PLCOm2012 (75.2%, N = 1109 at 0.8% 6-year risk), LCRAT (74.7%, N = 1101 at 0.8% 5-year risk), LLPv3 (70.5%, N = 1039 at 0.7% 5- year risk), and LLPv2 (69.2%, N = 1020 at 1.3% 5-year risk). Results for lung cancer deaths were similar. Lung cancer screening has the potential to substantially reduce lung cancer mortality among people with a heavy smoking history. In the United Kingdom, the success of the Targeted Lung Health Checks will depend partially on whether the programme can be implemented efﬁciently and cost-effectively. The protocol currently recommends the use of the PLCOm2012 and LLPv2 risk models to identify screening-eligible individuals.14 In this study, we compared the performance of these two models along with others that have performed well in other high-income settings. We found that the LLPv2 model had worst calibration and classiﬁed the lowest proportion of future lung cancer cases as eligible for screening. The PLCOm2012 model had better calibration, though all models predicted more cases than were observed. The LCDRAT was able to classify the highest proportion of future lung cancer cases as eligible for screening, with very good performance also observed for the LCRAT, PLCOm2012, and Bach models. g We analysed individuals aged 40–80 years, but the NHS England protocol restricts eligibility to ages 55–74 years. When we repeated our analysis after restricting to individuals aged 55–74 years in UK Biobank (n.b. there were no participants in UK Biobank aged >74 years), AUCs decreased as expected due to the loss in prediction derived from age variation. However, the rank order of AUCs and the calibration results were not affected (Table 4). Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. 0.70 0.75 0.80 0.85 0.90 LLPv2 Bach LCDRAT LCRAT LLPv3 PLCOm2012 Risk models AUC Cohorts UKB EPIC GS Discrimination Fig. 2 Discrimination of lung cancer risk models in the UK Biobank, EPIC-UK, and Generations Study cohorts, as measured by the area under the ROC curve (AUC). UKB UK Biobank, GS Generations Study. Estimates for UK Biobank also appear in Supplementary Tables 2 and 3. Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. 30 2030 PLCOm2012 LLPv3 Fig. 2 Discrimination of lung cancer risk models in the UK Biobank, EPIC-UK, and Generations Study cohorts, as measured by the area under the ROC curve (AUC). UKB UK Biobank, GS Generations Study. Estimates for UK Biobank also appear in Supplementary Tables 2 and 3. METHODS Applying risk models and the thresholds described above to deﬁne the screened population identiﬁed higher proportions of future cases: the LCDRAT and LCRAT identiﬁed the highest proportion of future cases as screening eligible (each 60.9%, N = 897), followed by PLCOm2012 (58.3%, N = 859), Bach (58.0%, N = 855), LLPv3 (56.6%, N = 835), and LLPv2 (53.7%, N = 791). For lung cancer deaths, the ranking of models was similar, with LCDRAT identifying 63.2% of lung cancer deaths as screening eligible (N = 522), followed by LCRAT (62.8%, N = 519), Bach (60.7%, N = 501), PLCOm2012 (59.3%, N = 490), LLPv3 (59.2%, N = 489), and LLPv2 (56.3%, N = 465). y Using USPSTF 2013 guidelines to deﬁne screening eligibility, 50.7% of future lung cancer cases (N = 747) would be classiﬁed as eligible for screening (Table 3). Similarly, 50.2% of future lung Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. DISCUSSION L The models evaluated in our study were previously validated in multiple USA cohorts, including the NIH-AARP and CPS-II,21 as well as the NLST and PLCO trials.22 Taken together, these studies showed good calibration for the Bach model, LCRAT, LCDRAT, and PLCOm2012 but overestimation of risks for the LLP model. In our study, all models overestimated risks; the extent was greatest for LLPv2. For discrimination, prior results in NIH-AARP and CPS-II showed best performance for LCDRAT, followed sequentially by LCRAT, PLCOm2012, Bach, and LLP.21 The study analysing PLCO and NLST found higher discrimination for PLCOm2012 and Bach compared with LLP.22 The order in which models ranked in our study was similar, with best performance for the LCDRAT, LCRAT, Bach, and PLCOm2012 models. The likely explanation for inferior discrimination of the LLP/LLPv2/LLPv3 models is that they Supplementary Table 3 describes the characteristics of lung cancer cases that are not identiﬁed as screening eligible (are “missed”) by USPSTF 2013, USPSTF 2020, and each risk model at the risk thresholds identiﬁed in Table 3. Compared with USPSTF 2013, the cases missed by risk models, while fewer in number, were more commonly former smokers (62% of cases missed by USPSTF vs. 69–80% for risk models). They also tended to be slightly younger (median age at baseline 63 years for cases missed by USPSTF vs. 60–61 years for risk models) and slightly more frequently female (53% of cases missed by USPSTF vs. 54–57% for risk models). Patterns using thresholds based on USPTF 2020 were similar or more pronounced. Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. 2031 Table 2. Calibration estimates for lung cancer risk models in the subgroups of UK Biobank, as measured by the ratio of expected to observed cases. DISCUSSION L Quartiles of the Townsend deprivation index were deﬁned such that UK Biobank participants were divided equally, using the following cutpoints: −6.26 (minimum), −3.42, −1.75, 1.21, 11.0 (maximum). Body mass index categories were deﬁned as follows: <18.5 underweight, 18.5–24.9 normal weight, 25–29.9 overweight, and ≥30 obese. CPD cigarettes per day, SES socioeconomic status. incorporate only smoking duration (omitting intensity and quit- years) and use categorical instead of continuous parameterisa- tions of age and smoking.20 Overall, the magnitude of AUCs in our study (often exceeding 0.80) was higher than in prior reports (typically ranging from 0.75 to 0.80).21,22 This is likely caused by a wider age distribution in our analysis, which included more younger individuals. incorporate only smoking duration (omitting intensity and quit- years) and use categorical instead of continuous parameterisa- tions of age and smoking.20 Overall, the magnitude of AUCs in our study (often exceeding 0.80) was higher than in prior reports (typically ranging from 0.75 to 0.80).21,22 This is likely caused by a wider age distribution in our analysis, which included more younger individuals. have overrepresentation of high-SES individuals and are likely inﬂuenced by “healthy volunteer” effects. In UK Biobank, all-cause mortality among 70–74-year-olds is half that in the general population (although the difference in cancer incidence is smaller).33 EPIC-Oxford is partially comprised of “health-conscious” individuals. The Generations Study is a volunteer cohort with recruitment based on engagement in a health issue (ﬁnding the causes of breast cancer), and cancer incidence is estimated to be 16% lower than in the general UK population (unpublished data). These inﬂuences, taken together with the overrepresentation of high-SES individuals in whom overestimation is highest, suggest that model calibration would be better in the overall UK population of ever-smokers than in the research cohorts we analysed. In UK screening studies, lung cancer detection rates have commonly been higher than in the NLST.3,6,7,11 Lung cancer detection over 2 screens was 4.5% in the Manchester Lung Health Checks, compared with 1.7% in NLST.3,6,7 Detection rates in single- screen UK studies are commonly approximately 2%.11,12 These observations might have been taken as evidence that USA-based lung cancer risk models would predict too few cases in UK populations, but we found the opposite result. Unlike the screening studies, which commonly comprised individuals living in low-SES communities,6,11,12 the research cohorts we analysed y There is a troubling consequence to the correlation between risk model overestimation and SES. DISCUSSION L Calibration estimate (expected/observed) by risk model Bach LCDRAT LCRAT LLPv2 LLPv3 PLCOm2012 All participants 1.39 1.52 1.44 2.16 1.20 1.30 Sex Male 1.50 1.56 1.50 2.46 1.20 1.36 Female 1.25 1.47 1.37 1.81 1.20 1.23 Age, years 40–49 1.69 2.16 2.51 1.31 0.73 2.20 50–59 1.36 1.32 1.47 1.73 1.00 1.28 60–69 1.38 1.56 1.38 2.32 1.28 1.25 70–74 1.89 2.61 1.93 3.52 1.92 2.12 Area-level SES Q1 (highest SES) 1.89 2.03 1.93 3.06 1.69 1.64 Q2 1.79 1.95 1.84 2.78 1.54 1.57 Q3 1.51 1.61 1.57 2.20 1.22 1.39 Q4 (lowest SES) 1.14 1.26 1.21 1.49 0.82 1.06 Smoking status Current 1.28 1.39 1.44 1.60 0.88 1.10 Former 1.48 1.65 1.44 2.64 1.47 1.47 Smoking intensity ≤10 CPD 1.17 1.40 1.50 2.96 1.70 0.65 11–29 CPD 1.43 1.52 1.41 2.09 1.16 1.44 ≥30 CPD 1.48 1.64 1.48 1.63 0.86 1.48 Education Less than secondary 1.10 1.31 1.20 1.64 0.91 1.10 Secondary degree 1.58 1.82 1.70 2.41 1.36 1.51 Some post-secondary training 1.45 1.52 1.48 2.30 1.29 1.33 Some university 1.42 1.21 1.20 2.13 1.88 1.10 University graduate 1.97 2.01 1.96 3.39 1.13 1.71 Body mass index Underweight 0.98 1.52 1.64 1.40 0.84 1.20 Normal weight 1.09 1.41 1.36 1.76 1.00 1.17 Overweight 1.50 1.61 1.55 2.41 1.31 1.39 Obese 1.59 1.52 1.38 2.31 1.28 1.31 Estimates are provided for UK Biobank only due to the small size of the other cohorts. SES is measured using the Townsend deprivation index, an area-level measure that is applied to individuals based on their place of residence. Quartiles of the Townsend deprivation index were deﬁned such that UK Biobank participants were divided equally, using the following cutpoints: −6.26 (minimum), −3.42, −1.75, 1.21, 11.0 (maximum). Body mass index categories were deﬁned as follows: <18.5 underweight, 18.5–24.9 normal weight, 25–29.9 overweight, and ≥30 obese. CPD cigarettes per day, SES socioeconomic status. cancer risk models in the subgroups of UK Biobank, as measured by the ratio of expected to observed cases. Table 2. Calibration estimates for lung cancer risk models in the subgroups of UK Biobank, as measured by the ratio of expected to observed cases. Estimates are provided for UK Biobank only due to the small size of the other cohorts. SES is measured using the Townsend deprivation index, an area-level measure that is applied to individuals based on their place of residence. DISCUSSION L Table 3. Performance of lung cancer risk models for deﬁning lung cancer screening eligibility among current and former smokers in the combined UK Biobank, EPIC-UK, and Generations Study cohorts. Important limitations of risk-based eligibility for lung screening are receiving increased recognition. Risk models preferentially select older individuals, reducing life-years gained and cost- effectiveness, as well as individuals with comorbidities such as COPD who may have lower screening beneﬁts.19,35–37 To address Important limitations of risk-based eligibility for lung screening are receiving increased recognition. Risk models preferentially select older individuals, reducing life-years gained and cost- effectiveness, as well as individuals with comorbidities such as COPD who may have lower screening beneﬁts.19,35–37 To address these issues, a model to deﬁne eligibility based on predicted life- years gained from screening has been proposed,37 which incorporates LCDRAT and an additional prediction model for overall mortality. Important evidence for the comparative performance of PLCOm2012 and LLPv2 will be provided by the Yorkshire Lung Screening Trial, which is enrolling participants based on eligibility by either model to compare their performance directly.38 The trial is also collecting sufﬁcient information to validate the LCRAT, LCDRAT, and Bach models retrospectively. y g these issues, a model to deﬁne eligibility based on predicted life- years gained from screening has been proposed,37 which incorporates LCDRAT and an additional prediction model for overall mortality. Important evidence for the comparative performance of PLCOm2012 and LLPv2 will be provided by the Yorkshire Lung Screening Trial, which is enrolling participants based on eligibility by either model to compare their performance directly.38 The trial is also collecting sufﬁcient information to validate the LCRAT, LCDRAT, and Bach models retrospectively. Our study has important limitations that result from its approach of analysing cohort data. Our ﬁndings cannot be assumed to be nationally representative for the UK, though the rank-order performance of lung cancer risk prediction models is likely generalisable. There was also a substantial amount of missing data for key smoking variables, which we handled by multiple imputation. For comparison, we calculated E/O statistics and AUCs using the subset of individuals in UK Biobank who had complete data on required variables (63% of the cohort). The degree of overestimation was reduced for all models (Supple- mentary Fig. 2), while AUCs were not affected (Supplementary Fig. 3). By contrast, our approach of analysing multiple cohorts is a strength, because it allows for evaluating whether results are consistent across studies. DISCUSSION L Inclusion of the Generations Study, despite its small size, is important due to the underrepresentation of women in the European screening trial literature. Women in the Generations Study had lower smoking intensity and longer periods of cessation compared with participants in the other studies, highlighting potential equity issues around screening eligibility. Models are listed in order of their performance for identifying future lung cancer cases (USPSTF 2013). “Risk” refers to lung cancer death risk for LCDRAT and to lung cancer risk for all other models. Results are based on a combined data set, which uses a single imputation for missing data. USPSTF 2013 criteria classify 41,107 (15.0%) current and former smokers in the 3 cohorts combined as screening eligible, and USPSTF 2020 criteria classify 71,387 (26.1%) as eligible. These percentages differ from Table 1 as they are calculated after imputation, whereas Table 1 omits missing data. NA not applicable. Models are listed in order of their performance for identifying future lung cancer cases (USPSTF 2013). “Risk” refers to lung cancer death risk for LCDRAT and to lung cancer risk for all other models. Results are based on a combined data set, which uses a single imputation for missing data. USPSTF 2013 criteria classify 41,107 (15.0%) current and former smokers in the 3 cohorts combined as screening eligible, and USPSTF 2020 criteria classify 71,387 (26.1%) as eligible. These percentages differ from Table 1 as they are calculated after imputation, whereas Table 1 omits missing data. NA not applicable. g y The question of which risk model optimally deﬁnes screening eligibility is somewhat distinct from the question of which model can most practically be implemented. Web-based tools are available for some risk models, including the Risk-based NLST Outcomes Tool for LCDRAT and LCRAT39 and MyLungRisk for LLPv2,40 and a spreadsheet tool is available for PLCOm2012.41 It is possible to integrate these tools into electronic medical records to facilitate calculations, but input information would ﬁrst need to be veriﬁed with the patient. A practical solution may be to use a simpliﬁed model or algorithm applied to electronic medical records to initially identify people who are potentially eligible, followed by a more precise assessment of risk using data collected in person or by phone. The goal to automate further the calculation of lung cancer risk and the classiﬁcation of individual screening eligibility represents an ongoing challenge. DISCUSSION L Considering four individuals with the same true risk of lung cancer, one in each SES quartile, Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. 2032 Table 3. Performance of lung cancer risk models for deﬁning lung cancer screening eligibility among current and former smokers in the combined UK Biobank, EPIC-UK, and Generations Study cohorts. Risk model Threshold to screen the same number of participants as USPSTF guidelines Lung cancer cases eligible for screening over 5 years Lung cancer deaths eligible for screening over 5 years USPSTF 2013 guidelines (age 55–80 years, at least 30 pack-years, no more than 15 quit-years) Total NA 1474 (100%) 826 (100%) USPSTF criteria NA 747 (50.7%) 415 (50.2%) LCDRAT 0.8% 5-year risk 897 (60.9%) 522 (63.2%) LCRAT 1.4% 5-year risk 897 (60.9%) 519 (62.8%) PLCOm2012 1.5% 6-year risk 859 (58.3%) 490 (59.3%) Bach 1.6% 5-year risk 855 (58.0%) 501 (60.7%) LLPv3 1.3% 5-year risk 835 (56.6%) 489 (59.2%) LLPv2 2.3% 5-year risk 791 (53.7%) 465 (56.3%) USPSTF 2020 guidelines (age 50–80 years, at least 20 pack-years, no more than 15 quit-years) Total NA 1474 (100%) 826 (100%) USPSTF criteria NA 979 (66.4%) 560 (67.8%) LCDRAT 0.4% 5-year risk 1135 (77.0%) 655 (79.3%) LCRAT 0.8% 5-year risk 1101 (74.7%) 637 (77.1%) PLCOm2012 0.8% 6-year risk 1109 (75.2%) 629 (76.2%) Bach 0.8% 5-year risk 1131 (76.7%) 652 (78.9%) LLPv3 0.7% 5-year risk 1039 (70.5%) 604 (73.1%) LLPv2 1.3% 5-year risk 1020 (69.2%) 590 (71.4%) Models are listed in order of their performance for identifying future lung cancer cases (USPSTF 2013). “Risk” refers to lung cancer death risk for LCDRAT and to lung cancer risk for all other models. Results are based on a combined data set, which uses a single imputation for missing data. USPSTF 2013 criteria classify 41,107 (15.0%) current and former smokers in the 3 cohorts combined as screening eligible, and USPSTF 2020 criteria classify 71,387 (26.1%) as eligible. These percentages differ from Table 1 as they are calculated after imputation, whereas Table 1 omits missing data. NA not applicable. 032 between the threshold we identiﬁed for LCDRAT (0.8% 5-year lung cancer death risk) and previously proposed thresholds (1.2, 1.33, 1.7%).19,31 Thresholds identiﬁed based on USPSTF 2020 criteria, which broadened eligibility substantially, were much lower, and it is not clear whether all individuals meeting these thresholds would have a favourable trade-off of screening beneﬁts and harms. DISCUSSION L the individual with the highest SES will have her risk over- estimated the most and will be most likely to be classiﬁed as eligible for screening. This could exacerbate disparities in lung screening.34 Our ﬁndings suggest that there are factors related to SES that increase lung cancer risk but are not captured by the variables in risk models or are related to differential measurement error for the variables in risk models. Further, the effects of USA educational and ethnicity categories on lung cancer risk are unlikely to align with these effects in the UK. Any future efforts to develop risk models for use in lung screening in the UK should focus carefully on the role of SES and the accurate estimation of risk within subgroups. In conclusion, we analysed the performance of lung cancer risk models in three UK cohorts, including the PLCOm2012 and LLPv2 models that are recommended for use in lung screening by the NHS protocol for Targeted Lung Health Checks. We found that the LLPv2 model had worst calibration and classiﬁed the lowest proportion of future lung cancer cases as eligible for screening. The LLPv3 model had best calibration (but poor discrimination), while the LCDRAT was best able to identify individuals at high risk of lung cancer. All models strongly over-predicted risk in groups with high SES, raising concerns about exacerbation of disparities in lung cancer screening. Taken together, our results suggest potential revisions to the list of models endorsed by the NHS lung g The choice of what risk threshold to use for screening eligibility depends on multiple factors, including the accepted trade-off of beneﬁts and harms and the capacity of the health system. We did not address these issues here, but we did identify thresholds that would classify the same number of individuals as screening eligible as USPSTF criteria. The thresholds selected by this approach, when considering USPSTF 2013 criteria, aligned with those already proposed for the PLCOm2012 (1.5% in our study vs. 1.51% in the NHS protocol) and the LLPv2 (2.3% in our study vs. 2.5% in the NHS protocol).14 There was a larger difference Comparative performance of lung cancer risk models to deﬁne lung. . . HA Robbins et al. Table 4. Calibration and discrimination estimates for lung cancer risk models in UK Biobank current and former smoking participants, restricted to ages 55–74 years. ACKNOWLEDGEMENTS 1. Cancer Research UK. Cancer Statistics for the UK. https://www.cancerresearchuk. org/health-professional/cancer-statistics-for-the-uk (2020). We thank all study participants, study staff, and the doctors, nurses and other health care staff and data providers who contributed to the UK Biobank, EPIC-UK, and Generations Study. This research has been conducted using the UK Biobank Resource under application number 15825. We thank Dr. Paul Brennan for his feedback on the manuscript. 2. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A. & Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality world- wide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, 394–424 (2018). 3. National Lung Screening Trial Research Team, Aberle, D. R., Adams, A. M., Berg, C. D., Black, W. C., Clapp, J. D. et al. Reduced lung-cancer mortality with low-dose computed tomographic screening. N. Engl. J. Med. 365, 395–409 (2011). DISCLAIMER Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41416-021-01278-0. Where authors are identiﬁed as personnel of the International Agency for Research on Cancer/World Health Organisation, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organisation. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. AUTHOR CONTRIBUTIONS 4. de Koning, H. J., van der Aalst, C. M., de Jong, P. A., Scholten, E. T., Nackaerts, K., Heuvelmans, M. A. et al. Reduced lung-cancer mortality with volume CT screening in a randomized trial. N. Engl. J. Med. 382, 503–513 (2020). H.A.R. and M.J. conceived of the study and its design. A.J.S., M.J.S., N.W., and R.C.T. provided study data. K.A. and H.A.R. completed statistical analyses. H.A.R., K.A., A.J.S., M.J.S., N.W., R.C.T., M.C., R.L., P.A.J.C., D.R.B., and M.J. interpreted the results. H.A.R. drafted the manuscript. H.A.R., K.A., A.J.S., M.J.S., N.W., R.C.T., M.C., R.L., P.A.J.C., D.R.B., and M.J. critically reviewed the draft manuscript and approved the ﬁnal version. H.A.R. and M.J. conceived of the study and its design. A.J.S., M.J.S., N.W., and R.C.T. provided study data. K.A. and H.A.R. completed statistical analyses. H.A.R., K.A., A.J.S., M.J.S., N.W., R.C.T., M.C., R.L., P.A.J.C., D.R.B., and M.J. interpreted the results. H.A.R. drafted the manuscript. H.A.R., K.A., A.J.S., M.J.S., N.W., R.C.T., M.C., R.L., P.A.J.C., D.R.B., and M.J. critically reviewed the draft manuscript and approved the ﬁnal version. 5. Moyer, V. A. Screening for lung cancer: U.S. Preventive Services Task Force recommendation statement. Ann. Intern. Med. 160, 330–338 (2014). 6. Crosbie, P. A., Balata, H., Evison, M., Atack, M., Bayliss-Brideaux, V., Colligan, D. et al. Implementing lung cancer screening: baseline results from a community- based “Lung Health Check” pilot in deprived areas of Manchester. Thorax 74, 405–409 (2018). DISCUSSION L Lung cancer risk model Bach LCDRAT LCRAT LLPv2 LLPv3 PLCOm2012 Calibration (E/O) Among age 40–74 years 1.39 (1.31–1.47) 1.52 (1.41–1.64) 1.44 (1.36–1.52) 2.16 (2.05–2.28) 1.20 (1.14–1.27) 1.30 (1.23–1.36) Among age 55–74 years 1.38 (1.30–1.47) 1.52 (1.41–1.64) 1.40 (1.32–1.48) 2.25 (2.13–2.39) 1.25 (1.18–1.33) 1.26 (1.19–1.33) Discrimination (AUC) Among age 40–74 years 0.80 (0.79–0.81) 0.82 (0.81–0.84) 0.81 (0.79–0.82) 0.77 (0.76–0.78) 0.78 (0.76–0.79) 0.79 (0.78–0.80) Among age 55–74 years 0.76 (0.75–0.78) 0.79 (0.77–0.81) 0.77 (0.76–0.79) 0.73 (0.72–0.75) 0.74 (0.73–0.75) 0.75 (0.74–0.77) The E/O statistic is the ratio of cases expected (predicted by the model) to the cases observed. AUC is the area under the receiver operating curve. The estimates for ages 40–74 years correspond with the primary estimates presented in Fig. 1 (n.b. there were no participants in UK Biobank aged >74 years). Table 4. Calibration and discrimination estimates for lung cancer risk models in UK Biobank current and former smoking participants, restricted to ages 55–74 years. of Cancer Research. The ICR acknowledges NHS funding to the Royal Marsden/ICR NIHR Biomedical Research Centre. P.A.J.C. is supported by the NIHR Manchester Biomedical Research Centre. M.C. is co-chief investigator of the Yorkshire Lung Screening Trial, which is funded by Yorkshire Cancer Research. R.L. is supported by the Intramural Research Program of the US National Institutes of Health/National Cancer Institute. The funders had no role in the design, analysis, or reporting of the study. of Cancer Research. The ICR acknowledges NHS funding to the Royal Marsden/ICR NIHR Biomedical Research Centre. P.A.J.C. is supported by the NIHR Manchester Biomedical Research Centre. M.C. is co-chief investigator of the Yorkshire Lung Screening Trial, which is funded by Yorkshire Cancer Research. R.L. is supported by the Intramural Research Program of the US National Institutes of Health/National Cancer Institute. The funders had no role in the design, analysis, or reporting of the study. screening protocol and that further work may be needed to ensure that eligibility for lung cancer screening can be deﬁned equitably in the UK population. More generally, they highlight the importance of carefully validating risk prediction models in speciﬁc contexts before they are applied in practice. ADDITIONAL INFORMATION Screening for lung cancer: CHEST guideline and expert panel report. Chest 153, 954–985 (2018). 20. Cassidy, A., Myles, J. P., van Tongeren, M., Page, R. D., Liloglou, T., Duffy, S. W. et al. The LLP risk model: an individual risk prediction model for lung cancer. Br. J. 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Assess. 20, 1–146 (2016). Competing interests H.A.R., K.A., A.J.S., M.J.S., N.W., R.C.T., M.C., R.L., M.J.: None. P.A.J. C.: consultancy and share options, Everest Detection. D.R.B.: reimbursement for participation on advisory boards, AstraZeneca, and MSD. 12. Grover, H., Ross, T. & Fuller, E. Implementation of targeted screening for lung cancer in a high-risk population within routine NHS practice using low-dose computed tomography. Thorax 75, 348–350 (2020). Funding information This study was funded in part by the US National Cancer Institute (R03 CA245979 and U19 CA203654) and Cancer Research UK (C18281/ A19169). 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https://openalex.org/W4312045317	https://link.springer.com/content/pdf/10.1007/s44186-022-00089-y.pdf	English	null	Overlapping worlds of art and plastic surgery: developing a concept model and its implications in surgical education	Global surgical education	2,022	cc-by	7,513	Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 https://doi.org/10.1007/s44186-022-00089-y Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 https://doi.org/10.1007/s44186-022-00089-y Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 https://doi.org/10.1007/s44186-022-00089-y ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract Purpose Editorials speculate on the relationship between art and plastic surgery, and studies of limited art education in surgical training show intriguing benefits. Identifying the shared concepts and skills in art and plastic surgery could advance incorporating artistic skills and concepts into plastic surgery training and curriculum.i Methods Using a grounded theory approach, we interviewed plastic surgeons and formally trained or self-identified artists and then analyzed the transcripts. During the process, we used a constant comparison approach while coding along with data collection. The team developed the codebook from initial transcripts; 2 members coded each transcript. We reconciled codes and summarized codes into themes based on discussion among the team. Results 15 plastic surgeons aged 36–80 years and 16 artists aged 19–62 years were interviewed. We then developed a concept model, “Ways of Making,” to illustrate the shared aspects of the artistic and surgical process through their ways of doing, knowing, seeing, and thinking. Both plastic surgeons and artists recognized that strong technical foundational skills are key to developing competency. Both groups spoke about the Elements of Art and Principles of Design, though artists know this formally. Artists and plastic surgeons shared that awareness to one’s surroundings or to human features facilitates identifying problems or ideas. They described how technical skills, manual dexterity, and three-dimensional thinking can be taught and nurtured. Both groups also recognized that creativity played a major role in their work. While creativity was seen as innate, participants can learn to be innovative through critical thinking. Conclusion This study provides a model for how plastic surgery and art overlap using data from interviews. Though there are differences between the two fields, the ways of doing, knowing, seeing, and thinking are key components of the artistic and surgical processes. Identifying the shared concepts and skills in art and plastic surgery could help enhance curricula seeking to incorporate artistic skills and concepts into plastic surgery training. Keywords Education · Plastic surgery · Arts and humanities · Visual arts · Curriculum Overlapping worlds of art and plastic surgery: developing a concept model and its implications in surgical education Audrey Nguyen1 · Dawn Duong1 · Patricia O’Sullivan2 Received: 19 May 2022 / Revised: 23 November 2022 / Accepted: 27 November 2022 / Published online: 10 December 2022 © The Author(s) 2022 1 Department of Surgery, Division of Plastic and Reconstructive Surgery, University of California San Francisco (UCSF), 505 Parnassus Ave, Suite M‑593, San Francisco, CA 94143, USA * Audrey Nguyen Audrey.Nguyen@ucsf.edu 2 School of Medicine, Departments of Medicine and Surgery, University of California San Francisco, San Francisco, USA Introduction shown intriguing benefits. Surgeons have published opin- ions in editorials and correspondence about art and plastic surgery. These opinions describe the importance of under- standing beauty, grace, and esthetics [6, 7], how knowledge of art helps with plastic surgeons’ three-dimensional vis- ual-spatial skills [8], or how the fundamental difference is that art is a form of self-expression whereas plastic surgery is a science that “seeks to uncover knowledge (and argu- ably beauty)” [2]. In one survey study of 26 plastic surgery trainees in England, who were asked whether they felt that artistic skills were relevant in plastic surgery; 58% agreed [7]. Notably, many of the published comments were about the ability to draw, and one of the trainees who disagreed wrote, “Just because you can draw does not mean you can operate competently.” The relationship between art and plastic surgery has inter- ested plastic surgeons for almost fifty years [1–6]. Pub- lished studies about this relationship are mostly editorial in nature, but limited art education in surgical training has (0121 3456789) 3 Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 9 Page 2 of 9 9 Page 2 of 9 However, art goes beyond the act of drawing. A study from the Giesel School of Medicine in Dartmouth, New Hampshire, introduced art in a different way [9]. The authors showed a painting titled, In Hospital (The Operating Room), by Bernard Perlin (1918–2014) to surgical trainees and academic surgeons and asked them to describe what they saw. The results were interesting; the trainees described the painting in clinical terms and focused on anatomy and tech- nique while the surgeons focused on the primary surgeon and his role in the painting. The authors concluded that the understanding of perspective and mature clinical gaze can be established through observations and analyses of works of art [9]. The Pennsylvania Academy of Fine Arts offers art courses to medical students and personnel with the goal of “improving visual literacy” and observational skills [10]. Casey Lesser, Creativity Editor of Artsy, an online platform for collecting and discovering art, writes that medical stu- dents have been required to take art classes to help improve communication skills, perspective, and even, compassion [11]. Introduction Though anecdotal, these opinion pieces approach an appealing idea—that an arts education is beneficial to surgi- cal training.i unsure how concepts from artists and surgeons would overlap and because there is sparse literature and under- lying theories related to this topic. There are editorial and opinion pieces in the plastic surgery literature that suggest a relationship between the two fields that improves plastic surgery skill as previously discussed [1–8]. However, there is a lack of a model that describes how art and plastic sur- gery are related. We chose interviews as a data collection method because they provide rich data for the develop- ment of a model for this grounded theory study. An inter- view “attempts to understand the world from the subjects’ point of view, to unfold the meaning of their experience, to uncover their lived world” [16]. The grounded theory methodology allows us to identify and understand a way in which the concepts that are important to both artists and plastic surgeons can be interrelated. We recruited practicing plastic surgeons and practicing artists to interview for this study. Plastic surgeons were eli- gible to participate if they were board-certified or board- eligible. Artists were eligible if they identified as an artist for their primary occupation or if they had formal visual arts training and education. Many of the recruited surgeons and artists were considered experts in their field. They were considered experts based on academic or social status, years in practice, and productivity. The presented sample size is typical of studies using grounded theory approaches, as the purpose of the study is to achieve an understanding and satu- rate the underlying theory model [15]. We sampled until we believed information sufficiency had been obtained, which fell in the range of our goal sample size [15]. Exploring the shared core concepts and skills in the fields of art and plastic surgery could inform plastic surgery train- ing programs about what aspects of artistic skills and con- cepts can be applied to training. Art education courses and artistic skill are not a requirement of plastic surgery trainees, nor are they assessed in the current model of competency based medical education (CBME), which is set as the struc- ture for assessing performance in United States Plastic Sur- gery Programs accredited by the Accreditation Council of Graduate Medical Education (ACGME) [12, 13]. Introduction However, plastic surgery programs aspire to educate residents who can demonstrate skills resulting in aesthetically pleasing results, who are experts in human anatomy, and who have the obser- vational and surgical skills to design and complete a suc- cessful surgery [14]. The goal of this study is to describe the core concepts and skills that art and plastic surgery share to provide the language and a new framework necessary to enrich the curriculum and competencies for plastic surgery trainees. The findings may provide guidance for educating surgeons by incorporating the skills and practices of the related field of visual arts. g g p [ ] Using purposeful sampling [17], we recruited academic and community plastic surgeons who represent all subspe- cialities within the field using the network of the principal investigator, AN. She started with her local network of aca- demic plastic surgeons whom she trained with at UCSF and also engaged in artistic activities (sculpture, photography, digital art). She also interviewed plastic surgeons who were considered experts and with whom she had a relationship. AN also approached plastic surgeons via email who have presented at conferences and expressed an interest in the visual arts based on their lecture. AN contacted all plastic surgeons personally and did not use any other recruitment methods. AN also knew some artists and used a snowball sampling approach by asking artist participants to recom- mend other participants. Like the plastic surgeons, we selected the participants based on their background and specialty to include a representative sample of visual artists who use different media and/or are in varying types of prac- tices. We also included artists who were educators, as this was comparable to plastic surgeon educators in academia. This study was approved by the UCSF Institutional Review Board (IRB). Methods We conducted interviews and qualitative analyses of the interview transcripts using grounded theory—a qualita- tive research method used by investigators to generate a theory that explains a process or action based on “data from participants who have experienced the process” [15]. We identified that this approach was useful since we were 3 Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 Page 3 of 9 9 9 Demographic information A total of 31 in-depth interviews were conducted with 15 plastic surgeons and 16 artists (Table 1). The plastic sur- geons ranged from 36 to 80 years of age, and the artists ranged from 19 to 60 years. In both groups, most participants identified as White. There were three artists who identified as Latinx. There was an even spread between gender in the artist group, with plastic surgery having slightly more par- ticipants who identified as men (n = 9) than women. Both groups interviewed represented a variety of subspecialties in their respective fields. Eight of the plastic surgeons were educators at a university or university-affiliate, and four art- ists were educators at the junior high, high school or college level. While we interviewed this large number of partici- pants, we felt information sufficiency had been reached in both groups around the 10th interview. Interviews: We conducted single, semi-structured inter- views that lasted 60–120 min. Each interviewee provided demographic data such as age, gender, and specialty. The initial five interviews were performed in-person and recorded. However, at the start of the COVID-19 pandemic, we switched to Zoom interviews for safety. These interviews were audio and video recorded on Zoom. The recordings allowed for verbatim transcription of the interview through artificial intelligence (Rev.com). Review of the conversation and video by the investigators and transcriptionist permitted checks for accuracy and extraction of narratives, themes, and quotations. Each interviewee received a $75 Amazon gift card for their participation. Table 1 Demographic and practice information about the two groups of participants, plastic surgeon and artist * More than 1 type of media (i.e., ceramics, photography, drawing, and painting) Plastic surgeon (N = 15) Artist (N = 16) Age, years 36–80 19–60 Gender, N Male 9 8 Female 6 8 Race, N White 13 12 Asian 2 1 Latinx 0 3 Specialty, N Cosmetic Surgery, 5 Multimedia*, 9 Microsurgery, 5 Painting, 3 Craniofacial Surgery, 3 Drawing, 1 Hand Surgery, 2 Sculpture, 1 General Reconstruction Only, 1 Graphic Design, 1 Practice Type Academic, 8 Educator, 4 Private Practice, 7 Personal Business, 12 Table 1 Demographic and practice information about the two groups of participants, plastic surgeon and artist Data collection each transcript was coded by a single researcher who was blinded to the other researcher’s proposed codes. The researchers then reviewed each assigned code and came to an agreement on the codes through discussion and reference to the codebook if the codes were divergent. We memoed throughout the interview and coding process. We then sum- marized our codes into themes based on discussion among the team. Interview Guide: We developed a preliminary interview guide based on literature review and the experiential knowl- edge of AN. We used two framing questions. First, we asked participants to describe in detail the core concepts, princi- ples, and competencies that they feel are important to their field, including those directly taught in training and those developed via extramural learning. Second, we asked if there were specific skills that they developed or were developing to become a competent or master surgeon or artist. During the interviews, we probed participants to further explore the themes that the research team was identifying from prior interviews. We used this semi-structured approach since it “can help ensure the comparability of data across individu- als, times, settings and researchers, and are particularly use- ful in answering questions that deal with differences between people or settings” [17]. Concept model The concept model that we developed based on our intensive interviews revealed a framework for understanding how art and plastic surgery overlap that we have titled, “Ways of Making” (Fig. 1). “Making” referred to the finished product or work. For visual artists, this was their artwork. For plastic surgeons, this was the surgery and its results. The model was organized into five interrelated categories: ways of knowing, ways of doing, ways of seeing what is, ways of seeing what could be, and ways of thinking. Table 2 highlights quotes supporting each concept. Analysis The research team developed a codebook using in vivo codes from the initial transcripts. The team used a constant com- parison approach and met throughout the data collection, coding, and analysis processes. Each member’s views were discussed and considered as the analysis progressed. AN is a plastic surgeon with some experience in art courses but limited formal training, and she has illustrated plastic sur- gery articles and texts. She is completing a Master of Art in Education. AN completed the plastic surgery interviews. DD has also taken art classes and has been involved in other qualitative research studies. DD was comfortable talking with educators and performing interviews. AN and DD did the artist interviews. PO’S provided expertise in qualitative analysis. All team members served to add perspective and to challenge AN to clarify how her perspective influences the interpretation of the results. We used the software Dedoose (SocioCultural Research Consultants, LLC, Los Angeles, CA) to code and to organ- ize the analysis of the interview transcripts. Using Dedoose, 1 9 Page 4 of 9 Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 Of these shared ways of knowing, the plastic surgeons understood and knew anatomy at an extremely detailed level. We defined anatomy as the study and knowledge of the human body. This was necessary for patient safety, and a core com- ponent of reconstruction was knowing the baseline anatomy to be altered. However, artists also studied anatomy to under- stand the underlying structures that are important for overlying contour, shape, and movement. Both groups recognized the importance of knowing surface anatomy—how the surfaces of something are shaped but what lies beneath. The other impor- tant way of knowing for plastic surgeons also came from the elements of art and principles of design, though informally and not through art classes. Plastic surgeons used many of the terms within the elements of art and principles of design as they described their work. Some of these terms included “color”, “symmetry”, “shadow,” “light intensity” and “bal- ance.” These basic terms were used in addition to the scien- tific, medical, and anatomical terms to describe their process and surgical design. Ways of doing Artists and plastic surgeons also shared ways of doing, which described how each group goes through the process of achiev- ing a work—either a surgical case or a piece of artwork. This process was centered around technical skill, which referred to the use of tools, methods and techniques in each field. For visual artists, one of the most important skills was drawing and mark-making because it developed the artistic eye and hand–eye coordination. Other skills included mixing paint and understanding the chemistry, how to use the instruments and how to use one’s hands. For plastic surgeons, the basic skills were suturing, working and handling human tissue and its underlying structures, and using instruments while doing so. There were certainly differences in dissecting tissue and suturing versus preparing paint colors, canvases, and paint- ing. However, it was clear from the interviews that manual dexterity and tactile sensation were integral to the success of both artists and plastic surgeons and was developed early in training. Both groups strongly believed that practice was a key to improvement in the field and many participants from both groups quoted that 10,000 h of practice was necessary. Fig. 1 Concept Model of Ways of Making – a framework that demon- strates the artistic and plastic surgery process The ways of knowing and ways of doing represented the foundations of the concept model, even though all aspects were interrelated. These foundations were important because once they were developed, they can be pushed and broken, allowing for freedom and creativity. Ways of knowing Both groups shared ways of knowing which was based on shared knowledge of anatomy and fundamental art concepts: elements of art and principles of design. Artists learned the elements of art and the principles of design in foundational art courses, and those guided everything in making art. The elements of art are the building blocks used by artists to cre- ate a work of art and include line, shape, form, color, value, space, and texture [18]. The principles of design describe the way artists use the elements of art in their work: bal- ance, emphasis, movement, pattern, repetition, proportion, rhythm, variety, and unity [19]. For plastic surgeons, under- standing anatomy was one of the most important ways of knowing. Fundamental concepts for this field were knowing the blood flow and anatomy and replacing like with like. Ways of seeing The ways of knowing and doing were related to ways of see- ing. This notion described how artists and plastic surgeons see what is and what could be. Fig. 1 Concept Model of Ways of Making – a framework that demon- strates the artistic and plastic surgery process 3 3 Page 5 of 9 9 Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 Page 5 of 9 9 Global Surgical Education Journal of the Association for Surgical Education (2023) 2:9 1 Page 5 of 9 Table 2 Exemplary quotes from artists and plastic surgeons as it relates to the concept model by concept. A refers to artists, and PS refers to plastic surgeon Artist Plastic surgeon Ways of doing Technical Skill: the use of tools, methods, and techniques “So, I think that learning how to draw from life is very important to be able to see and translate what you see in reality, the physical reality, and that's why figure drawing is important. The anatomy is important, because it's a huge instructional thing about drawing the world. Learning how to draw from what you see, you learn the abstract relation- ships between objects, between form.” (A6) “Dexterity. I'm watching people sew, watching people do stuff.” (PS5) “So I think that that can be taught, in how you motion, how you move, how you handle things, the instruments that you use, and why or not.” (PS6) Ways of knowing Elements of art: building blocks used by artists to create a work of art and include line, shape, form, color, value, space, and texture Principles of design: the way artists use the elements of art in their work: balance, emphasis, movement, pattern, repeti- tion, proportion, rhythm, variety, and unity Anatomy: the study and knowledge of the human body “The first one is value. And if you take white at one end and black at the other end, and then you have nine shades in between or eight shades in between, you establish what are called values…And whenever we paint something, it's a matter of breaking it down into shapes.” (A13) “Classes that teach you how to draw a human form, classes that teach you about color, about the principle of design, classes that teach you how to work with your hands, and classes that teach you how to work computer programs. Ways of seeing All classes that will give you your core values and what people are expecting from you in terms of your knowledge of art.” (A15) “In anatomy class for instance, there was a lot of drawing live skeletons and that kind of stuff. And one of my professors was always over the top, so in that particular class, we would go and unwrap cadavers and we would draw from that. And I actually found that very fascinating.” (A16) “So, if I'm dealing with a human body, and I understand those things, I understand then physically, the relationship of one part to the next, and how it fits into a volume. And that to me seems like that would be an incredibly valuable for some- body who was dealing with human bodies.” (A11) “So, I think I improved my anatomy a ton and learning about art and art techniques made me look at things differently. All of a sudden, I realized things were there that I never knew were there, like shadows and different light intensities and shades, and I didn’t even know what I didn’t know, and it was nice to learn that and I think it helped me understand anatomy better. It helped me understand, for instance, cheekbones and the points of shining on light and lips and ears and things like that. Ways of seeing So, when I was learning how to do it, it was very excit- ing for me because I thought I was getting better at surgery because I was learning this art knowledge.” (PS4) “Well again, the aesthetic eye is just it’s seeing any part of the human body and knowing what looks right or what looks wrong, what looks esthetic or not, what looks pretty, what looks symmetrical, what looks balanced, what looks propor- tional.” (PS7) “I think it's important that you have a basic understanding of anatomy, and principles, and basic principles of flap design, and stuff like that, and then when you see a problem, you can figure out how to apply that in a specific case that may be novel or may be a little different from the way something else was done.” (PS5) “So, I think a good plastic surgeon has a great knowledge of anatomy, has a creative mind and then has the technical skills to execute something difficult in rearranging tissue.” (PS9) Ways of seeing what is Environmental Awareness: being in tune and attentive to one’s surroundings Subject Sensitivity: the ability to see the subtleties of the subject matter than an untrained eye would not “Because even when I walk, I walk daily, I can't help but notice patterns and the sunlight coming through the trees and the shadows and all that. I see designs. I see that all the time.” (A16) “I think artistic people see things that normal people don’t look at or they are hypersensitive to esthetics. They have sensibilities that are very different from people who are outside of the arts. They’re sensitive to form and arts and principles of design. They notice the things in between the line. The message in between the lines.” (A8) “One of the things that happens to lots of people in drawing classes is, the teachers, they would be telling you, you have to look, you have to look, you have to look, and you have to see.” (A11) "When I was trying to learn what is the earlobe shape, what does the back of the earlobe really look like? How do we inset the earlobe and a facelift, why, what is this angle the earlobe makes with the jawline. Ways of seeing I would be in the elevator, and I would just stare at the back of people's heads.” (PS11) “And when people look at an ear, they don't look the way we look at an ear. They don’t dissect by helix, antihelix, scapha, inferior crus, concha, that kind of stuff. We do that, and so we’re seeing things that other people don’t see.” (PS1) “I see better and if I can see better and see more things, I think I can communicate with the patient better. If I'm looking at somebody and I just see two or three things versus 20 things, I think you having 20 things that I'm seeing makes my diagno- sis better and my planning better.” (PS4) 1 Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 9 Page 6 of 9 9 Th b f ll i th l tt i th ti i t i d h id tifi Artist Plastic surgeon Ways of seeing what could be Visualization: thinking ahead by picturing the final product and going through the process in one’s mind Intention: setting the goal that the artist or surgeon is trying to achieve “I guess again, it depends on when I set out, I have certain goals of the level I want to get it to. For me, myself, that is I know what, in a way, the endpoint is going to be.” (A12) “You're thinking two steps ahead, or three steps ahead, because it's just natural, the way you're doing things.” (A16) “To train awareness, but also once you have awareness of something and language for describing it, then you can have command over it. And I think a lot of artists aren't able to put that into words maybe, but they are actively using it. Ways of seeing So that's the difference, is intentionality.” (A8) “Usually, I'm thinking about the end where I want to get and then thinking about steps on how to get there, but probably when I'm walking into the operating room, the literal image I have in my head is what the patient looks like when we're done.” (PS4) “You have to have the image in mind as well and you kind of know what you're aiming for.” (PS11) ""Okay, he's got all these little dots on the lip." I said, "Okay, so what are the goals of our operation?" He starts talking through the dots, and I broke it down and was like, "No, no, what are we really trying to accomplish here?"” (PS15) Ways of thinking Critical Thinking: problem or puzzle solving along with find- ing creative solutions Creativity: pushing the limits or boundaries, thinking outside the box, and being original or new by going beyond the normal, average, or expected “It's kind of like how engineers invent things to solve prob- lems. We're artistic engineers. We're inventing designs to solve people's needs with marketing and things like that. A core value or something you would need to become a designer is the desire to solve a problem.” (A15) “When I say learning how to think, it's learning how to take your successes and your failures in particular and building on those. There's an old cliché that you can succeed without failure, and that's true. You go through life, and you fall on your face, and you get up, you learn from it, if you're smart you learn from it, and you build on that, and out of those mistakes, or that chaos comes something new, and these discoveries.” (A6) “I would say putting yourself in uncomfortable situations forces you to be creative because usually when something's uncomfortable, it’s sometimes unprecedented.” (A9) “It's a surgical discipline where essentially no two operations are ever completely the same…you have a problem; the patient has a problem, and that problem needs to be solved and it's a way of using tissue to solve those problems crea- tively.” (PS12) “I think it is that you have a set of rules that create your framework, but you're not bound by them. Ways of seeing You get to use that framework to solve a problem, and I think most… If you think about problem-solving in any field, whether it's engineering or literature, it's about being creative. In plastic surgery, it seems like it's all about problem-solving, so that's where that creativ- ity comes in.” (P15) “Creativity is the ability to produce, see, think or in any other way, something new, different and astounding or surprising. That's creativity… the big C is where you're actually doing something that has intrinsically more value or more power to the person that's observing or to yourself” (PS7)_ Page 7 of 9 9 Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 Page 7 of 9 9 9 Artists and plastic surgeons have ways of seeing what is, which we have equated to observational ability, and we described this concept through two terms: environmental awareness and subject sensitivity. From the interviews, we defined environmental awareness as being in tune and atten- tive to one’s surroundings. The ability to observe, or to see or to look, took practice and over time, the observer learned to be aware of subtleties that the untrained eye would not, and thus, have developed subject sensitivity. Both groups were constantly observing their surroundings and had developed this skill acutely. Visual artists looked to the environment for inspiration and motivation. Plastic surgeons reported observing human features in their daily lives to understand the nuances of such features and the anatomy to replicate normal, reconstruct, or attempt to capture beauty surgically. Artists did the same, but their subject matter could be quite broad, sometimes they were human and for others they were landscapes, animals, or creations from their imagination. into play during the process of making. Both groups agree that creativity was a key component of mastery for their respective fields. Artists and plastic surgeons used critical thinking to improve their ways of doing, knowing and seeing, so that they could ultimately create and be creative. Art versus science Though there are important similarities in the “Ways of Making” for artists and plastic surgeons, we discovered through the in vivo code “Art vs. Science” an important difference in self-expression versus patient safety. We have demonstrated that plastic surgery is connected to art, but it still originates from the medical sciences. Though there were more similarities than not, the tension between art versus science made it clear that there are boundaries to using artis- tic concepts in plastic surgery, namely, around the form of self-expression. To describe art, artists and plastic surgeons used words such as “emotion”, “self-expression”, and “right brain”. To describe science, both groups used words such as “logic,” “measurement,” and “left brain.” Science in plastic surgery relates to the medicine of the field; its complications related to patient safety, anatomy, physiology, and human tissue. Art is described as a form of self-expression, where science is not. For example, in visual arts, one can play, paint, or sculpt the human body in unnatural ways and push the human body to extremes as the artists Pablo Picasso and Egon Schiele do. Plastic surgeons cannot because human safety is a high priority in plastic surgery where in art, it is not. The ways of seeing what could be related to how each group saw the future in their mind through intention setting and visualization. Intention meant setting the goals of what the artist or surgeon was trying to achieve. We defined visu- alization as thinking ahead by picturing the final product and going through the process in one’s mind. This visualization also helped with identifying or anticipating issues ahead of time. It was important to set a goal for the work, even if the process changed the end goal. Another key component of visualization was the ability to think spatially and in three dimensions. Setting the intention allowed for visualiza- tion. Artists and surgeons described this as going through the steps of the procedure in their minds, considering one’s choices, and imagining the final product. Ways of thinking The curriculum included six hours of general art courses and three hands- on sessions with clay sculpture. After taking the classes, the authors observed that the trainees had improved in the following areas: medical charting of anatomical problems, predicting the result of a surgery based on preoperative markings, and applying more thorough and detailed surgi- cal dressings [21]. Guneron concluded that the art classes helped with overall attention-to-detail. He did not include how these improvements were measured. Furthermore, the authors did not measure improvement in surgical technique, though they concluded that trainees should continue art pro- jects because practice leads to improvement, and trainees should use the learned art concepts to “clearly define esthetic surgical problems” [21].i p p g y Other studies have also stressed the importance of art and humanities in medical education [22–24]. Though these studies included a broad range of topics beyond vis- ual arts such as creative writing and philosophy, Moniz et al. developed a prism model based off Dennhardt’s pre- vious findings to describe four beneficial epistemic func- tions of arts and humanities [22, 24]. These are: master- ing skills, perspective taking, personal insight and social advocacy [24]. Moniz et al. describe an inability to report a typology of skills due to lack of coherence and broad- ness of the term in the arts and humanities [24]. Our study, however, which is focused on the visual arts, was able to delineate and describe why technical skills are important. Because Moniz and Denhardt’s models are applicable to the broader world of medical education and arts and humanities, there are differences with our model. Where we describe the importance of goal-setting through visu- alization and environmental awareness, they describe per- spective taking as “engaging in art…to facilitate students to relate to and reflect upon their and others’ experiences and thus improve the doctor-patient relationship” [22]. Our model does not include aspects of social advocacy. However, the prism model and Denhardt’s model do not include how arts and humanities enhances creativity and critical thinking skills, where it is extremely important in our model and may be more specific to plastic surgery and correlated with obtaining mastery. The limitations of the study are that the surgeons mostly came from one geographic area that might influence their practice. The artists were more varied in their location. Ways of thinking We have defined creativity as pushing the limits or boundaries, thinking out- side the box, and being original or new by going beyond the normal, average, or expected. Creativity thrives in the face of boundaries, constraints, or rules because it is in pushing those limits that one can be creative. In understanding these “Ways of Making,” specifically ways of thinking, we may be able to teach creativity to our trainees which is strongly correlated with mastery. include a fundamental or foundational lesson that teaches the elements of art and principles of design and how artists learn anatomy. Another lesson could focus on manual dex- terity and technical skill as these authors have designed in the clay and sculpture classes. Any lesson or course would also emphasize the importance of visualization, purposeful action, environmental awareness and subject sensitivity. As an example, this could be done through observations of paintings or even descriptions of human features. Finally, incorporating a component of creativity and problem-solv- ing would be necessary as this represents the highest level on the model. This can be achieved through developing an art practice that is related to plastic surgery. The potential overlapping concepts and skills have led some surgeons to suggest inclusion of artistic training in standard plastic surgery education [1, 2, 5, 20]. These plas- tic surgeons believe that artistic training leads to a better understanding of the concepts of symmetry, proportion, and dimensionality and skills such as anatomic drawing and hand–eye coordination [1, 2, 5–8, 20–22]. Two stud- ies specifically investigated the effect of art education on plastic surgery training. In 1969, Craig Gosling, an artist in the Department of Medical Illustration at Indiana Univer- sity, designed an art course for plastic surgery trainees that included 16 h of drawing, clay modeling, and molding and casting [1]. He and Lewis Thompson, Assistant Director of the Plastic Surgery Section at that same university, found marked improvement in trainees’ concepts of human sym- metry and proportion and better descriptions of patients and communication about surgical techniques. In 2005, another plastic surgeon, Ethem Guneron, emphasized the importance of art education within the field of plastic surgery because of the joint emphasis on “proportion, line, balance, harmony” [21]. With the help of the Department of Art Education, he designed an art course for plastic surgery trainees at Ondokuz Mayis University in Turkey [21]. Ways of thinking Using a methodologically rigorous approach, we have con- structed a model to illustrate how art and plastic surgery share concepts and skills. Our model incorporates data from interviews with practicing visual artists in addition to the perspectives of plastic surgeons. This model is a framework that underpins the work and practices of plastic surgeons and artists and illustrates how they are intimately connected. Our discussion will review the similarity and differences with existing literature, new insights, and impact on curriculum. Finally, these ways were all related to ways of thinking which involved critical thinking and creativity. Both artists and surgeons had repeatedly stressed the importance of these two concepts in their interviews. From analysis of the coded transcripts, we defined critical thinking as synonymous with problem or puzzle solving along with finding creative solu- tions. It involved constant self-assessment and self-criticism. Both artists and plastic surgeons had an intense inner dialog often involving the question, “Why is it this way?”. The goal of critical thinking was to innovate, create, improve and understand the self and the work. We defined creativity as pushing the limits or boundaries, thinking outside the box, and being original or new by going beyond the normal, average or expected. Creativity was also closely linked with critical thinking and was born from it. In exploring creativ- ity, we also found a tension between being technically skilled while maintaining innovation. We discovered a push and pull between the technical and the creative and how each comes Our data are consistent with the current literature that report how these two fields are thought to share similar concepts, such as aesthetics and proportion, and skills, such as anatomic drawing and manipulation of physical objects, required for occupational mastery [2, 6–9]. We add to these similarities by expanding on these foundational concepts and include ways of doing and ways of knowing. We also go beyond the physical skills by incorporating the ways of seeing to which both artists and plastic surgeons subscribe. Finally, we identified ways of thinking and specified two key 1 3 Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 9 Page 8 of 9 9 Page 8 of 9 9 components of this: critical thinking and creativity. Critical thinking for these participants is synonymous with prob- lem-solving and involves self-assessment. Competing interests The authors have no other financial disclosures or competing interests. 15. Creswell JW. Qualitative Inquiry and Research Design: Choosing Among Five Approaches. 3rd ed. Thousand Oaks, CA: SAGE Publications; 2013. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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/. 16. Brinkmann S, Kvale S. InterViews: Learning the craft of qualita- tive research interviewing. 3rd ed. Sage; 2015. 17. Maxwell JA. Chapter 5: Methods. In: Qualitative Research Design: An Interactive Approach. 3rd ed. Sage; 2013:87–120. 18. Elements of Art. In: Understanding Formal Analysis. Education: The J. Paul Getty Museum. https://www.getty.edu/education/ teachers/building_lessons/elements_art.pdf. Accessed February 24, 2022. 19. Principles of Design. In: Understanding Formal Analysis. Edu- cation: The J. Paul Getty Museum. https://www.getty.edu/educa tion/teachers/building_lessons/principles_design.pdf. Accessed February 24, 2022. 20. Fernandes JW, Metka S. Art and Plastic Surgery. Aesth Plast Surg. 2016;40(2):331–3. https://doi.org/10.1007/s00266-016-0616-y. Conclusion 7. Eng JS. Can aesthetic surgeons be true artists? Aesth Plast Surg. 2008;33(2):137–8. https://doi.org/10.1007/s00266-008-9291-y. 7. Eng JS. Can aesthetic surgeons be true artists? Aesth Plast Surg. 2008;33(2):137–8. https://doi.org/10.1007/s00266-008-9291-y. 2008;33(2):137–8. https://doi.org/10.1007/s00266-008-9291-y. 8. Sepehripour S, Patel AJK. Art, artistry, and plastic surgery. Plast Reconstr Surg. 2012;130(4):638e–40e. https://doi.org/10.1097/ prs.0b013e318262f646. This study provides a model for how plastic surgery and art overlap using data from interviews. Though there are differ- ences between the two fields, the ways of doing, knowing, seeing, and thinking are key components of the artistic and surgical processes. This model of “Ways of Making” may offer guidance for educators in plastic surgery to enhance curricula. p 9. Kirkland KB, Craig SR. Exploring the surgical gaze through lit- erature and art. JAMA. 2018;319(15):1532–4. https://doi.org/10. 1001/jama.2018.0396. 10. Zimmerman M. Art and Medicine at PAFA. Pennsylvania Acad- emy of Fine Arts. https://www.pafa.org/adult-programs/art-and- medicine-programs. Accessed November 30, 2018. 11. Lesser C. Why Med Schools Are Requiring Art Classes. Artsy. https://www.artsy.net/article/artsy-editorial-med-schools-requi ring-art-classes. Accessed December 1, 2018. Acknowledgements This research is funded by the Plastic Surgery Foundation (Grant #627051) through the Association for Surgical Edu- cation/Plastic Surgery Foundation Combined Research Grant. 12. Eno C, Correa R, Stewart NH, Lim J, Holmboe ES, Edgar L (2022) The Milestones Guidebook. Accreditation Council of Graduate Medical Education. 2020. https://www.acgme.org/globa lassets/pdfs/milestones/milestonesguidebookforresidentsfellows. pdp. Accessed May 19, 2022. Funding This research is funded by the Plastic Surgery Foundation (Grant #627051) through the Association for Surgical Education/Plas- tic Surgery Foundation Combined Research Grant. 13. Plastic Surgery Milestones: The Accreditation Council of Gradu- ate Medical Education. Implementation July 2022. https://www. acgme.org/globalassets/pdfs/milestones/plasticsurgerymilest ones2.0.pdfAccessed May 19, 2022. Data availability The datasets generated during and/or analyzed dur- ing the current study are available from the corresponding author on reasonable request. 14. ACGME Program Requirements for Graduate Medical Education in Plastic Surgery (Integrated and Independent). Effective July 1, 2020. https://www.acgme.org/globalassets/PFAssets/ProgramReq uirements/360-362_PlasticSurgery_2020.pdf?ver=2020-06-29- 163732-893&ver=2020-06-29-163732-893 Accessed May 19, 2022. Ways of thinking However, many participants in both groups did receive training in classes or programs across the country. Another limitation is that the interviewers were untrained in the visual arts and thus potentially probed differently than experienced artists would. This may have been an asset since this helped in the development of a model with less bias. Further work is needed to explore how the concept model is relevant to other disciplines such as general sur- gery and head and neck surgery. Both studies could benefit from the model we have pre- sented by aligning their curricular goals with the levels on the model and ensuring that the shared concepts of the model are incorporated. For example, the courses could 3 3 Page 9 of 9 Global Surgical Education - Journal of the Association for Surgical Education (2023) 2:9 9 Declarations Competing interests The authors have no other financial disclosures or competing interests. References 21. Guneron E, Kivrak N, Koyuncu S, Tuncer S, Uysal A. Aesthetic surgery training: The role of art education. Aesthetic Surg J. 2005;25(1):84–6. https://doi.org/10.1016/j.asj.2004.10.003.i 1. Thompson LW, Gosling CG, Epstein LI, Zook EG. A sim- plified art course for the plastic surgeon. Plast Reconstr Surg. 1972;49(4):408–10. 22. Dennhardt S, Apramian T, Lingard L, Torabi N, Arntfield S. Rethinking research in the medical humanities: a scoping review and narrative synthesis of quantitative outcome studies. Med Educ. 2016;50:285–99. https://doi.org/10.1111/medu.12812. 2. Swanson E. The plastic surgeon: artist or scientist? Plast Reconstr Surg. 2013;131(1):182–4. https://doi.org/10.1097/prs.0b013e3182 72a0f3. 3. Goldwyn RM. The plastic surgeon as an artist. Plast Reconstr Surg. 2003;112(1):327. https://doi.org/10.1097/01.prs.00000 66012.99511.ea. 23. Moniz, T., Golafshani M, Gaspar CM, Adams NE, et al. How Are the Arts and Humanities Used in Medical Education? Results of a Scoping Review. Acad Med. 2021. Publish Ahead of Print. https:// doi.org/10.1097/acm.0000000000004118 4. Colon GA. The plastic surgeon as an artist. Plast Reconstr Surg. 2004;113(5):1513–20. https://doi.org/10.1097/01.prs.00001 10764.50411.2d. 24. Moniz T, Golafshani M, Gaspar CM, Adams NE, et al. The prism model: advancing a theory of practice for arts and humanities in medical education. Perspectives Medical Educ. 2021;10:207–14. https://doi.org/10.1007/s40037-021-00661-0. 5. Morani AD. Plastic surgeons and art. Plast Reconstr Surg. 1979;64(4):550. 6. Fernandes JW. The legacy of art in plastic surgery. Plastic Recon- str Surg Global Open. 2021;9: e3519. https://doi.org/10.1097/gox. 0000000000003519. 6. Fernandes JW. The legacy of art in plastic surgery. Plastic Recon- str Surg Global Open. 2021;9: e3519. https://doi.org/10.1097/gox. 0000000000003519. 1 3
https://openalex.org/W2771317192	https://curis.ku.dk/ws/files/190731341/10.1007_2Fs00226_017_0977_7.pdf	English	null	Experimental techniques for characterising water in wood covering the range from dry to fully water-saturated	Wood science and technology	2,017	cc-by	20,601	Citation for published version (APA): Thybring, E. E., Kymäläinen, M., & Rautkari, L. (2018). Experimental techniques for characterising water in wood covering the range from dry to fully water-saturated. Wood Science and Technology, 52(2), 297-329. https://doi.org/10.1007/s00226-017-0977-7 university of copenhagen university of copenhagen Experimental techniques for characterising water in wood covering the range from dry to fully water-saturated Thybring, Emil Engelund; Kymäläinen, Maija; Rautkari, Lauri university of copenhagen Experimental techniques for characterising water in wood covering the range from dry to fully water-saturated Thybring, Emil Engelund; Kymäläinen, Maija; Rautkari, Lauri Download date: 24. Oct. 2024 Thybring, Emil Engelund; Kymäläinen, Maija; Rautkari, Lauri Published in: Wood Science and Technology Published in: Wood Science and Technology DOI: 10.1007/s00226-017-0977-7 Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Document license: CC BY Citation for published version (APA): Thybring, E. E., Kymäläinen, M., & Rautkari, L. (2018). Experimental techniques for characterising water in wood covering the range from dry to fully water-saturated. Wood Science and Technology, 52(2), 297-329. https://doi.org/10.1007/s00226-017-0977-7 Download date: 24. Oct. 2024 Wood Sci Technol (2018) 52:297–329 https://doi.org/10.1007/s00226-017-0977-7 ORIGINAL ORIGINAL Experimental techniques for characterising water in wood covering the range from dry to fully water- saturated Emil Engelund Thybring1 • Maija Kyma¨la¨inen2 • Lauri Rautkari2 Received: 14 February 2017 / Published online: 4 December 2017 The Author(s) 2017. This article is an open access publication Abstract Water plays a central role in wood research, since it affects all material properties relevant to the performance of wood materials. Therefore, experimental techniques for characterising water within wood are an essential part of nearly all scientiﬁc investigations of wood materials. This review focuses on selected experimental techniques that can give deeper insights into various aspects of water in wood in the entire moisture domain from dry to fully water-saturated. These techniques fall into three broad categories: (1) gravimetric techniques that deter- mine how much water is absorbed, (2) ﬁbre saturation techniques that determine the amount of water within cell walls, and (3) spectroscopic techniques that provide insights into chemical wood–water interactions as well as yield information on water distribution in the macro-void wood structure. For all techniques, the general measurement concept is explained, its history in wood science as well as advantages and limitations. 1 Department of Geosciences and Natural Resource Management, University of Copenhagen, Rolighedsvej 23, 1958 Frederiksberg C, Denmark 2 Department of Bioproducts and Biosystems, Aalto University, PO BOX 6300, 00076 Aalto, Finland & Emil Engelund Thybring eet@ign.ku.dk 2 Department of Bioproducts and Biosystems, Aalto University, PO BOX 6300, 00076 Aalto, Finland Introduction Wood is a hygroscopic material which means that it absorbs and exchanges water molecules with the surroundings. Water molecules found within cell walls interfere with internal secondary wood–wood bonds, for example hydrogen bonds. Therefore, it is no surprise that absorbed water, often termed moisture, has a profound effect on 12 123 123 Wood Sci Technol (2018) 52:297–329 298 wood properties dependent on these bonds, for example strength and stiffness (Tiemann 1906; Wagner et al. 2015). However, moisture also inﬂuences the performance of wood and wood products in other ways important for the structural application of these materials such as durability, i.e. resistance to biodegradation (Ammer 1963a; Meyer and Brischke 2015; Stamm and Baechler 1960; Stienen et al. 2014; Thybring 2013). The central role of moisture in the performance of wood materials makes the wood–water interaction an obvious target for enhancing material performance to speciﬁc applications. Thus, a wide range of physicochem- ical modiﬁcation processes exist aimed at improving the performance of wood materials. Common targets for wood modiﬁcation include improved durability and dimensional stability (Stamm and Baechler 1960). However, recent years have seen an increase in advanced modiﬁcations adding novel functionalities to wood such as stimuli-responsive properties, for example ‘‘sweating wood’’ for cooling purposes or stimuli-responsive ﬁltration (Keplinger et al. 2016). For all these types of modiﬁcations, wood–water interactions within the modiﬁed material are important for the performance. For instance, the resistance to biological decay is directly linked to the water capacity of modiﬁed cell walls (Stamm and Baechler 1960; Thybring 2013), while advanced modiﬁcations such as stimuli-responsive hydrogel formation within the wood structure use water uptake or release for cooling purposes by water evaporation or for ﬁltration (Keplinger et al. 2016). To advance the understanding of the fundamental mechanisms of wood performance as affected by water as well as for tailoring wood modiﬁcations to speciﬁc applications, characterisation of the amount, distribution, and physicochemical interactions of water within wood materials is important. This review covers selected experimental techniques for characterising moisture in wood in the entire moisture content range from absolutely dry to fully water- saturated, i.e. where water takes up all available space in cell walls and voids (pits, lumina, vessels) in the wood structure. Furthermore, techniques which elucidate other fundamental aspects of wood–water interactions in untreated and modiﬁed wood are included. Experimental techniques which concern effects of water on, for example, mechanics or dimensions will not be discussed. Introduction The schematic sorption isotherm shown in Fig. 1 covering the entire relative humidity (RH) range provides an overview of the characterisation techniques covered in this review as well as the RH range to which each technique can be applied. Terminology One of the most fundamental and commonly used descriptors for moisture in wood is the moisture content (MC). In this review, it is described by water mass divided by dry wood mass. When dealing with physicochemically modiﬁed wood materials, it is often necessary to adjust MC for added dry mass due to the modiﬁcation process (Hill 2008; Thybring 2013) in order to correctly compare a given modiﬁed material with untreated or differently modiﬁed materials. This is done by multiplying MC of the modiﬁed material, MCmod [%], with (1 ? Rmod) as shown in (1): 123 299 Wood Sci Technol (2018) 52:297–329 Fig. 1 Schematic illustration of moisture uptake in wood in absorption from absolute dry state (lower black line) and desorption from fully water-saturated state (upper black line) in the entire relative humidity (RH) range from dry to water-saturated. Characterisation (gravimetric, ﬁbre saturation, and spectroscopic) techniques described in the present review are indicated along with the RH range they cover Fig. 1 Schematic illustration of moisture uptake in wood in absorption from absolute dry state (lower black line) and desorption from fully water-saturated state (upper black line) in the entire relative humidity (RH) range from dry to water-saturated. Characterisation (gravimetric, ﬁbre saturation, and spectroscopic) techniques described in the present review are indicated along with the RH range they cover Fig. 1 Schematic illustration of moisture uptake in wood in absorption from absolute dry state (lower black line) and desorption from fully water-saturated state (upper black line) in the entire relative humidity (RH) range from dry to water-saturated. Characterisation (gravimetric, ﬁbre saturation, and spectroscopic) techniques described in the present review are indicated along with the RH range they cover MCR ¼ mwater mwood;dry ¼ mwater mmod;dry 1 þ Rmod ð Þ1 ¼ MCmod 1 þ Rmod ð Þ ð1Þ ð1Þ where MCR [%] is adjusted moisture content also termed ‘‘reduced moisture con- tent’’, mwater [kg] is water mass, and mwood,dry [kg] and mmod,dry [kg] are dry masses of the wood itself and the modiﬁed wood material, respectively. Terminology Rmod [-] is the modiﬁcation ratio deﬁned as relative mass gain due to modiﬁcation Rmod ¼ mmod;dry mwood;dry mwood;dry ð2Þ ð2Þ Mass change is commonly used in the literature to describe the intensity of modi- ﬁcation either by the term ‘‘weight per cent gain’’ (WPG) [% WPG] in cases of increased mass due to modiﬁcation, for example acetylation, furfurylation, etc., or by the term ‘‘mass loss’’ (ML) [% ML] in cases of decreased mass due to modiﬁ- cation, for example thermal modiﬁcation. It was chosen to describe the mass change by the common term ‘‘modiﬁcation ratio’’ (Rmod) and deﬁne it as a dimensionless fraction to avoid confusion with the relative mass gain given in per cent (WPG). Correction of MC in (1) and following equations should, however, only be done in cases where Rmod is positive. Thus, MC should not be corrected for thermally modiﬁed wood where Rmod \ 0. As water molecules absorbed by wood are continuously exchanged with the surroundings as well as meandering around within the material, MC is often characterised in the equilibrium state where there is no net change in water with the surroundings. This alleviates issues of comparing various samples with differences in moisture gradients depending on sample geometry, wood density, etc. In case of equilibrium, it is common to use the terms ‘‘equilibrium moisture content’’ (EMC) 12 3 3 3 300 Wood Sci Technol (2018) 52:297–329 and ‘‘reduced equilibrium moisture content’’ (EMCR) in (1) instead of MC and MCR, respectively. In this review, the term ‘‘absorption’’ is used to describe the process of moisture uptake in wood instead of the term ‘‘adsorption’’ more commonly used in the wood research literature. The IUPAC deﬁnition of ‘‘absorption’’ relates to the uptake of one material by another, regardless of the mechanism of uptake. On the other hand, the term ‘‘adsorption’’ in the terminology suggested by IUPAC relates to the uptake of one material on surfaces/interphases with another material and thus implies that water molecules attach to surfaces within the wood structure. As a result of the developments in the ﬁeld of water uptake in wood cell walls in the last few decades, it was felt that the term ‘‘adsorption’’ is misleading as it relates to ‘‘internal surfaces’’, a concept which can be questioned when water molecules enter the solid wood cell walls and are taken up by the constituent polymers. Gravimetric techniques Wood can absorb signiﬁcant amounts of water in its structure, i.e. both within cell walls and in the void structure. The maximum amount depends on the wood density, which varies widely between species. As an example, a low-density wood species like balsa (Ochroma lagopus Sw.) with a bulk density around 100 kg/m3 can ﬁt a total amount of water of around 960–980 kg/m3 in its bulk volume, equal to a maximum MC of 960–980%. On the other hand, a high-density wood species such as ironwood (Lophira alata Banks ex. Gaertn.) with a bulk density around 1000 kg/ m3 can only ﬁt about 630–780 kg/m3 water inside its bulk volume, yielding a maximum MC of just 63–78%. The noticeable change in mass associated with absorption of water makes gravimetric techniques ideal for determining the amount of water inside the bulk material. Terminology Therefore, the more general term ‘‘absorption’’ was adopted to include both water uptake in cell walls and capillary condensation in the wood structure. This review covers the entire moisture range from dry to fully water-saturated in dependence of the surrounding climate. In line with common terminology in building materials research, the full RH range is divided into a hygroscopic and an over-hygroscopic range (Fig. 1). These two regimes intersect around 97–98% RH although the limiting RH is not well deﬁned (Espinosa and Franke 2006). The differentiation reﬂects a change in water uptake in the material, where the hygroscopic range is dominated by cell wall absorption and the over-hygroscopic range is dominated by capillary condensation outside cell walls, i.e. in pits, lumina, vessels, and other voids in the wood structure. Early-stage sorption balances, salt solutions, and climate chambers In the early scientiﬁc investigations of wood materials, it is recognised that the MC of wood exposed to climatic conditions changes with changes in temperature and RH (Forest Products Laboratory 1919; Glass et al. 2014; Zeller 1920). In the early days of wood research, it was, however, a common practice to achieve a speciﬁc 123 301 Wood Sci Technol (2018) 52:297–329 MC in wood samples by drying the material from green or water-saturated conditions (Carrington 1922; Record 1914; Tiemann 1906; Volbehr 1896) and then maybe leaving the wood for moisture to redistribute within the material before further testing, for example mechanical. Alternatively, wood samples were kept under laboratory conditions for a period of time to obtain an MC around 12% (Carrington 1921). The ﬁrst published study in a scientiﬁc journal on wood MC in equilibrium with a controlled, stable climate appears to be the work of Zeller (1920), who used sulphuric acid solutions of various concentrations to generate various levels of RH in a specially designed climate chamber. Similar investigations performed at the Forest Products Laboratory in the USA might, however, predate this publication (Glass et al. 2014). In these early investigations, the now well- known effect of sorption history, i.e. that conditioning wood to a speciﬁc RH through moisture uptake (absorption) or through moisture release (desorption) will not produce similar MC, was not considered. Therefore, the relation between wood MC and RH at constant temperature was described by a single curve, i.e. sorption isotherm. The seemingly ﬁrst absorption and desorption isotherms were reported by Larian and co-workers (Larian et al. 1930; Lavine and Gauger 1930) who conditioned birch shavings (about 1.5 g each) in desiccators over saturated salt solutions generating different RH levels. Initially, all samples were in the green condition, and after equilibrium with the speciﬁc RH had been obtained, they were dried over pure sulphuric acid (0% RH) followed by conditioning by absorption to the same level of RH as before. These data can therefore be regarded as the ﬁrst desorption and absorption isotherms for wood, where the previous is initiated from a condition close to fully water-saturated. The same year, Pidgeon and Maass (1930) reported on absorption and desorption isotherms for white spruce and jack pine found by exposing small samples (150 mg) suspended from a quartz helix spring inside a vacuum tube system to various levels of RH. Early-stage sorption balances, salt solutions, and climate chambers Desorption was, however, initiated from hygroscopic conditions (\ 98% RH), the effect of which will be discussed later. RH in the vacuum tube system was controlled by tuning the vapour pressure above a water bath via controlling the bath temperature while keeping the rest of the system at constant temperature. Moisture uptake was measured by the spring elongation, and the system is a precursor to modern-day automated sorption balances. Several later studies on wood use this kind of technique for studying absorption and desorption in wood (Christensen 1959, 1960; Christensen and Kelsey 1959; Grace and Maass 1932; Kelly and Hart 1970; Spalt 1958). For modiﬁed wood, the ﬁrst sorption isotherm appears to be due to Mo¨rath (1931) who steamed beech at 150 and 190 C and used sulphuric acid solutions for conditioning the material at different RH levels. The second reported sorption isotherm for modiﬁed wood seems to be the absorption and desorption isotherms reported by Spalt (1958) for acetylated (Rmod = 0.32) white pine, although studies from the intervening period of time mention reduced hygroscopicities of modiﬁed wood (Buro 1954; Seborg et al. 1953) without showing sorption isotherms. Of the different methods for generating speciﬁc RH levels detailed above, one of MC in wood samples by drying the material from green or water-saturated conditions (Carrington 1922; Record 1914; Tiemann 1906; Volbehr 1896) and then maybe leaving the wood for moisture to redistribute within the material before further testing, for example mechanical. Alternatively, wood samples were kept under laboratory conditions for a period of time to obtain an MC around 12% (Carrington 1921). The ﬁrst published study in a scientiﬁc journal on wood MC in equilibrium with a controlled, stable climate appears to be the work of Zeller (1920), who used sulphuric acid solutions of various concentrations to generate various levels of RH in a specially designed climate chamber. Similar investigations performed at the Forest Products Laboratory in the USA might, however, predate this publication (Glass et al. 2014). In these early investigations, the now well- known effect of sorption history, i.e. that conditioning wood to a speciﬁc RH through moisture uptake (absorption) or through moisture release (desorption) will not produce similar MC, was not considered. Therefore, the relation between wood MC and RH at constant temperature was described by a single curve, i.e. sorption isotherm. Early-stage sorption balances, salt solutions, and climate chambers The seemingly ﬁrst absorption and desorption isotherms were reported by Larian and co-workers (Larian et al. 1930; Lavine and Gauger 1930) who conditioned birch shavings (about 1.5 g each) in desiccators over saturated salt solutions generating different RH levels. Initially, all samples were in the green condition, and after equilibrium with the speciﬁc RH had been obtained, they were dried over pure sulphuric acid (0% RH) followed by conditioning by absorption to the same level of RH as before. These data can therefore be regarded as the ﬁrst desorption and absorption isotherms for wood, where the previous is initiated from a condition close to fully water-saturated. The same year, Pidgeon and Maass (1930) reported on absorption and desorption isotherms for white spruce and jack pine found by exposing small samples (150 mg) suspended from a quartz helix spring inside a vacuum tube system to various levels of RH. Desorption was, however, initiated from hygroscopic conditions (\ 98% RH), the effect of which will be discussed later. RH in the vacuum tube system was controlled by tuning the vapour pressure above a water bath via controlling the bath temperature while keeping the rest of the system at constant temperature. Moisture uptake was measured by the spring elongation, and the system is a precursor to modern-day automated sorption balances. Several later studies on wood use this kind of technique for studying absorption and desorption in wood (Christensen 1959, 1960; Christensen and Kelsey 1959; Grace and Maass 1932; Kelly and Hart 1970; Spalt 1958). For modiﬁed wood, the ﬁrst sorption isotherm appears to be due to Mo¨rath (1931) who steamed beech at 150 and 190 C and used sulphuric acid solutions for conditioning the material at different RH levels. The second reported sorption isotherm for modiﬁed wood seems to be the absorption and desorption isotherms reported by Spalt (1958) for acetylated (Rmod = 0.32) white pine, although studies from the intervening period of time mention reduced hygroscopicities of modiﬁed wood (Buro 1954; Seborg et al. 1953) without showing sorption isotherms. Of the different methods for generating speciﬁc RH levels detailed above, one of Of the different methods for generating speciﬁc RH levels detailed above, one of the most common methods is by saturated salt solutions (Ammer 1963b; Eriksson and Noren 1965; Forster 1998; Higgins 1957; Nearn 1955; Papadopoulos and Hill 2003; Wangaard and Granados 1967; Yasuda et al. Early-stage sorption balances, salt solutions, and climate chambers 2002; Meyer and Brischke 2015; Williams and Hale 2003), but the resultant EMC after such conditioning is questionable. This is due to the great difﬁculty with obtaining saturated water vapour as discussed by Strømdahl (2000). Differences in temperature between vapour and surrounding surfaces will result in either water condensation on surfaces if these are cooler or a non-saturated vapour if the surfaces are warmer. Even slight temperature ﬂuctuations can give noticeable effects. Thus, ﬂuctuations of 0.5 C around room temperature cause a ‘‘saturated RH’’ around 97%, while even small changes of 0.2 C limit the maximum RH to 99%. As shown by Hoffmeyer et al. (2011), prolonged conditioning of untreated wood in such presumably saturated water vapour will not give the same isotherm upon subsequent desorption as when the material is desorbing from the fully water- saturated state. Moreover, as discussed later under ‘‘Fibre saturation techniques’’, it is highly uncertain that conditioning wood in presumably saturated water vapour will actually saturate cell walls. ) Whether using early-stage sorption balances, saturated salt solutions, or climate chambers for conditioning wooden samples, it is important to select appropriate sample masses taking into consideration the balance uncertainty and the stability of the conditioning climate in order to minimise the uncertainty in determined MC. The ASTM C1498 (ASTM 2016) and ISO 12571 (ISO 2013) standards which concern the determination of hygroscopic moisture sorption isotherms require the balance accuracy to be at least 0.01–0.1% of the sample mass. However, these standards also require a sample mass of 10 g or more, which is often not feasible or wanted in scientiﬁc studies, for example on sorption kinetics. To determine EMC, it is of course important that equilibrium has been obtained as marked by a constant sample mass after conditioning at constant RH for a given time. Many studies employ stability criteria adopted from the ASTM C1498 (ASTM 2016) and ISO 12571 (ISO 2013) standards to specify when the variation in sample mass is low enough for equilibrium to be assumed obtained. These standards require mass variations of \ 0.1% over a period of 5 days after daily mass readings or over 7 days in two consecutive mass readings, respectively. Other stability criteria have been proposed in the literature, for example the one by Wadso¨ et al. (2004) which also takes into account the change in RH from the previous to the current climate. Early-stage sorption balances, salt solutions, and climate chambers 1995) since they are relatively 12 3 Wood Sci Technol (2018) 52:297–329 302 simple to use and cover a wide range of RH levels (see, for example, Greenspan 1977) for solutions in the range 3–98% RH. Their use limits, however, which RH levels can be achieved. This problem is avoided when RH is generated by mixing of wet and dry air streams to produce any RH level desired in the range 0–97% RH. Such method has been employed in climate chambers (Strømdahl 2000; Thygesen et al. 2010) and has even be used below the freezing temperature of water (Hedlin 1968). 1968). Whether using early-stage sorption balances, saturated salt solutions, or climate chambers for conditioning wooden samples, it is important to select appropriate sample masses taking into consideration the balance uncertainty and the stability of the conditioning climate in order to minimise the uncertainty in determined MC. The ASTM C1498 (ASTM 2016) and ISO 12571 (ISO 2013) standards which concern the determination of hygroscopic moisture sorption isotherms require the balance accuracy to be at least 0.01–0.1% of the sample mass. However, these standards also require a sample mass of 10 g or more, which is often not feasible or wanted in scientiﬁc studies, for example on sorption kinetics. To determine EMC, it is of course important that equilibrium has been obtained as marked by a constant sample mass after conditioning at constant RH for a given time. Many studies employ stability criteria adopted from the ASTM C1498 (ASTM 2016) and ISO 12571 (ISO 2013) standards to specify when the variation in sample mass is low enough for equilibrium to be assumed obtained. These standards require mass variations of \ 0.1% over a period of 5 days after daily mass readings or over 7 days in two consecutive mass readings, respectively. Other stability criteria have been proposed in the literature, for example the one by Wadso¨ et al. (2004) which also takes into account the change in RH from the previous to the current climate. One important thing to note about the use of early-stage sorption balances, saturated salt solutions, and climate chambers is that these methods are unsuitable for conditioning wood samples to saturation. Nonetheless, several studies condition wood samples in desiccators over liquid water as a way of saturating the material (Boonstra et al. 2007; Feist and Sell 1987; Harju et al. 2002; Kamdem et al. Early-stage sorption balances, salt solutions, and climate chambers One important thing to note about the use of early-stage sorption balances, saturated salt solutions, and climate chambers is that these methods are unsuitable for conditioning wood samples to saturation. Nonetheless, several studies condition wood samples in desiccators over liquid water as a way of saturating the material (Boonstra et al. 2007; Feist and Sell 1987; Harju et al. 2002; Kamdem et al. 2002; Meyer and Brischke 2015; Williams and Hale 2003), but the resultant EMC after such conditioning is questionable. This is due to the great difﬁculty with obtaining saturated water vapour as discussed by Strømdahl (2000). Differences in temperature between vapour and surrounding surfaces will result in either water condensation on surfaces if these are cooler or a non-saturated vapour if the surfaces are warmer. Even slight temperature ﬂuctuations can give noticeable effects. Thus, ﬂuctuations of 0.5 C around room temperature cause a ‘‘saturated RH’’ around 97%, while even small changes of 0.2 C limit the maximum RH to 99%. As shown by Hoffmeyer et al. (2011), prolonged conditioning of untreated wood in such presumably saturated water vapour will not give the same isotherm upon subsequent desorption as when the material is desorbing from the fully water- saturated state. Moreover, as discussed later under ‘‘Fibre saturation techniques’’, it is highly uncertain that conditioning wood in presumably saturated water vapour will actually saturate cell walls. 123 123 Wood Sci Technol (2018) 52:297–329 303 Automated sorption balances This type of equipment, often referred to as dynamic vapour sorption (DVS), combines automated climate control in the range 0–97% RH with continuous high- resolution mass measurements. RH is generated by mixing wet and dry gas streams, for example air or nitrogen. As only a small sample mass (typically in the 5–20 mg range) is needed like in the quartz helix spring systems, a faster approach to equilibrium is observed compared with conditioning larger samples over saturated salt solutions or in climate chambers. As automated sorption balances are becoming a common research tool in wood research laboratories, the literature is abundant with sorption isotherms determined with such equipment, also for various types of modiﬁed wood (Hill et al. 2013; Jalaludin et al. 2010a, b; Popescu and Hill 2013; Xie et al. 2010, 2011). While the low sample mass makes possible detailed studies of differences in water sorption between earlywood and latewood (Fredriksson and Thygesen 2017), even at the growth ring level (Hill et al. 2015; Song et al. 2014), it also requires the user to consider how to collect representative samples for the study in question. For generating representative samples of larger wood volumes, several studies have used milled wood (Hill et al. 2009; Himmel and Mai 2015; Xie et al. 2010; Zaihan et al. 2009, 2011), but it could be speculated if this gives a representative material or might produce unwanted side effects, for example a change in sample chemistry. Milling is not as intense a method for subdividing wood as ball milling which has been shown to change the material chemistry (Mao et al. 2006) and internal chemical bonding (Schwanninger et al. 2004). Nonetheless, different particle size fractions generated from milling might not have identical chemical composition if some anatomical fractions (e.g. lignin-rich middle lamella fragments) are over-represented in smaller size fractions that are more easily lost upon handling the milled material. Instead of milling, several studies have used wood slices cut with a microtome or razor blade to produce a large sample surface area while preserving the microscopic wood structure (Fredriksson and Thygesen 2017; Fredriksson et al. 2010; Glass et al. 2017; Hill et al. 2015; Hosseinpourpia et al. 2016; Song et al. 2014), while a few use small wood blocks (Fredriksson and Johansson 2016). Automated sorption balances All of these studies use the automated humidity control by which RH is automatically changed upon reaching a speciﬁed mass stability (dm/dt) criterion. If such dm/dt criterion is not selected carefully, however, it can lead to inaccurate results. If the stability criteria of the ASTM C1498 (ASTM 2016) and ISO 12571 (ISO 2013) standards are converted into %/min, i.e. per cent mass change per minute, employed in most automated sorption balances, the dm/dt is around 0.00001%/min. While the stability criteria of the standards are based on mass change relative to the previously observed mass, the dm/dt of automated sorption balances is typically based on mass change relative to the dry sample mass which is often determined by initial drying. Thus, it would seem that the latter gives stricter stability requirements. However, the dm/dt suggested by the producer of one of the most common types of automated sorption balances is 0.002%/min for 10–20 mg samples and 0.0005%/min for 100 mg samples, i.e. about 50–200 times higher than required by the standards for larger ([ 10 g) samples. The other important parameter for establishing if equilibrium is obtained is the ‘‘stability 12 3 3 304 Wood Sci Technol (2018) 52:297–329 window’’, i.e. the time period over which the dm/dt needs to meet the stability criterion. In the ASTM C1498 (ASTM 2016) and ISO 12571 (ISO 2013) standards, the mass change is calculated over 5 and 7 days, respectively, whereas the default stability window in the most popular automated sorption balance in wood research is 15 min. The 0.002%/min over 15-min stability criterion is commonly employed for wood materials, but Glass et al. (2017) recently demonstrated that it gives too low EMC values in absorption and too high in desorption. The error was up to 0.7% MC for southern pine wood and up to 2.9% MC for isolated lignin for changes in the high-RH regime. Moreover, Glass et al. (2017) were unable to ﬁnd literature data to support the claim that a dm/dt of 0.002%/min is sufﬁcient for accurate determinations of EMC, even in publications often cited to document this claim. The use of a dm/dt criterion becomes critical when considering sample drying which in many studies is performed initially. The results of Christensen and Hergt (1969) show that the longer the time under constant climate before a given change in RH, the slower the rate of sorption. Automated sorption balances Thus, if a sample is initially dried out using a dm/dt criterion after experiencing stable conditions for a long while, it will exhibit a slower rate of desorption than in a subsequent second drying stage after re-wetting. This will affect the determined dry mass in the two drying stages as the dm/dt criterion is met at higher residual MC in the ﬁrst stage, thus causing an overestimation of the dry mass. Examining the mass curves in the literature when a dm/dt criterion of 0.002%/min is used, it is often clearly seen that the sample material is far from having reached equilibrium with 0% RH before subsequent re- wetting is initiated. The overestimated dry mass is problematic as it shifts the entire sorption isotherm downwards, partly due to the increased dry mass but mainly due to decreased estimated water masses. Moreover, a second drying stage will yield a lower dry mass, perhaps leading to speculations about ‘‘trapped water’’ in the ﬁrst drying stage (Hill et al. 2015). It is recognised that Hergt and Christensen (1965) did ﬁnd that up to 1% MC is retained in the wood after vacuum-drying if the material is not rapidly dried from the water-saturated state, i.e. conditioning to any level of RH before drying results in incomplete drying even at a drying temperature of 65 C. However, their experiments used thin, microtomed wood sections and drying times of 1–5 days which is considerably longer than the drying duration if using a dm/dt criterion of 0.002%/min. In the authors’ experience, drying for 6 h at 60 C in dry nitrogen gas of a 20-mg wood block sample gives a reproducible dry mass within 0.05% even when several cycles of re-wetting to various RH (10–90%) and drying are used. For samples cut into thin slices, this drying protocol (6 h at 60 C, 0% RH) should be more than enough to secure complete drying. Therefore, it is recommended that longer, ﬁxed drying times, preferably at slightly elevated temperatures, are used in automated sorption balances in order to secure accurate dry mass determinations. For getting closer to the ‘‘true’’ EMC upon conditioning at a speciﬁc RH, it might be useful to lower the dm/dt as well as increase the stability window. However, further investigations are needed for establishing guidelines for the appropriate dm/dt settings for wood. Automated sorption balances Until such have been established, a degree of uncertainty exists as to the accuracy of reported EMC and sorption isotherms by 123 123 Wood Sci Technol (2018) 52:297–329 305 The difference in MC in equilibrium with a given RH depends on whether equilibrium is reached by absorption or desorption (Urquhart 1929). This phenomenon is termed sorption hysteresis and can be seen as a difference between the absorption isotherm from dry state and desorption isotherm from water-saturated state (see Fig. 2). It has been speculated that it is linked to non-elastic mechanical responses during swelling, but the exact mechanism is not yet understood (Engelund et al. 2013). Nonetheless, wood modiﬁcation can have different effects on sorption hysteresis. For instance, acetylation decreases the absolute hysteresis (Popescu et al. 2014), i.e. MC difference between the absorption and desorption isotherms, but this result appears to be correlated with the general reduction in MC at given RH. A similar general reduction in MC is found for thermal modiﬁcation of wood; however, this treatment appears to increase the absolute sorption hysteresis (Olek et al. 2013). Automated sorption balances are useful for studies of sorption hysteresis, and several published studies have focused on this phenomenon. In most studies, absolute sorption hysteresis has a maximum around 70–75% RH and decreases for higher RH (Hill et al. 2009, 2012b; Himmel and Mai 2015; Hosseinpourpia et al. 2016; Popescu et al. 2014; Simon et al. 2017; Xie et al. 2011). The decreasing sorption hysteresis at high RH might, however, be an artefact due to desorption being initiated from a non-saturated state (often by conditioning to 95% RH). Thus, in recent results by Fredriksson and co-workers (Fredriksson and Johansson 2016; Fredriksson and Thygesen 2017), sorption hysteresis in untreated Norway spruce gradually increases up to at least 95% RH (see Fig. 2). Furthermore, as shown by Hoffmeyer et al. (2011), desorption from a non-saturated state will yield a lower desorption isotherm than for water-saturated wood until around 75% RH. Therefore, most desorption isotherms using automated sorption balances reported in the literature can be regarded as scanning curves above 70–75% RH Fig. 2 a Sorption isotherms for desorption from water-saturated state and absorption from dry state for untreated and acetylated (Rmod = 0.18) Norway spruce in the range of 0–97% relative humidity (RH). Based on data from Fredriksson and Johansson (2016), Fredriksson et al. Automated sorption balances (2013) at 20 C and from authors at 25 C (dm/dt = 0.0005%/min). Note that the desorption and absorption isotherms do not coalesce at 97% RH; b absolute sorption hysteresis of untreated wood from moisture content data acquired with automated sorption balances. Based on data for Norway spruce (Fredriksson and Johansson 2016; Fredriksson and Thygesen 2017; Ka¨llbom 2015; Sedighi-Gilani and Schwarze 2015) and Sitka spruce (Hill et al. 2009). Note that in all studies except (Fredriksson and Johansson 2016; Fredriksson and Thygesen 2017) desorption is initiated from a non-saturated state (conditioning to 95% RH) Fig. 2 a Sorption isotherms for desorption from water-saturated state and absorption from dry state for untreated and acetylated (Rmod = 0.18) Norway spruce in the range of 0–97% relative humidity (RH). Based on data from Fredriksson and Johansson (2016), Fredriksson et al. (2013) at 20 C and from authors at 25 C (dm/dt = 0.0005%/min). Note that the desorption and absorption isotherms do not coalesce at 97% RH; b absolute sorption hysteresis of untreated wood from moisture content data acquired with automated sorption balances. Based on data for Norway spruce (Fredriksson and Johansson 2016; Fredriksson and Thygesen 2017; Ka¨llbom 2015; Sedighi-Gilani and Schwarze 2015) and Sitka spruce (Hill et al. 2009). Note that in all studies except (Fredriksson and Johansson 2016; Fredriksson and Thygesen 2017) desorption is initiated from a non-saturated state (conditioning to 95% RH) 12 3 3 306 Wood Sci Technol (2018) 52:297–329 produced by a reversal in sorption direction (absorption/desorption) at intermediate RH, i.e. away from the dry or saturated states. Besides deriving MC in equilibrium with various climates, automated sorption balances have been used for studying the kinetics of sorption. In many polymeric materials, the sorption process exhibits two-stage behaviour (Christensen and Kelsey 1959; Crank 1953; Kelly and Hart 1970; Newns 1956) and cannot be explained by Fickian diffusion into the material. Thus, sorption is often a much slower process than expected for diffusion through the solid material which is also the case for wood (Christensen 1959; Kelly and Hart 1970). Instead, sorption might be controlled by relaxation of stresses arising from swelling of the dense and stiff wood cell walls (Christensen 1959, 1960; Christensen and Kelsey 1959). This also explains why samples conditioned for longer times after a change in RH exhibit slower rate of sorption as observed by Christensen and Hergt (1969). Automated sorption balances Due to the two-stage nature of the sorption process, it has often been analysed by the parallel exponential kinetics (PEK) model featuring two moisture sorption processes (fast and slow exponentials) using data for both untreated (Hill et al. 2010) and modiﬁed wood (Hill et al. 2012a; Himmel and Mai 2016; Jalaludin et al. 2010a; Popescu et al. 2014; Xie et al. 2010). It may be difﬁcult to compare the results across studies due to differences in sample mass and geometry as diffusion into the sample at some point becomes relevant, at least in the low-RH regime (Christensen 1965). More importantly, however, the recent results by Glass et al. (2017) demonstrate that the PEK model is not capable of capturing the long-term kinetics of untreated wood if the data acquisition at a given RH is disrupted when a dm/dt stability criterion of 0.002%/min is met. This seriously questions whether results derived using PEK model ﬁttings on sorption data with a 0.002%/min dm/dt criterion are correct. Adding to this uncertainty, the PEK model is quite sensitive to initial guesses of parameter values of the two moisture sorption processes (Himmel and Mai 2016). More details about wood–water interactions can be gained from automated sorption balances using heavy water, i.e. deuterium oxide (2H2O or D2O), instead of normal water (1H2O or H2O) as conditioning vapour. Hereby, hydrogen of the hydroxyl (OH) groups interacting with heavy water will be exchanged for deuterium if the material is continuously exposed to 2H2O vapour for sufﬁcient time. As deuterium (2H) is about 1 g/mol heavier than protium (1H), the material dry mass increases slightly upon deuteration (* 1% for untreated wood), which can only be measured with enough accuracy using very sensitive balances. Therefore, vapour- phase deuteration has been used on untreated wood in an early-stage sorption balance (Taniguchi et al. 1978) as well as recently on untreated (Thybring et al. 2017) and modiﬁed wood (Popescu et al. 2014; Rautkari et al. 2013) using modern automated sorption balances, yielding information about how many hydroxyls are accessible for water. It should be noted, however, that not all accessible hydroxyls will have their hydrogen exchanged. For instance, Lindh et al. (2016) found that only two out of three surface hydroxyls on cellulose microﬁbrils are accessible for deuteration as O(3)H hydroxyls act solely as hydrogen bond acceptors, i.e. interacting exclusively with water via the O(3) (Lindh et al. 2016). Automated sorption balances Therefore, if assuming a conservative estimate of 50% higher concentration of heavy water than of normal water after the drying stage, the measurement errors in determined hydroxyl accessibility for residual water contents of 1 and 0.1% MC would be 66–133 and 7–13%, respectively. This clearly demonstrates the need for sufﬁcient drying of the material to eliminate residual water as a signiﬁcant source of error. Therefore, the use of a dm/dt stability criterion for determining hydroxyl accessibility is not rec- ommended due to the uncertainties it involves as discussed previously for gener- ating sorption isotherm data. As mass differences involved in the determination of hydroxyl accessibility are much lower than those for sorption isotherms, the potential errors introduced are much higher. where Dmdry,true [g] is the ‘‘true’’ dry mass increase due to hydroxyl deuteration, m1,water and m2,water [g] are residual water masses after ﬁrst and last drying stages, respectively, c [mol/g] is molar concentration of residual water per gram of wood, M [g/mol] is molar mass, and indices ‘‘D2O’’ and ‘‘H2O’’ refer to heavy water and normal water, respectively. Even if the concentrations are similar after both drying stages, the dry mass is still increased due to deuterium associated with the heavy water. Thus, a residual water content corresponding to 1% MC of normal water will add 0.11% mass. Compared with an expected dry mass increase due to deuteration in the range of 0.5–1.0%, this will yield an error of 11–22%. If only 0.1% MC residual water is left after drying, the error is one order of magnitude lower. However, this is only for similar concentrations of normal and heavy water after drying, which is an unlikely situation, even after similar drying conditions and durations in both ﬁrst and last drying stages, since transport of heavy water is about 20% slower than that of normal water (Mills 1973). Therefore, if assuming a conservative estimate of 50% higher concentration of heavy water than of normal water after the drying stage, the measurement errors in determined hydroxyl accessibility for residual water contents of 1 and 0.1% MC would be 66–133 and 7–13%, respectively. This clearly demonstrates the need for sufﬁcient drying of the material to eliminate residual water as a signiﬁcant source of error. Automated sorption balances Therefore, out of around 6 mmol/g accessible hydroxyls in wood cellulose (Engelund et al. 2013), only 4 mmol/g will contribute to the increased dry mass after deuteration. In order 123 Wood Sci Technol (2018) 52:297–329 307 to determine the hydroxyl accessibility, the sample has to be dried initially and after deuteration for determining the change in dry mass. Hereafter, the hydroxyl accessibility, OHaccess [mol/g], can be calculated as OHaccess ¼ Dmdry m0;dry DMhydrogen 1 þ Rmod ð Þ1 ð3Þ ð3Þ where Dmdry [g] is the change in dry mass due to deuteration, m0,dry [g] is initial dry mass, DMhydrogen [g/mol] is molar mass difference between deuterium and protium (1.006 g/mol), and (1 ? Rmod)-1 [-] corrects for mass gain due to modiﬁcation if present. The change in dry mass is dependent on not only the added deuterium on hydroxyls, but also the residual normal and heavy water in the ﬁrst and last drying stages, respectively, i.e. Dmdry ¼ Dmdry;true þ m2;water m1;water ¼ Dmdry;true þ m0;dry cD2OMD2O cH2OMH2O ð Þ ð4Þ ð4Þ where Dmdry,true [g] is the ‘‘true’’ dry mass increase due to hydroxyl deuteration, m1,water and m2,water [g] are residual water masses after ﬁrst and last drying stages, respectively, c [mol/g] is molar concentration of residual water per gram of wood, M [g/mol] is molar mass, and indices ‘‘D2O’’ and ‘‘H2O’’ refer to heavy water and normal water, respectively. Even if the concentrations are similar after both drying stages, the dry mass is still increased due to deuterium associated with the heavy water. Thus, a residual water content corresponding to 1% MC of normal water will add 0.11% mass. Compared with an expected dry mass increase due to deuteration in the range of 0.5–1.0%, this will yield an error of 11–22%. If only 0.1% MC residual water is left after drying, the error is one order of magnitude lower. However, this is only for similar concentrations of normal and heavy water after drying, which is an unlikely situation, even after similar drying conditions and durations in both ﬁrst and last drying stages, since transport of heavy water is about 20% slower than that of normal water (Mills 1973). Automated sorption balances Therefore, the use of a dm/dt stability criterion for determining hydroxyl accessibility is not rec- ommended due to the uncertainties it involves as discussed previously for gener- ating sorption isotherm data. As mass differences involved in the determination of hydroxyl accessibility are much lower than those for sorption isotherms, the potential errors introduced are much higher. The following is a short guideline for using heavy water in automated sorption balances. In general, it is advisable to use at least three but preferably more replicates for each sample batch due to measurement uncertainties. Only one conditioning stage in D2O vapour is needed as the determined accessibility depends on time of exposure and not on number of repeated conditioning cycles (Po¨nni et al. 2013). Moreover, the carrier gas (often nitrogen) should be dried out before the gas 12 123 Wood Sci Technol (2018) 52:297–329 308 inlet to the automated sorption balance, e.g. with molecular sieves to get rid of tracer amounts of normal water. Even high-purity gas (like grade 5) has enough water to slightly reverse deuteration in the last drying stage. Lastly, one important issue should be mentioned concerning the RH level(s) used for D2O vapour conditioning or their evaluation by RH sensors. For automated sorption balances, the calibration of generated RH or built-in RH sensors is commonly done using saturated salt solutions which generate known equilibrium RH at a given temperature. However, two issues complicate the use of D2O in a similar way: the saturated vapour pressure of D2O is lower than of H2O, and saturated salt solutions with D2O generate a slightly (1–3% RH) lower RH than their H2O counterparts (Becker et al. 1969). These differences are important to remember if the deuteration technique is used to generate D2O sorption isotherms or deuterate a material at a speciﬁc RH or vapour pressure. Pressure plate technique As previously discussed, it is very difﬁcult to condition samples close to saturation due to problems with keeping the temperature stable enough to achieve equilibrium in the over-hygroscopic RH range (98–100% RH). Therefore, in this range the pressure plate technique is often employed for conditioning, a method in which one or several samples are placed within an extractor where pressures up to 100 bar (10 MPa) can be applied. Typically, samples are cuboids or cylindrical discs with side length/diameter in the range 10–60 mm and a thickness of 2–20 mm (Almeida and Hernandez 2006, 2007; Cloutier and Fortin 1991; Fredriksson and Johansson 2016; Fredriksson et al. 2013; Thygesen et al. 2010; Zauer et al. 2016); however, Stone and Scallan (1967) used the pressure plate technique to condition microtome sections of 100 lm thickness and a cross-sectional area of 2 cm2. After water- saturating samples and the supporting substrate (ceramic plate or cellulose membrane, dependent on pressure range), pressure is applied to the extractor using dry gas, whereby water is pressed out of pores in which capillary forces are lower than the applied pressure. From a combination of the Young–Laplace and Kelvin equations, a correlation between applied pressure in the extractor and RH can be found: lnðRHÞ ¼ DP Mw RTqw ð5Þ ð5Þ where DP [Pa] is applied pressure (above atmospheric pressure), Mw [kg/mol] is molar mass of water (0.018 kg/mol), R [J/mol K] is the universal gas constant (8.3145 J/mol K), T [K] is temperature, and qw [kg/m3] is density of water. By adjusting the applied pressure, it is possible to tune RH much more precisely in the 98–100% RH range than with other methods. For instance, an applied pressure of 15 bar corresponds to 98.90% RH, while 9 bar is equivalent to 99.34% RH. When equilibrium is reached as monitored on the amount of water pressed out over time, samples are taken out and weighed on a balance of sufﬁcient mass resolution. 123 12 Wood Sci Technol (2018) 52:297–329 309 According to standard ISO 11274 (ISO 1998) equilibrium is obtained when two consecutive weekly mass readings show \ 0.1% variation in sample mass. According to standard ISO 11274 (ISO 1998) equilibrium is obtained when two consecutive weekly mass readings show \ 0.1% variation in sample mass. Pressure plate technique The technique has primarily been used to condition untreated wood (Almeida and Hernandez 2006, 2007; Cloutier and Fortin 1991; Fortin 1979; Fredriksson and Johansson 2016; Fredriksson et al. 2013; Grifﬁn 1977; Stone and Scallan 1967), but some studies of modiﬁed wood have been published (Thygesen et al. 2010; Zauer et al. 2016) (see Fig. 3). The traditional use of the technique has been for conditioning in desorption from water-saturated state; however, Fredriksson and Johansson (2016) used a rebuilt extractor for absorption measurements which avoids problems of previous modiﬁcations of the technique to handle absorption (Cloutier and Fortin 1991; Fortin 1979). EMC in the over-hygroscopic RH range depends on the size of voids in the wood structure such as pits, lumina, vessels, etc., and the sorption isotherms therefore depend heavily on wood species due to the high variability of these anatomical characteristics. Moreover, while the over-hygroscopic absorption isotherms depend on water condensation in voids and pores of gradually increasing void sizes (Engelund et al. 2010), desorption isotherms are governed by the size of bottleneck pits connecting lumina/vessels to the surrounding climate (Fredriksson and Johansson 2016; Salin 2008). During absorption, lumina/vessels take up moisture when an RH corresponding to equilibrium with their lumen/vessel radius is reached, but during desorption they will empty at a lower RH corresponding to radius of their largest pits facing the surroundings. This is known as the ‘‘ink bottle effect’’ (Ravikovitch and Neimark 2002) and might explain the large sorption hysteresis in the over-hygroscopic regime. In an interesting study by Zelinka et al. (2016), mercury intrusion porosimetry is explored as a complementary technique for studying over-hygroscopic moisture sorption in wood. The technique is based on applying pressure to push liquid mercury into thinly cut transverse wood sections. The applied pressure correlates with the radius of voids ﬁlled with mercury as described by the Kelvin equation. Therefore, after adjusting for differences in contact angle and surface tension between mercury and liquid water, the volumetric Fig. 3 a Desorption isotherms in the over-hygroscopic range determined after conditioning with the pressure plate technique and saturated salt solutions for untreated and modiﬁed Norway spruce (Thygesen et al. 2010; Zauer et al. 2016). Data points in the range of 94–98% RH from climate chamber measurements have been omitted from the results of Thygesen et al. (2010) for clarity. b Zoom of (a) in the 99.5–100.0% RH range Fig. Pressure plate technique 3 a Desorption isotherms in the over-hygroscopic range determined after conditioning with the pressure plate technique and saturated salt solutions for untreated and modiﬁed Norway spruce (Thygesen et al. 2010; Zauer et al. 2016). Data points in the range of 94–98% RH from climate chamber measurements have been omitted from the results of Thygesen et al. (2010) for clarity. b Zoom of (a) in the 99.5–100.0% RH range 12 3 310 Wood Sci Technol (2018) 52:297–329 uptake of mercury as a function of applied pressure can be used to calculate the capillary water in the wood void structure as a function of RH. The order of mercury intrusion into the wood structure, i.e. vessels-lumina-pits, makes the calculated capillary water curves correspond to the desorption isotherms of thin wood sections. For thicker wood sections without direct access to cell lumina, the order of emptying of voids as well as ﬁlling of voids with mercury changes. Mercury intrusion porosimetry would therefore need to be performed on such thicker samples in order to correctly represent over-hygroscopic desorption isotherms of capillary water. Although wood modiﬁcations rarely cause signiﬁcant changes to the void geometry unless modiﬁcation agent ﬁlls up lumina or pits, they may considerably affect the over-hygroscopic sorption isotherms. This is due to potential changes after modiﬁcation in the contact angle between wood surface and wetting liquid. Such change will affect EMC in the over-hygroscopic range as predicted by Engelund et al. (2010). The contact angle of liquids with modiﬁed wood has been investigated in several studies by the Wilhelmy and sessile drop methods. The ﬁrst determines the force required during immersion and withdrawal from a probe liquid, for example water, while in the latter the contact angle is determined over time after dropping a liquid droplet onto the surface. Despite the shortcomings of both methods as discussed by Wa˚linder and co-workers (Wa˚linder and Johansson 2001; Wa˚linder and Stro¨m 2001), they both show that modiﬁcation can alter the contact angle with water. For instance, both thermal modiﬁcation (Bakar et al. 2013; Hakkou et al. 2006; Kocaefe et al. 2008) and acetylation (Bryne and Wa˚linder 2010) appear to increase the contact angle with water, while furfurylation (Bryne and Wa˚linder 2010) slightly decreases it. The effect of these changes on the over- hygroscopic sorption isotherms has not been investigated. 123 Fibre saturation techniques The original deﬁnition of the ﬁbre saturation point (FSP) was suggested by Tiemann (1906) as the MC reached upon drying where lumina are empty of liquid water, cell walls begin to dry, and the mechanical properties start increasing. As shown by Stamm (1971), however, these three phenomena do not occur at the same MC. FSP was later suggested to be determined as MC at the ﬁrst shrinkage upon drying from green or water-saturated states (Koehler and Thelen 1926). However, due to inconsistencies in the previous deﬁnitions, Stone and Scallan (1967) focused on the emptying of lumina in the over-hygroscopic RH range and deﬁned FSP as the MC where lumina are empty, but cell walls are still water-saturated. Using the pressure plate technique, they found FSP for black spruce at 99.75% RH, while Grifﬁn (1977) added his own experimental results and adjusted it to 99.93% RH. This deﬁnition is also problematic as liquid water may still be present in some cell lumina even at equilibrium conditions where cell walls in other parts of the same sample are not saturated (Passarini et al. 2015). Although the traditional FSP concept originated more than a century ago, it is still widely used to describe the transition point imagined between cell wall 123 311 Wood Sci Technol (2018) 52:297–329 saturation and (signiﬁcant) moisture uptake in the wood void structure in the over- hygroscopic regime. For practical purposes, for example for structural engineering applications of wood with a strong focus on mechanical performance, the traditional concept of FSP works well. However, from a scientiﬁc standpoint the concept is problematic (Babiak and Kudela 1995; Feist and Tarkow 1967), and it mischar- acterises the wood–water relations. Instead, in this review, FSP is regarded as the maximum cell wall water capacity measured in the fully water-saturated state (Hill 2008). The ﬁrst part of this deﬁnition is in line with the intension behind the original deﬁnition by Tiemann (1906), i.e. the amount of water found within cell walls when these are water-saturated. By determining FSP in the fully water-saturated state, i.e. where water takes up all available space in cell walls and voids (pits, lumina, vessels) in the wood structure, uncertainties about partial drying of any cell walls are avoided. However, investigating FSP in the fully water-saturated state requires experimental techniques that can differentiate between water within and outside cell walls. Differential scanning calorimetry The cell wall moisture content, MCcell-wall [%], can then be found as 12 123 312 Wood Sci Technol (2018) 52:297–329 MCcellwall ¼ mwater mtotDHtot Hf m0;dry 1 þ Rmod ð Þ1 ð6Þ ð6Þ where mwater [g] is total mass of water, mtot [g] is total wet sample mass, m0,dry [g] is dry sample mass, DHtot [J/g] is measured speciﬁc enthalpy of melting per total wet sample mass, Hf [J/g] is melting enthalpy (often termed enthalpy of fusion) of pure water (333.6 J/g) (Lide 2013), and (1 ? Rmod)-1 [-] corrects for mass gain due to modiﬁcation if present. If the sample is fully water-saturated, MCcell-wall corre- sponds to FSP, and using such samples avoids problems of non-saturation of any parts of the wood volume examined. The technique can, however, also be used to differentiate cell wall water from liquid water outside cell walls in the over-hy- groscopic RH range as long as a sufﬁcient amount of freezable water is present. Although the DSC technique removes the sample from its conditioning temperature and FSP has been reported to change with temperature (Babiak and Kudela 1995; Stamm and Loughborough 1935), it would be unreasonable to relate the determined FSP to a temperature around 0 C since the experimental run in the DSC is much shorter than the time to equilibrate MC. Therefore, it can be assumed that the determined FSP relates to the conditioning temperature. Besides the above-de- scribed method for determining FSP, an additional one has been reported in several studies (Simpson and Barton 1991; Zauer et al. 2014; Zelinka et al. 2012) in which samples within a wide range of MC have been used and a linear regression is constructed between MC and speciﬁc enthalpy measured. The inﬂection point of this linear regression is then FSP, and the slope of the line is the enthalpy of water associated with the wood structure (Hager and Macrury 1980). In some studies (Simpson and Barton 1991; Zauer et al. 2014; Zelinka et al. 2012), it is stated that this method is more correct as it does not assume any speciﬁc value of the melting enthalpy. However, all studies show that the melting enthalpies determined are close to the generally accepted value of 333.6 J/g (Magne et al. 1947; Nelson 1977; Zauer et al. 2014) as shown in Fig. Differential scanning calorimetry One technique which is capable of separating cell wall MC from the total MC is differential scanning calorimetry (DSC). This type of equipment measures the energy involved in changing the temperature of a small sample over a wide temperature range. The amount of sample depends on the geometry of the crucible of the speciﬁc DSC instrument, but typical sample masses and sizes are in the range 5–15 mg (Park et al. 2006; Weise et al. 1996) and 1-mm discs of 3–4 mm diameter (Simpson and Barton 1991; Zauer et al. 2014), respectively. The technique is particularly useful for determining the energy involved in exothermic or endother- mic reactions occurring during an experiment, for example phase transitions such as freezing or thawing of water. As cell wall water is tightly associated with the chemical constituents, it will not exhibit a phase change at least down to - 70 C (Berthold et al. 1994). This was ﬁrst used by Magne et al. (1947) to determine the amount of cell wall water in water-saturated textile ﬁbres (cotton, rayon, nylon, glass ﬁbres) by measuring the energy for freezing water at - 4 C. Since then, DSC techniques have been used to determine cell wall water within, for example, pulp and paper ﬁbres (Maloney 2000; Nelson 1977; Park et al. 2006; Weise et al. 1996), untreated wood (Simpson and Barton 1991; Zauer et al. 2014; Zelinka et al. 2012), and modiﬁed wood (Repellin and Guyonnet 2005; Zauer et al. 2014). By cooling or quenching a wet sample to a given sub-zero temperature and ramping the temperature at constant rate to a speciﬁed temperature above the freezing point of water, the energy input (enthalpy) required to melt the ice formed can be measured. Temperature ranges and ramping rates employed vary between studies, but are most often found within the - 40 to 40 C range (Park et al. 2006; Repellin and Guyonnet 2005; Simpson and Barton 1991; Weise et al. 1996; Zauer et al. 2014) and from 1 to 3 C/min (Park et al. 2006; Repellin and Guyonnet 2005; Weise et al. 1996; Zauer et al. 2014) to 5 C/min (Zelinka et al. 2012) or even 10 C/min (Nelson 1977; Simpson and Barton 1991), respectively. Differential scanning calorimetry 4, considering the experimental uncertainties (small sample masses, high/low temperature ramping rates, heat ﬂow accuracy, etc.) involved in the measurements. The only exception to this is the study by Zelinka et al. (2012) in which a very low melting enthalpy of 240 J/g is found, leading the Fig. 4 Melting enthalpy as a function of reduced moisture content (corrected for added mass of modiﬁcation) for untreated and variously modiﬁed Norway spruce samples. Some of the samples had excess water on surfaces, resulting in very high calculated moisture contents, even though this water was not physically inside the material. Authors’ own data Fig. 4 Melting enthalpy as a function of reduced moisture content (corrected for added mass of modiﬁcation) for untreated and variously modiﬁed Norway spruce samples. Some of the samples had excess water on surfaces, resulting in very high calculated moisture contents, even though this water was not physically inside the material. Authors’ own data 123 123 313 Wood Sci Technol (2018) 52:297–329 authors to speculate about temperature buffering from the sample heat capacity. However, this effect should be mostly caught by baseline correction of the recorded energy ﬂow. That the melting enthalpy can differ from that of bulk water is clear for water found in nano-pores (Ja¨hnert et al. 2008; Shimizu et al. 2015). A vast majority of liquid water in wood is, however, found in voids larger than 0.1 lm. This is, for instance, seen in the negligible capillary water uptake at 98% RH (Engelund et al. 2010) corresponding to a void size of 0.1 lm. For voids larger than this size, the melting enthalpy reduction due to conﬁnement is \ 1% (Ja¨hnert et al. 2008). If freezable water was found in wood nano-pores in detectable amounts, it would exhibit a signiﬁcant freezing/melting point depression (Shimizu et al. 2015); how- ever, no peak of appreciable lower melting point has been found for solid wood. Besides the effect of heavy conﬁnement on melting enthalpy of liquids, there are no results found in the literature to suggest that surface chemistry or other factors should affect the melting enthalpy. Therefore, determining FSP by the DSC method using fully water-saturated samples is deemed appropriate, whereas conditioning to lower MC might not result in fully saturated cell walls and as a result yield a lower determined FSP. Solute exclusion technique However, the incubation time employed in the literature varies dramatically from a couple of minutes for pulp samples (Stone and Scallan 1968b) to 1–14 days for solid wood samples (Farahani 2003; Flournoy et al. 1991; Forster 1998). Clearly, more research is needed concerning the approach to equilibrium and appropriate incubation times as a function of diffusion pathways into samples. When selecting solutes for SET, it is important that they are chemically inert towards the sample and do not adsorb to surfaces within cell walls (Stone and Scallan 1967) as this would shift the equilibrium between pore solution and surrounding solution. The solutes most often used are small sugars, PEG, and dextrans. Their size is determined from the hydrodynamic diameter calculated from the Stokes–Einstein equation based on their diffusivity. The smallest solutes are saccharides such as glucose (0.8 nm) (Flournoy et al. 1991; Kuga 1981; Lin et al. 1987; Stone and Scallan 1968a; Wang et al. 2011), mannitol (0.8 nm) (Carpita et al. 1979), sucrose (1.0 nm) (Carpita et al. 1979; Kuga 1981; Van Dyke 1972), cellobiose (1.0 nm) (Lin et al. 1987), maltose (1.0 nm) (Flournoy et al. 1991; Stone and Scallan 1968a), rafﬁnose (1.2 nm) (Flournoy et al. 1991; Kuga 1981; Stone and Scallan 1968a; Van Dyke 1972), and stachyose (1.4 nm) (Carpita et al. 1979; Stone and Scallan 1968a). PEGs range in size from 0.44 nm for ethylene glycol (Kuga 1981) to 13.0 nm (Lin et al. 1987; Van Dyke 1972), while dextrans have been reported in the literature from 1.8 nm in diameter (Ishizawa et al. 2007) up to 586 nm (Arond and Frank 1954). Figure 5 shows how the solute diameter correlates with molecular mass for both PEGs and dextrans. Fig. 5 a Diameter of dextran and PEG probe molecules as a function of molecular weight (based on data from Arond and Frank 1954; Carpita et al. 1979; Day et al. 1978; Flournoy et al. 1991; Granath and Kvist 1967; Hill et al. 2005; Ishizawa et al. 2007; Kuga 1981; Lin et al. 1987; Peters 1986; Stone et al. 1969; Stone and Scallan 1968a; Van Dyke 1972; Wang et al. 2011; Wong et al. 1988). b Inaccessible cell wall water volume as a function of size of different molecular probes. Data for untreated and acetylated (Rmod = 0.21) Corsican pine from Forster (1998). Solute exclusion technique The amount of different pools of water in very small voids can be investigated by the solute exclusion technique (SET). The method was originally developed by Aggebrandt and Samuelson (1964) and modiﬁed by Stone and co-workers (Stone et al. 1969; Stone and Scallan 1967, 1968a, b) to characterise pore volumes and pore size distributions in pulps in the water-swollen state. Since then, SET has been used to characterise both untreated (Ahlgren et al. 1972; Farahani 2003; Feist and Tarkow 1967; Flournoy et al. 1991, 1993; Forster 1998) and modiﬁed wood (Farahani 2003; Forster 1998). In SET, a water-saturated sample with known mass of water is placed in an aqueous solution of molecular probe solutes of a known size and concentration. The typical sample is a cuboid of 20–30 mm side length and a thickness of 3–5 mm (Farahani 2003; Flournoy et al. 1991, 1993; Forster 1998), although several older studies have used shavings, pulp, or milled wood (Ahlgren et al. 1972; Feist and Tarkow 1967; Stone and Scallan 1968b). As solutes diffuse into the water within the sample, their concentration in the surrounding solution decreases. The change in concentration is related to the cell wall water volume that can be penetrated by solutes, and from this change in concentration the inaccessible moisture content, MCinaccess [% g/g], can be calculated: MCinaccess ¼ mwater cinitial þ Dc ð Þ þ msolDc mdry cinitial þ Dc ð Þ 1 þ Rmod ð Þ1 ð7Þ ð7Þ where mwater [g] is total mass of water in the sample before immersion, msol [g] is mass of solution added, mdry [g] is dry mass of the sample, cinitial [mol/g] is initial concentration of solutes in the solution, Dc [mol/g] is change in solute concentration after immersion of the sample, and (1 ? Rmod)-1 [-] corrects for mass gain due to modiﬁcation if present. Concentration differences are small, and a HPLC or 314 Wood Sci Technol (2018) 52:297–329 calibrated refractometer is needed for determining the concentration before and after immersion of the wet sample. To maximise the concentration difference, solution volumes used to immerse samples in are typically not more than 3–10 times the sample volume (Farahani 2003; Flournoy et al. 1991; Forster 1998; Stone and Scallan 1968b). It is important that equilibrium has been achieved before the change in concentration is determined. Solute exclusion technique Note that the ﬁbre saturation point (FSP) can be determined from the plateau in inaccessible cell wall volume for probe sizes too large to enter cell walls Fig. 5 a Diameter of dextran and PEG probe molecules as a function of molecular weight (based on data from Arond and Frank 1954; Carpita et al. 1979; Day et al. 1978; Flournoy et al. 1991; Granath and Kvist 1967; Hill et al. 2005; Ishizawa et al. 2007; Kuga 1981; Lin et al. 1987; Peters 1986; Stone et al. 1969; Stone and Scallan 1968a; Van Dyke 1972; Wang et al. 2011; Wong et al. 1988). b Inaccessible cell wall water volume as a function of size of different molecular probes. Data for untreated and acetylated (Rmod = 0.21) Corsican pine from Forster (1998). Note that the ﬁbre saturation point (FSP) can be determined from the plateau in inaccessible cell wall volume for probe sizes too large to enter cell walls Spectroscopic techniques While the gravimetric and ﬁbre saturation techniques are useful for determining total amount of water in wood or amount of cell wall water in the over-hygroscopic range, they are not able to provide a deeper insight into wood–water interactions or the distribution of water within the wood structure in the entire RH range. For this, spectroscopic techniques are needed that can distinguish water in different environments or provide more detail about molecular interactions and chemistry within cell walls. A wide range of spectroscopic techniques are available, but here two of the most important ones are highlighted. 123 315 Wood Sci Technol (2018) 52:297–329 The inaccessible MC describes the mass of water inaccessible for solutes of a given size. Thus, by using several differently sized solutes it is in principle possible to determine the pore size distribution within cell walls as indicated in Fig. 5. However, the underlying assumption of (6) which allows calculation of probed water mass is that solute concentration within accessible pores is the same as in the surrounding solution (Lin et al. 1985). This assumption has been shown to be incorrect, since solute concentration within accessible pores decreases as the sizes of probe solutes and accessible pores get closer (Alince 1991; Day et al. 1978, 1979). Thus, for solutes about half as big as the accessible pore, the solute concentration within the pore is around 30% of that in the surrounding solution (Alince 1991), while it decreases to nearly zero for solute sizes close to the pore size. For this reason, SET does not provide absolute pore size distributions, but can only hint at it. However, for solutes too large to enter cell walls, the voids they occupy (pits, lumina, vessels, etc.) are much larger than the solute size. Therefore, the assumption of equal solute concentration in these voids to the ones in the surrounding solution is valid, and SET can offer a good estimate of total pore volume, i.e. FSP (Alince 1991). If several sizes of solutes are used which are too big to enter cell walls, a plateau corresponding to FSP is found for MCinaccess as a function of solute size (see Fig. 5). Low-ﬁeld nuclear magnetic resonance spectroscopy and magnetic resonance imaging Low-ﬁeld (LF) nuclear magnetic resonance (NMR) spectroscopy, also known as time-domain NMR (Thygesen and Elder 2008) or NMR relaxometry (Kekkonen et al. 2014), can distinguish between water-bound hydrogen located in different physical and chemical environments. Consequently, it is a good technique for determining the distribution of water within different parts of the wood structure (Araujo et al. 1992). The technique uses the very, very slight magnetic polarisation in a material caused by a permanent magnet within the spectrometer (typically up to a few tesla in strength). The polarisation arises because the permanent magnetic ﬁeld B0 gives rise to two energy states for the spins (along and against the ﬁeld) of hydrogen nuclei within the sample with a tiny difference in abundance given by the Boltzmann distribution. This difference is what causes a polarisation vector M of the 12 3 123 316 Wood Sci Technol (2018) 52:297–329 material aligned with B0. The spectrometer is also equipped with an electromagnetic coil which can generate short-lived radiofrequency pulses that result in a magnetic ﬁeld B1 ( B0) oriented perpendicular to B0. These pulses will change the orientation of M, and the spectrometer will afterwards record the decay or relaxation of M back to its equilibrium value, realigned along B0. This relaxation is characterised by two different relaxation times termed T1 (longitudinal or spin– lattice relaxation) and T2 (transverse or spin–spin relaxation). T1 relaxation is studied as the increase in M in the direction along the permanent ﬁeld B0, while T2 relaxation is studied in the transverse plane, i.e. perpendicular to B0, as the decrease in the component of M in this plane. This latter process does not only depend on the realignment of M along B0, but also on the dephasing of spins in the transverse plane due to transfer of spins between neighbouring nuclei. T1 and T2 relaxations are both ﬁrst-order rate processes and can therefore be described by exponential decay functions with characteristic relaxation times T1 and T2, respectively. For ﬂuids in porous materials, the relaxation times depend on the size of pores conﬁning the ﬂuid as well as interactions between pore wall and ﬂuid (Mitchell et al. 2005). If a material contains ﬂuid in micro- and macro-pores of various sizes, the ﬂuid will exhibit several relaxation times characteristic of pore sizes and chemistry. Low-ﬁeld nuclear magnetic resonance spectroscopy and magnetic resonance imaging 6 Low-ﬁeld NMR characterisation of water in untreated and acetylated (Rmod = 0.20) Norway spruce in a fully water-saturated state: a zoom of CPMG decay curves for three replicates of untreated and acetylated spruce, b average T2 relaxation distribution curves for the three replicates derived from non- negative least square analysis with regularisation using Prospa version 3.1 (Magritek, NZ) software. Note that upon acetylation, the T2 relaxation times increase for water in pits and lumina due to weakened wood–water interactions. The cell wall water appears to split into two distinct pools after acetylation. The amount of moisture (MCR) found in lumina remains the same, while the amounts in pits, small cavities, and cell walls are reduced. Original LFNMR data from Thygesen and Elder (2008) Fig. 6 Low-ﬁeld NMR characterisation of water in untreated and acetylated (Rmod = 0.20) Norway spruce in a fully water-saturated state: a zoom of CPMG decay curves for three replicates of untreated and acetylated spruce, b average T2 relaxation distribution curves for the three replicates derived from non- negative least square analysis with regularisation using Prospa version 3.1 (Magritek, NZ) software. Note that upon acetylation, the T2 relaxation times increase for water in pits and lumina due to weakened wood–water interactions. The cell wall water appears to split into two distinct pools after acetylation. The amount of moisture (MCR) found in lumina remains the same, while the amounts in pits, small cavities, and cell walls are reduced. Original LFNMR data from Thygesen and Elder (2008) and the areas of respective T2 peaks assigned to these voids for water-saturated samples (Fredriksson and Thygesen 2017). Even more details about the chemical environment of water in pores within wood can be gained from T1 to T2 2D LFNMR correlation spectroscopy as reported by Cox et al. (2010). In this method, T1 and T2 relaxations are determined simultaneously allowing an analysis of their correlation (Song 2009; Song et al. 2002). This is useful since T2 relaxation occurs both as a result of realignment of M, like T1 relaxation, and due to local interactions independent of T1 relaxation. For a material with a spatially homogenous chemistry, relaxation times depend only on pore sizes which would result in similar T1/T2 ratios for the peaks observed (Song et al. 2002). On the other hand, if surface interactions vary between differently sized pores, this would show up as a markedly different T1/ T2 ratio. Low-ﬁeld nuclear magnetic resonance spectroscopy and magnetic resonance imaging By measuring the decay of the low-ﬁeld (LF) NMR signal and deconvoluting it, the relaxation times of the different components, i.e. ﬂuid in variously sized pores, can be determined and the distribution of ﬂuid in different environments (pore sizes and chemistry) can be analysed. The deconvolution can be done by ﬁtting discrete exponential functions to the decaying signal, for example to add components until residuals are randomly distributed. Another way is to ﬁt continuous relaxation time distributions to the signal by non-negative least square ﬁtting (Whittall et al. 1991) using algorithms often found in computer software for LFNMR data analysis, for example the CONTIN algorithm by Provencher (1982a, b). Before decay of the LFNMR signal can be measured, the spins of the sample need to be perturbed by one or more B1 pulses. For this, several pulse sequences have been developed. For determining T1 relaxation times, one of the pulse sequences termed ‘‘inversion recovery’’ and ‘‘saturation recovery’’ is used, while T2 relaxation times are determined after free induction decay (FID) or the Carr–Purcell–Meiboom–Gill (CPMG) sequence (Carr and Purcell 1954; Meiboom and Gill 1958). In particular, CPMG is a commonly used sequence for determining T2 relaxation as it reduces effects of magnet inhomogeneity by multiple refocusing of the spins in the transverse plane. Several studies have employed LFNMR for characterising water in both untreated (Almeida et al. 2007; Araujo et al. 1992, 1994; Cox et al. 2010; Flibotte et al. 1990; Fredriksson and Thygesen 2017; Labbe´ et al. 2002, 2006; Menon et al. 1987; Passarini et al. 2015; Telkki et al. 2013; Thygesen and Elder 2008, 2009) and modiﬁed wood (Elder et al. 2006; Hietala et al. 2002; Javed et al. 2015; Kekkonen et al. 2014; Thygesen and Elder 2008, 2009). Based on a deconvolution of the decay curves, signals from water in different compartments within the wood structure have been analysed (Almeida et al. 2007; Araujo et al. 1992, 1993; Fredriksson and Thygesen 2017; Passarini et al. 2015) (see Fig. 6). A comparison of void geometries and volumes shows good correspondence between pore volumes of pits and lumina Wood Sci Technol (2018) 52:297–329 317 Fig. Low-ﬁeld nuclear magnetic resonance spectroscopy and magnetic resonance imaging In the results of Cox et al. (2010), the T2 peak assigned to water in cell walls by using a regular CPMG splits up into two components in the T1 T2 correlation diagram implying two different environments for this water. There are a number of important issues to remember when using LFNMR to characterise water in wood. First of all, recorded decay curves should all be fully decayed within the duration of a scan, i.e. one decay measurement, in order to retrieve meaningful results from the data analysis algorithm. Secondly, temporal resolution should be adequate for capturing the shortest expected relaxation time. It is recommended that at least ﬁve data points are acquired before the shortest expected characteristic relaxation time in order to yield reasonable curve ﬁts. Thus, the shortest expected relaxation time should be divided by ﬁve to ﬁnd the maximum temporal spacing between equidistant pulses in a CPMG sequence. Since water in wood exhibits typical T2 relaxation times in the range from milliseconds to seconds as shown in Fig. 6, each scan needs to capture a decaying signal over a range of [ 1 s with a resolution of * 1/5 ms. As several scans are needed for adequate signal-to-noise ratio, each wood sample is exposed to a large number of 12 123 123 318 Wood Sci Technol (2018) 52:297–329 radiofrequency pulses. Although the amount of energy put into the sample by each pulse is tiny, the accumulated number of pulses can heat up the sample considerably in extreme cases. This is problematic as isothermal conditions are required for LFNMR since the magnitude of M is temperature dependent (Dinesh and Rogers 1971). To limit this excessive heating, the waiting time between scans can be increased. Magnetic resonance (MR) imaging uses the same principle as LFNMR although typically with considerably stronger permanent magnetic (2–14 tesla) and electro- magnetic ﬁelds. By employing CPMG sequences, relaxation spectra from different parts of the wood structure can be analysed. This has been done on both annual ring level (Dvinskikh et al. 2011; Quick et al. 1990) and cell level (Almeida et al. 2008; Hernandez and Caceres 2010; Meder et al. 2003; Passarini et al. 2015), allowing a visualisation of water distributions in both the hygroscopic (Hernandez and Caceres 2010; Passarini et al. 2015) and over-hygroscopic RH range (Almeida et al. 2008). Low-ﬁeld nuclear magnetic resonance spectroscopy and magnetic resonance imaging MR imaging is mainly appropriate for water with longer relaxation times than bound water (Hernandez and Caceres 2010). Vibrational spectroscopy Authors’ own data Fig. 7 Infrared vibrational spectra determined with ATR-FTIR equipment on untreated and acetylated (Rmod = 0.13) Norway spruce after deuteration in liquid D2O. Average curves based on 4–5 replicates are shown. Note that for acetylated wood both O–H stretching (* 3400 cm-1) and O–D stretching (* 2400 cm-1) vibrations decrease, while the C=O stretching (* 1750 cm-1) and CH3 deformations (* 1350–1250 cm-1) increase. Authors’ own data sorption of water from the molecular vibrations of water (Christensen et al. 2006; Inagaki et al. 2008; Tsuchikawa and Tsutsumi 1998). One of the advantages of vibrational spectroscopy is, however, the insights it can provide into chemical interactions of wood materials with water. By deuteration of a material, i.e. exchanging protium (1H) for deuterium (2H) in chemical groups hydrogen-bonding with liquid D2O or D2O vapour (Lindh et al. 2016), a marked shift in vibrational wavenumbers of affected molecular bonds is seen. For instance, deuteration changes the fundamental stretching vibrations of O–H, N–H, and C–H by around 1000, 900, and 800 cm-1, respectively, which is due to the near-doubling of the vibrating hydrogen mass. If the extra neutron of deuterium instead was added to the heavier vibrating atom of the O–H, N–H, and C–H bonds, for example exchanging 16O for 17O, the shift in vibrational wavenumber would hardly be detectable with vibrational spectroscopy as the relative mass increase in O, N, and C would be signiﬁcantly lower. Compared with gravimetric techniques using deuteration, the advantage of vibrational spectroscopy is that deuteration can be achieved without drying the material prior to exposure to D2O, unlike gravimetric techniques which require determination of initial dry mass. From vibrational spectroscopic studies, it can be seen that wood–water interactions are dominated by interactions with O–H groups (hydroxyls), since deuteration only affects O–H vibrations, while C–H stretching vibrations remain unchanged and no C–D stretching vibrations are observed (Hofstetter et al. 2006; Mann and Marrinan 1956a; Schmidt et al. 2006; Taniguchi et al. 1966; Watanabe et al. 2006). After deuteration and drying, the relative accessibility of hydroxyls to water can be determined as the ratio of integrated areas of O–D to the sum of O–H and O–D vibrations (Suchy et al. 2010b; Taniguchi et al. 1966) or alternatively as the ratio of weighted intensities of O–D to the sum of O–H and O–D vibrations (Mann and Marrinan 1956b; Sepall and Mason 1961). Vibrational spectroscopy Molecular bonds in a material vibrate at certain frequencies dependent on involved atomic masses and stiffness (elastic force constant) of the bond in question. For historic reasons, vibrational frequencies are often given in wavenumbers (cm-1) determined as frequency divided by speed of light (3 9 108 m/s). As an example, the covalent bonds C=C, C–C, and C–H all have different vibrational wavenumbers due to differences in either bond stiffness (single or double bond) or in masses of the atoms involved. In vibrational spectroscopy, this is used to probe the chemistry of materials as different chemical bonds are seen to vibrate at different wavenumbers. For instance, infrared (IR) spectroscopy detects the absorption of photon (light) energy at different wavelengths from an incoming infrared beam containing all wavelengths in the range 4000–400 cm-1, while near-infrared (NIR) spectroscopy uses a similar principle, but a different wavenumber range (14.000–4000 cm-1) in which mainly vibrational overtones and coupling of vibrations are found. Unlike these techniques, Raman spectroscopy detects the shift in scattered photon energy from a material which occurs for 10-6–10-8 of scattered photons from an incoming laser beam of ﬁxed wavelength. This shift corresponds to a change in vibrational energy of molecular bonds in the material. Thus, Raman spectroscopy probes the same molecular bonds as IR and NIR spectroscopy, but is based on a different physical phenomenon. Vibrational spectroscopy has been widely used to characterise chemical changes during wood modiﬁcation (see, for example, Gonza´lez-Pen˜a and Hale 2011; Kotilainen et al. 2000; Mitsui et al. 2008; Nuopponen et al. 2005; Stefke et al. 2008; Sun and Sun 2002; Temiz et al. 2007; Tserki et al. 2005; Vena˚s et al. 2006; and Fig. 7). Moreover, spatial chemical distributions in advanced modiﬁed materials have been mapped on the cell wall level using confocal Raman microscopy (Cabane et al. 2014, 2016; Keplinger et al. 2015, 2016). For studying the relation with water, vibrational spectroscopy has been used to examine the presence of water or the 319 Wood Sci Technol (2018) 52:297–329 Fig. 7 Infrared vibrational spectra determined with ATR-FTIR equipment on untreated and acetylated (Rmod = 0.13) Norway spruce after deuteration in liquid D2O. Average curves based on 4–5 replicates are shown. Note that for acetylated wood both O–H stretching (* 3400 cm-1) and O–D stretching (* 2400 cm-1) vibrations decrease, while the C=O stretching (* 1750 cm-1) and CH3 deformations (* 1350–1250 cm-1) increase. Vibrational spectroscopy These calculations are based on the Beer–Lambert law which states that the absorbance of a given vibration is given by the product of the three following parameters: abundance of the vibration 12 123 123 320 Wood Sci Technol (2018) 52:297–329 in the material, molar absorptivity of the vibration, and path length of the light beam through the material. For calculating the relative accessibility of hydroxyls to water, it is implicitly assumed that molar absorptivities of O–H and O–D vibrations are equal. This is not necessarily correct as O–D stretching vibrations in cellulose, starch, and wood have been reported to be about 10% more intense than O–H stretching vibrations (Mann and Marrinan 1956b; Nara et al. 1981; Thybring et al. 2017). From theoretical calculations, O–D stretching vibrations are expected to be less intense than O–H stretching vibrations (Crawford 1952; Swenson 1965), in line with studies on liquid normal and heavy water showing 29% lower intensity for O– D stretching vibrations (Venyaminov and Prendergast 1997). Moreover, it should be noted, as discussed for gravimetric techniques, that deuteration will not exchange hydrogens on all hydroxyls interacting with water as shown by Lindh et al. (2016), only those capable of acting as hydrogen bond donor, i.e. in which the hydrogen of hydroxyls is involved in a hydrogen bond with a water molecule. Water-interacting hydroxyls restricted to act as hydrogen bond acceptors, i.e. only interacting with water through their oxygen, are presumably only found on the surfaces of the stiff, aggregated cellulose microﬁbrils. Before the effect of these issues on the determined hydroxyl accessibility has been resolved for wood and plant materials, it is uncertain whether vibrational spectroscopy can yield quantitative results. Nonetheless, vibrational spectroscopy can be a useful technique for getting qualitative results for interactions between water and wood hydroxyls (see Fig. 7). Both IR and NIR spectroscopy have been applied to study untreated wood (Altaner et al. 2006; Chow 1972; Fackler and Schwanninger 2011; Fernandes et al. 2011; Suchy et al. 2010a, b; Taniguchi et al. 1966; Tsuchikawa and Siesler 2003a, b), while no published data on modiﬁed wood are available. Similarly, no known study uses Raman spectroscopy to probe the wood–water interactions by deuteration, whereas a few have been published on deuteration of proteins (Bolton and Scherer 1989; Sa´nchez-Gonza´lez et al. 2008) and lactose (Whiteside et al. 2008). Vibrational spectroscopy Even more information about hydroxyls can be gained from 2D correlation infrared spectroscopy (Noda 1990, 2006; Noda et al. 1993). By this technique, the IR spectrometer is coupled with a dynamic excitation which could be a change in climate (RH or temperature) or a mechanical excitation. This has, for instance, been done using dynamic mechanical analysis (Hinterstoisser and Salme´n 2000; Hofstetter et al. 2006; Stevanic and Salme´n 2006; A˚ kerholm and Salme´n 2003). From a correlation analysis between obtained spectra and external excitation, information about which vibrations in the rather broad and unstructured O–H stretching region (3700–3000 cm-1) are affected by external stimulus can be gained, for example the location and orientation of hydroxyls in cellulose. Conclusion The interaction of water with wood is highly important for the performance of wood materials. This review describes a range of experimental techniques for character- ising water within wood in the entire moisture domain from dry to fully water- saturated. The techniques fall into three broad categories: (1) gravimetric techniques 123 Wood Sci Technol (2018) 52:297–329 321 that determine how much water is absorbed, (2) ﬁbre saturation techniques that determine the amount of water within cell walls, and (3) spectroscopic techniques that provide insights into chemical wood–water interactions as well as yield information on water distribution in the macro-void wood structure. For each technique, advantages and limitations are discussed. The experimental data obtained from the various techniques show the many different aspects of wood–water interactions which can be probed. Thus, by combination of several experimental techniques, a wealth of information can be extracted about the state and distribution of water in wood, if the techniques are properly employed. The aim of this review is that more researchers become aware of the wide suite of available experimental techniques and by taking advantage of these techniques, will provide new insights into water interacting with wood materials. Acknowledgements EET gratefully acknowledges the ﬁnancial support from the VILLUM FONDEN postdoc programme and Innovation Fund Denmark. KM and LR gratefully acknowledge ﬁnancial support from European Regional Development Fund through South Savo Regional Council from Finland and industrial partners. 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, dis- tribution, 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. 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https://openalex.org/W3006374576	https://europepmc.org/articles/pmc7018810?pdf=render	English	null	Author Correction: Relationship between body mass index and renal function deterioration among the Taiwanese chronic kidney disease population	Scientific reports	2,020	cc-by	300	www.nature.com/scientificreports www.nature.com/scientificreports Author Correction: Relationship between body mass index and renal function deterioration among the Taiwanese chronic kidney disease population OPEN Published: xx xx xxxx Published: xx xx xxxx population Tian-Jong Chang, Cai-Mei Zheng, Mei-Yi Wu, Tzu-Ting Chen, Yun-Chun Wu , Yi-Lien Wu, Hsin-Ting Lin, Jing-Quan Zheng, Nain-Feng Chu, Yu-Me Lin, Sui-Lung Su, Kuo-Cheng Lu, Jin-Shuen Chen, Fung-Chang Sung, Chien-Te Lee, Yu Yang, Shang-Jyh Hwang, Ming- Cheng Wang, Yung-Ho Hsu, Hung-Yi Chiou, Senyeong Kao & Yuh-Feng Lin Correction to: Scientific Reports https://doi.org/10.1038/s41598-018-24757-6, published online 02 May 2018 The original version of this Article contained an error in Affiliation 3, which was incorrectly given as ‘Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan’. The correct affiliation is listed below: Division of Nephrology, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan This error has now been corrected in the HTML and PDF versions of the Article. 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 Cre- ative 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 per- mitted 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/. © The Author(s) 2020 Scientific Reports \| (2020) 10:2822 \| https://doi.org/10.1038/s41598-020-59783-w
https://openalex.org/W4286330890	https://hal.science/hal-03746965/file/Levy%20and%20Snir%20Oxford%20Open%20Economics%202022.pdf	English	null	Potterian economics	Oxford open economics	2,022	cc-by	55,746	Potterian Economics Daniel Levy, Avichai Snir To cite this version: Daniel Levy, Avichai Snir. Potterian Economics. Oxford Open Economics, 2022, 1 (1), pp.1-32. 10.1093/ooec/odac004. hal-03746965 To cite this version: Received: April 25, 2022. Revised: April 25, 2022. Accepted: June 2, 2022 © The Author(s) 2022. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. HAL Id: hal-03746965 https://hal.science/hal-03746965v1 Submitted on 6 Aug 2022 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. Oxford Open Economics, 2022, 1, 1–32 https://doi.org/10.1093/ooec/odac004 Advance access publication date 19 July 2022 Research Article Potterian economics Daniel Levy1,2,3,4,* and Avichai Snir5 1Department of Economics, Bar-Ilan University, Ramat Gan 5290002, Israel 2Department of Economics, Emory University, Atlanta, GA 30322, USA 3International School of Economics at Tbilisi State University, 0108 Tbilisi, Georgia 4ICEAand RCEA 5Department of Banking and Finance, Netanya Academic College, Netanya 42365, Israel Correspondence address. Department of Economics, Bar-Ilan University, Ramat Gan 5290002, Israel. E-mail: Daniel.Levy@biu 5Department of Banking and Finance, Netanya Academic College, Netanya 42365, Israel Correspondence address. Department of Economics, Bar-Ilan University, Ramat Gan 52900 5Department of Banking and Finance, Netanya Academic College, Netanya 42365, Israel *Correspondence address. Department of Economics, Bar-Ilan University, Ramat Gan 5290002, Israel. E-mail: Daniel.Levy@biu.ac.il Ariel Rubinstein (Econometrica, 2006, p. 881). Ariel Rubinstein (Econometrica, 2006, p. 881). ‘An investigation of novels and [economic] models . . . may help us better understand how the public thinks about economic issues.’ An investigation of novels and [economic] models . . Tyler Cowen (The Street Porter and the Philosopher: Conversations on Analytical Egalitarianism, 2008, p. 3 Keywords: popular opinion, Potterian economy, Harry Potter, economic and financial literacy, folk economics, social organization of economic activity Received: April 25, 2022. Revised: April 25, 2022. Accepted: June 2, 2022 © The Author(s) 2022. Published by Oxford University Press. Abstract Recent studies in psychology and neuroscience offer systematic evidence that fictional works exert a surprisingly strong influence on readers and have the power to shape their opinions and worldviews. Building on these findings, we study ‘Potterian economics’, the economic ideas, insights and structure, found in Harry Potter books, to assess how the books might affect economic literacy. A conservative estimate suggests that more than 7.3% of the world’s population has read the Harry Potter books, and millions more have seen their movie adaptations. These extraordinary figures underscore the importance of the messages the books convey. We explore the Potterian economic model and compare it to professional economic models to assess the consistency of the Potterian economic principles with the existing economic models. We find that some of the principles of Potterian economics are consistent with economists’ models. Many other principles, however, are distorted and contain numerous inaccuracies, contradicting professional economists’ views and insights. We conclude that Potterian economics can teach us about the formation and dissemination of folk economics—the intuitive notions of naïve individuals who see market transactions as a zero-sum game, who care about distribution but fail to understand incentives and efficiency and who think of prices as allocating wealth but not resources or their efficient use. ‘I think the writers [of popular literature] are not particularly sympathetic to or don’t understand how a market works. It’s not easy to convey that to a child. It’s not always easy to convey it to grown-ups.’ think the writers [of popular literature] are not particularly sympathetic to or don’t understand how a convey that to a child. It’s not always easy to convey it to grown-ups.’ Gary Becker (New York Times, August 21, 2011, p. SR5). Gary Becker (New York Times, August 21, 2011, p. SR5). ‘With all due respect to Richard Posner, Cass Sunstein, or Peter Schuck [reference to the books these scholars published in 2005], no book released in 2005 will have more influence on what kids and adults around the world think about government than [Rowling’s] The Half-Blood Prince.’ j i ( i i i ) Benjamin Barton (Michigan Law Review, 2006, p. 1525). Benjamin Barton (Michigan Law Review, 2006, p. 1525). ‘As economic theorists, we organize our thoughts using what we call models. The word “model” sounds more scientific than “fable” or “fairy tale” although I do not see much difference between them. The author of a fable draws a parallel to a situation in real life. He has some moral he wishes to impart to the reader. The fable is an imaginary situation that is somewhere between fantasy and reality. Any fable can be dismissed as being unrealistic or simplistic, but this is also the fable’s advantage. Being something between fantasy and reality, a fable is free of extraneous details and annoying diversions. In this unencumbered state, we can clearly discern what cannot always be seen in the real world. On our return to reality, we are in possession of some sound advice or a relevant argument that can be used in the real world.” INTRODUCTION For example, according to the July 13, 2007 NPR’s Morning Edition, ‘Faking Harry Potter books has become a cottage industry in China.’ Examples include ‘Harry Potter and the Golden Vase,’ ‘Rich Dad, Poor Dad and Harry Potter, etc.. . . Don’t worry if you’ve never heard of these books. They’re totally made up with no resemblance to the real thing.’ (Source: www.npr.org/ templates/story/story.php?storyId=11945354, accessed June 6, 2022.). people acquire their knowledge about the economy. It is widely accepted that literature, even fictional, is a mirror of culture and society (Albrecht, 1954). Recent studies in psychology and neuroscience, however, find that causality goes the other way as well, offering systematic evidence of fictional stories’ effects on the human mind (Hakemulder, 2000; Mar, 2004; Appel, 2008). We focus on the potential influence of literature on people’s views and opinions about the economy and its functioning, by studying the economic principles that Harry Potter books convey. We chose Harry Potter books because studies that focus specifically on these books find that they not only relate to and reflect the readers’ views but also have a powerful influence on them. It is widely accepted that literature, even fictional, is a mirror of culture and society (Albrecht, 1954). Recent studies in psychology and neuroscience, however, find that causality goes the other way as well, offering systematic evidence of fictional stories’ effects on the human mind (Hakemulder, 2000; Mar, 2004; Appel, 2008). We focus on the potential influence of literature on people’s views and opinions about the economy and its functioning, by studying the economic principles that Harry Potter books convey. We chose Harry Potter books because studies that focus specifically on these books find that they not only relate to and reflect the readers’ views but also have a powerful influence on them. Harry_Potter_in_translation#List_of_translations_by_ language, accessed June 6, 2022.) In some countries, the books have been translated into several local languages. There are also pirate and unofficial translations as well as counterfeit versions. For example, according to the July 13, 2007 NPR’s Morning Edition, ‘Faking Harry Potter books has become a cottage industry in China.’ Examples include ‘Harry Potter and the Golden Vase,’ ‘Rich Dad, Poor Dad and Harry Potter, etc.. . . Don’t worry if you’ve never heard of these books. They’re totally made up with no resemblance to the real thing.’ (Source: www.npr.org/ templates/story/story.php?storyId=11945354, accessed June 6, 2022.). INTRODUCTION analyze complex financial products . . . while 15 per- cent can, at best, make simple decisions about every- day spending.’ Similarly, to assess financial literacy, Tang et al. (2015) surveyed American young adults, asking them three simple intuitive questions, and find that the participants answered correctly only 1.8 questions, on average. According to Caplan (2007), ‘ . . . modern economic theories . . . begin by assuming that the typical citizen understands economics and votes accordingly.’ Empirical evidence, however, suggests that the economic literacy of the public is low. For example, OECD (2014, p. 1) reports that only ‘ . . . 10 percent of students can Oxford Open Economics, 2022, Vol. 1, No. 1 2 Similar findings were reported in 2014 in Israel: 82.2% of the Israeli adults could not identify the interest rate that the Bank of Israel sets monthly. This is despite the attention the media has paid to it with the rate cut to 0.25%, the lowest level ever (Source: www.ynet.co.il/ articles/0,7340,L-4568826,00.html (in Hebrew), accessed June 6, 2022.). Consistent with these findings, Nelson and Sheffrin (1991, p. 157) state that ‘The level of economic knowledge among most high school students is shocking.’ Second, among all such intermediaries, Harry Potter books are of particular importance because they are among the most popular books of our times. The books are popular among children and adults, men and women, irrespective of income and education. In sales, Harry Potter books rank fifth after The Holy Quran with over 3 billion copies sold, The King James Bible with over 2.5 billion copies sold, The Quotations of Chairman Mao Zedong with 800 million copies sold, and Don Quixote with 500 million copies sold (Source: www. stylist.co.uk/books/the-all-time-most-popular-books- in-the-world-revealed/127306, accessed June 6, 2022.). Rowling (2005) sold almost 7 million copies in the US and over two million copies in the UK just on the first weekend of its release (Source: news.bbc.co.uk/2/hi/ entertainment/4692093.stm, accessed June 6, 2022.). In total, over 450 million copies were sold in over 200 countries. For comparison, Tolkien’s The Hobbit was in print for more than 60 years and sold over 40 million copies, while Tolkien’s The Lord of the Rings was in print for almost 50 years and sold over 50 million copies (Shippey, 2002, p. xxiv). INTRODUCTION Downloaded from https://academic.oup.com/ooec/article/doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 There is evidence that better economic literacy improves economic decision-making, and therefore, the low level of economic literacy is worrisome as it may lead to poor financial planning (Note: See Bernheim et al. (2001), Boisclair et al. (2014), Gerardi et al. (2013), Brown et al. (2014), Grohmann et al. (2015), Lusardi & Mitchell (2014), Wiedrich et al. (2014) and Rubin (2018).). It may also lessen the effectiveness of economic policy (Bernanke, 2006). There is also evidence that the general public has biases and misconceptions about the economy, which influence and shape its views about the role of public policy (Rubin, 2003; Caplan, 2007; Hillman, 2010). These observations raise questions about how people acquire their knowledge about the economy. stylist.co.uk/books/the-all-time-most-popular-books- in-the-world-revealed/127306, accessed June 6, 2022.). Rowling (2005) sold almost 7 million copies in the US and over two million copies in the UK just on the first weekend of its release (Source: news.bbc.co.uk/2/hi/ entertainment/4692093.stm, accessed June 6, 2022.). In total, over 450 million copies were sold in over 200 countries. For comparison, Tolkien’s The Hobbit was in print for more than 60 years and sold over 40 million copies, while Tolkien’s The Lord of the Rings was in print for almost 50 years and sold over 50 million copies (Shippey, 2002, p. xxiv). Harry Potter books have been translated into 73 languages, including Afrikaans, Albanian and Arabic, all the way to Vietnamese, Welsh and Zulu. The books have even been translated into two dead languages, Latin (‘Harrius Potter’) and Ancient Greek (‘Aρειoσ oτιηρ’), the latter translation being the longest work in the language since the novels of Heliodorus of Emesa in the third century AD (Source: https://en.wikipedia.org/wiki/ Harry Potter in translation#List of translations by Harry Potter books have been translated into 73 languages, including Afrikaans, Albanian and Arabic, all the way to Vietnamese, Welsh and Zulu. The books have even been translated into two dead languages, Latin (‘Harrius Potter’) and Ancient Greek (‘Aρειoσ oτιηρ’), the latter translation being the longest work in the language since the novels of Heliodorus of Emesa in the third century AD (Source: https://en.wikipedia.org/wiki/ Harry_Potter_in_translation#List_of_translations_by_ language, accessed June 6, 2022.) In some countries, the books have been translated into several local languages. There are also pirate and unofficial translations as well as counterfeit versions. doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 Third, Potterian economics can teach us about the formation and dissemination of folk economics (Rubin, 2003)—the intuitive notions of naive individuals who care about distribution but fail to understand incentives and efficiency. Cowen (2008), for example, argues that literary works can help us understand what people think about economic issues. Following these arguments, our thesis is that Potterian economics may reveal some of the ideas of folk economics. Indeed, as far as we know, J.K. Rowling is not an economist (she majored in French and Classics), and therefore Potterian economic model may be viewed as a layman’s model and thus, it might reflect on the general public’s attitudes and understanding of the economy. y ) The importance of Harry Potter books has been rec- ognized by scholars in other fields besides literature. For example, they have been used to assess the completeness of tort theories (Hershovitz, 2010), and to shed light on the Anglo-American interpretation of rule of law (Liston, 2009; Thomas and Snyder, 2010). Barton (2006) studies governments’ legitimacy. Sheftell et al. (2007) identify the headache episodes found in the books as symptomatic of migraine. Woeste (2010) revisits the debate over free will and determinism. As of June 6, 2022, Amazon listed over 40 000 items with ‘Harry Potter’ in the title, over 20 000 of them under books. The titles include: The Psychology of Harry Potter, Harry Potter and History, Harry Potter and International Relations, Harry Potter and Philosophy, Ethics in the Bible and the World of Harry Potter, The Law and Harry Potter, and The Sociology of Harry Potter. Other Harry Potter titles (along with comparable economics titles) include, Who Killed Albus Dumbledore? by Wendy Harte 2006 (com- pare it to Who Killed John Maynard Keynes? by Carl Biven 1989), Rowling Revisited by James Thomas 2010 (compare it to Revisiting Keynes by Lorenzo Pecchi and Gustavo Piga 2008), The Complete Idiot’s Guide to the World of Harry Potter by Tere Stouffer 2007 (compare it to The Complete Idiot’s Guide to Economic Indicators by Mark Rogers 2009), Harry Potter and the Classical World by Richard Spencer 2016 (compare it to Keynes and the Classics Reconsidered by James Ahiakpor 1998), etc. INTRODUCTION The tour guide told me that I wouldn’t be able to see the university’s huge freshman dining hall as it was closed for the day, but to just imagine Hogwarts’s Great Hall in its place.’ (Source: Lauren Edelson, ‘Taking the Magic Out of College,’ New York Times (New York Edition), December 6, 2009, Op-Ed Contribution, p. WK12.). p A recent study by Facebook’s computer scientists reported that in a meme about ‘What Books Stayed with You,’ 130 000 participants (their average age was 37) ranked the Harry Potter books as the number 1. The Bible ranked sixth (Source: ‘Harry Potter Tops List of Facebook Users’ Favorite Books,’ The Telegraph, September 10, 2014. For details about the survey, see the Facebook post of the authors of the study: https://www.facebook.com/ notes/facebook-data-science/books-that-have-stayed- with-us/10152511240328859/, accessed June 6, 2022.). These extraordinary figures underscore the importance of the messages the books convey. p ) Literary scholars have also emphasized the unique importance of Harry Potter books. For example, according to John Pazdziora, speaking at Britain’s first academic conference on Harry Potter in 2012, ‘We cannot avoid the fact that Harry Potter is the main narrative experience of an entire generation, the children who quite literally grew up with Harry Potter. The Harry Potter novels are simply the most important and influential children’s books of the late 20th and early 21st centuries. For very many people, this is their first experience of literature, and of literary art . . . These are the most important, seminal texts for an entire generation of readers . . . In 100, 200 years’ time, when scholars want to understand the early 21st century, when they want to understand the ethos and culture of the generation that’s just breaking into adulthood, it is a safe bet that they’ll be looking at the Harry Potter novels.’ (See: A. Cramb, ‘Harry Potter and the Philosophers Conference at St Andrews University,’ The Telegraph, May 17, 2012, and A. Flood, ‘Harry Potter and the Order of the 60 Scholars Gets Mixed Initial Reception,’ The Guardian, May 18, 2012.) In that context, compare the Harry Potter books to the best-selling economics textbooks that were authored by Samuelson, selling over 4 million copies in 40 languages, and Mankiw, selling over one million copies in 17 lan- guages (Source: en.wikipedia.org/wiki/Paul_Samuelson, and en.wikipedia.org/wiki/Greg_Mankiw, accessed June 6, 2022.). INTRODUCTION Although Harry Potter books belong to the fantasy genre, Harry Potter lives not only in a social world but also in an economic one. Indeed, the Potterian world is rich with economic institutions and ideas, including monopolies, inefficient government, limited social mobil- ity, trade restrictions and other regulations, insufficient social capital, commodity money, prices, banking, etc. Not surprisingly, these economic ideas are unnoticed by most readers. But perhaps surprisingly, they are often unnoticed also by professional economists. Indeed, when we offered a colleague a list of the economic ideas we identified in the books, his response was, ‘Well, you are right, I completely missed that.’ By our conservative estimates, more than 7.3% of the world’s population of 6.08 billion has read the books (Source of population figures: www.infoplease.com/ipa/ A0762181.html, accessed June 6, 2022.). The estimate is conservative, and thus the actual share likely exceeds it because (1) the sales figure doesn’t include unauthorized translations and their sales, and (2) the actual number of readers likely exceeds the number of copies sold because a single copy is often read by many, e.g. by an entire family. Hundreds of millions have also seen their movie adaptations. In the US alone, Harry Potter movies sold 350 million tickets (Source: https://mrob.com/pub/film- video/topadj.html, accessed June 6, 2022.). Nine thou- sand FedEx trucks were used in the US, to deliver the initial release of The Goblet of Fire alone. The last four Studying what we term ‘Potterian economics’ is inter- esting for several reasons. First, as Lucas Jr. et al. (2002), Blinder & Krueger (2004), Giovannini & Malgarini (2012), and Cruijsen et al. (2010) note, the public acquires much of its knowledge about economics through popular inter- mediaries (books/newspapers, etc.), but the role of these intermediaries in shaping the public’s views and opinions about economics has received little attention. Levy and Snir 3 books in the Harry Potter series were the fastest-selling books in history, with the final book selling roughly 11 million copies in the US within 24 hours of its release. their school was to the Hogwarts. For example, ‘During a Harvard information session, the admissions officer compared the intramural sports competitions there to the Hogwarts House Cup. INTRODUCTION These figures, although impressive, are dwarfed by the sales figures of Harry Potter. Clearly, it is not fair to compare a popular fantasy book to academic textbooks, no matter how successful the latter are. The point, how- ever, is that to the extent that Harry Potter books teach millions of people of all ages, principles of economics, identifying these principles is informative and important. 4 \| Oxford Open Economics, 2022, Vol. 1, No. 1 4 \| Oxford Open Economics, 2022, Vol. 1, No. 1 In addition to books, there are also hundreds of papers, published and unpublished. For example, SSRN lists 63 Harry Potter studies in law, administrative science, polit- ical science, philosophy, history, marketing, etc. SciVerse- Scopus lists 46 items in engineering, neuroscience, psychology, etc. (Source: estrip.org/articles/read/tiny pliny/45318/Harry_Potter_in_Scientific_Literature.html, accessed June 6, 2022.). We also found 51 articles in PubMed. Finally, the Jstor database includes 159 papers, with ‘Harry Potter’ in their title (accessed June 6, 2022). We conclude that the Potterian model reflects folk economics. As such, although it is sometimes con- sistent with economists’ views, many of its deeper aspects of economics are distorted, containing numerous inaccuracies, which might potentially be absorbed by the public, and contribute to its lack of literacy on economic matters. This is particularly true for the young readers, who figure prominently among Harry Potter readers. The paper is organized as follows. In section 2, we review the economic literacy literature. In section 3, we discuss fiction’s influence. In section 4, we describe the setting. In section 5, we study money, credit, and banking. In section 6, we consider prices and their properties. In section 7, we focus on the foreign exchange rate and the commodity value of the Galleon. In section 8, we look at the Potterian government. In section 9, we discuss the law and order. In section 10, we focus on monopo- lies, oligarchies, and other pathologies. In section 11, we study income distribution in the Potterian economy. In section 12, we study international trade and migration. In section 13, we study war economics. In section 14, we study technological progress. In section 15, we discuss the Potterian education system and human capital. We conclude in section 16 by summarizing the implications of our findings, addressing possible objections to our interpretation, and discussing some caveats (Note: The online supplementary appendix contains a detailed ref- erence to all economic themes, topics and issues we have identified in the Harry Potter books, along with the quotations of the relevant texts from the books, and their exact locations.). In addition, numerous academic conferences were and are still devoted to these studies. 4 \| Oxford Open Economics, 2022, Vol. 1, No. 1 For example, in 2012, the University of California, San Diego, and the National Institute of Health, held a joint lecture series and an exhibit on ‘Harry Potter’s World: Renaissance Science, Magic, and Medicine.’ The lecture series featured UCSD scholars from medicine, mathematics, engineering and literature. According to the UCSD News center, the inter- est of the UCSD medical scholars in Harry Potter stems from the fact that ‘ . . . the magic depicted in the popular Harry Potter novels . . . can be traced to Renaissance traditions that played a pivotal role in the development of modern science and medicine.’ (Source: http:// ucsdnews.ucsd.edu/pressrelease/harry_potters_world_ renaissance_science_magic_and_medicine/, accessed June 6, 2022.) Chestnut Hill College in Philadelphia, PA, holds an annual conference on Harry Potter. The interdis- ciplinary conference includes several parallel sessions, on such topics as young adult literature, textual analysis, politics and justice, education and science, literature and education, psychology and philosophy, character studies, etc. (Source: https://www.chc.edu/events/10th- anniversary-harry-potter-academic-conference-hpac-x, accessed June 6, 2022.). doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 Fourth, if fiction’s influence is particularly strong on adolescents, and if a significant proportion of the readers of Harry Potter are children and teenagers, then understanding Potterian economics may also shed light on the sources of illiteracy that studies have documented among young adults (Tang et al., 2015) (Note: Lunt & Furnham (1996) contain essays on children’s and adolescents’ knowledge of economics and economic matters.) Indeed, many colleges and universities, includ- ing some Ivy League schools, recognize the influence of Harry Potter, and try to take advantage of the Harry Potter bandwagon in their sales’ pitch, in their efforts to make their institutions more attractive to the candidates of Harry Potter generation. According to Lauren Edelson, during her visits to various university campuses, she heard from numerous host tour guides, how similar ECONOMIC LITERACY . that are unsuitable, unnecessarily costly, or abusive.’ To learn economics, one could take college economics courses. However, ‘only about 10% of adults ever take college economics.’ It would be better to increase ‘ . . . the quality and quantity of discussion of economics in the mass media. When a TV show like “West Wing” considers the benefits and costs of free trade, it probably has more impact on the economic literacy . . . than all freshmen economic courses combined’ (Krueger, 2002, pp. 475–476). Indeed, some studies document the effect of TV shows on the political attitudes of the audience. See, for example, French & van Hoorn (1986), Lenart & McGraw (1989), and Mutz & Nir (2010). Mutz (2016) surveys some of the studies that find that fictional stories can influence political opinions, and offers evidence-based on the most recent data from the 2017 US presidential elections. The American Economic Association (AEA) has been involved in economic education since 1885. Hinshaw & Siegfried (1991) describe the AEA’s efforts ‘ . . . to edu- cate public . . . about economic questions and economic literature’ (p. 373). The AEA’s Committee on Economic Education has been active since the mid-1940s. The AEA routinely holds panels about teaching economics at its annual meetings. The establishment of the Journal of Economic Education in 1969 is also noteworthy. The US Fed has also been engaged in economic education for decades. For example, it offers teaching resources for K- 12 grades on finance, banking, monetary policy, etc. OECD is also involved in these efforts (Atkinson and Messy, 2011) (Note: Gleason & Van Scyoc (1995) offer evidence on economic literacy in the US. Jappelli (2010) offers international evidence. See also Nelson & Sheffrin (1991) and the studies cited therein. Lusardi & Mitchell (2014) offer a survey.). p Confirming Krueger’s (2002) assessments, Blinder & Krueger (2004) find that for US adults, print and elec- tronic mass media serve as the primary source of infor- mation about economic issues. Similarly, Cruijsen et al. (2010) find that the Dutch public learns about ECB’s mon- etary policy through the media. Caplan (2007) suggests that people are gullible and might believe what they read. A good analogy is offered by Gottschall (2012) in ana- lyzing the stubborn persistence of superstitions, which it turns out, are not limited to the uneducated. oi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 According to Gottschall (2012, p. 106), ‘Many conspir- acy theories would be funny except for the fact that stories . . . have consequences. For example, in Africa, many believe that AIDS is a racist hoax designed to . . . perpetrate a bloodless genocide. Believing this gets a lot of Africans killed.’ On July 21, 2014, Christiane Aman- pour ended her CNN show with a discussion of a denial of moon landing: ‘One of man’s greatest achievements [is] still . . . denied. 45 years ago . . . Armstrong and Aldrin walked on the moon . . . But almost from the start, there were those who said . . . that the whole thing had been staged . . . the deniers persist.’ (Source: edition.cnn.com/ TRANSCRIPTS/1407/21/ampr.01.html, accessed June 6, 2022.). Blinder & Krueger (2004) and Caplan (2001) find that educated people tend to think like economists. Jappelli (2010) finds that the overall economic literacy level is low, although he documents a positive correlation between education and economic literacy (Blinder et al., 2008). Education by itself, however, does not always guarantee economic literacy. For example, according to Blank (2002, p. 476), ‘A high share of . . . Congressional staff (never mind their bosses) do not understand basic economic principles.. . . in most meetings, my main role . . . was to lean forward and convincingly apply one of the follow- ing three concepts to the problem at hand: supply and demand, opportunity cost, or scarcity.’ ECONOMIC LITERACY ) However, economic studies of Harry Potter are scarce (Note: Economic analysis of literary works includes Watts (2002, 2003), Rockoff (1990) and Bookman & Bookman (2008).). Exceptions include Gouvin (2010) and Schooner (2010), who study the role of the Potterians’ only bank, the Gringotts, and Snir & Levy (2010), who try to under- stand the reasons for the lack of economic growth in the Potterian economy.Podemska-Mikluch & Deyo (2014) and Podemska-Mikluch et al. (2016) focus on the benefits of using Harry Potter series for undergraduate economics teaching. Salonikov et al. (2022) find evidence that the Harry Potter books affect readers’ perceptions of vari- ous economic issues including banks, bureaucracy and money. The topic of economic literacy is not new. Newcomb (1893, p. 395) wrote over 120 years ago about the need to educate the public because of the gap between ‘well- established economic conclusions on the one hand and the ideas of the public on the other.’ In 1948, at the First Workshop on Economic Education, Ernest Melby stated that economic education was a key to the survival of democracies: ‘Democracy will live if it works and . . . die if it does not...if it fails in the economic front it will . . . go down to defeat . . . [for] survival of our way of life, there is no kind of education more important than that which seeks to make the average American intelligent about our economic system’ (Troelstrup, 1954, p. 238). The importance of economic education was also emphasized by the US Fed. For example, the Fed Governor Ben Bernanke (2006) stated: ‘The Federal Reserve’s mission of conducting monetary policy and maintaining a stable financial system depends upon the participation and support of an educated public.’ Bernanke (2011) further emphasizes the individual benefits of economic literacy: ‘Well-informed consumers . . . are one of the best lines of defense against the proliferation of financial In this paper, we investigate the Potterian economy by analyzing its full structure. We find that it combines ingredients from various economic models but is not fully consistent with any particular model. Some fea- tures of the Potterian economy are in line with Marx- ist views, while others fit the public choice perspective. Prices in the Potterian economy are rigid in the Keynesian spirit, yet Potterians enjoy full employment as in the Classical model. 5 Levy and Snir products . . ECONOMIC LITERACY Indeed, according to Park (2008) and Aaronovitch (2010), many conspiracy theories originate and circulate among the educated. Many superstitions have survived long periods of time, and an incredible number of people seem to believe them. Despite these projects and efforts, the public rarely interprets economic ideas the same way as economists do (Alston et al. (1992) and Blendon et al. (1997).). Caplan (2007) argues that the public has various biases. (Note: Many biases of today’s public are remarkably similar to the biases noted more than a century ago by Newcomb (1893).), which Rubin (2003, pp. 157– 158) calls folk economics, and which ‘ . . . can explain the beliefs of naïve individuals regarding [economic] matters . . . [Folk economics] is the economics of wealth allocation, not production. Naive people . . . think of prices as allocating wealth but not . . . resources . . . The world of folk economics is zero-sum...if one person gets a job someone else must lose a job . . . Economists would do a better job of persuading others . . . if we paid explicit attention to folk economics.’ Krugman (1996) lists some popular books, and notes two features they share: ‘They all offer a view . . . of international trade as . . . “win-lose” competition . . . And they all contain little or nothing of what economists think . . . about international trade’ (p. viii). Referring to Lunt & Furnham (1996), Rubin (2003, p. 158) notes that . . . ‘the index to the book contains no entry for efficiency or productivity.’ Paldam & Nannestad (2000) find that Danish voters are myopic and have prediction biases. 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 6 The causal relationship between fiction and society, however, goes the other direction as well. The recognition that literature can influence society is not new. Consider, for example, the list of the books and writings that were banned throughout the history precisely because of the conviction that they had a strong and deep influence on the human mind. According to Newth (2010), the practice of censorship of books can be traced to the Romans, as far back as 443 BC, with the establishment of the Office of Censor: ‘In Rome, as in the ancient Greek communi- ties, the ideal of good governance included shaping the character of the people. Hence, censorship was regarded as an honorable task. In China, the first censorship law was introduced in 300 AD.’ to the US. For example, Cardinal Joseph Ratzinger, who later became Pope Benedict 16, in 2003 wrote a letter to the author of the book, Harry Potter: Good or Evil: ‘It is good that you enlighten us on the Harry Potter matter, for these are subtle seductions . . . barely noticeable, and precisely because of that have a deep effect and corrupt the Christian faith in souls even before it could properly grow.’ (Source: Pope Benedict XVI, March 2003 Letter, London Times, July 13, 2005.) y ) Confirming this view, the Vatican newspaper L’Osservatore Romano published an article on January 15, 2008, which stated that Cardinal Ratzinger was right to worry: ‘Despite the values that we come across in the narration, at the base of this story, witchcraft is proposed as a positive ideal. The violent manipulation of things and people, comes thanks to knowledge of the occult. The ends justify the means because the knowledgeable, the chosen ones, the intellectuals know how to control the dark powers and turn them into good. This a grave and deep lie, because it is the old Gnostic temptation of confusing salvation and truth with a secret knowledge. The characterization of common men who do not know magic as “muggles” who know nothing other than bad and wicked things is a truly diabolical attitude.’ (Source: M. Moore and N. Reynolds, ‘JK Rowling’s Harry Potter condemned in Vatican newspaper,’ The Telegraph, January 15, 2008.) Perhaps ironically, however, Amazon- UK ‘ . . . 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 revealed it had received advance orders for Harry Potter and the Half-Blood Prince from . . . the Vatican.’ (Source: ‘Pope Criticizes Harry Potter,’ R. Blakely, The Times, July 13, 2005.) The invention of the printing press in the 15th cen- tury increased the need for censorship further, primarily because of the emerging tensions between the Catholic Church and the Protestant movement, which led to the introduction of the first Index Librorum Prohibitorum (Index of Prohibited Books) by Pope Paul IV in 1559. According to Newth (2010), the Index was re-issued 20 times since then by different popes, and the last update was published in 1948. However, book censorship was not limited to religious authorities, and throughout the history, it served other goals and motives, primarily political. USSR and its allied countries took book censorship to new levels (recall the books of Solzhenitsyn, Sakharov, etc.). Perhaps surpris- ingly, libraries played an important role in supporting censorship: Public libraries were expected to act as the benevolent guardians of literature, particularly books for young readers. Consequently, this gave teachers and librarians license to censor a wide range of books in libraries, under the pretext of protecting readers from morally destructive and offensive literature. The perceived dangers that Harry Potter books pose, have been noted by other religious authorities as well. For example, in orthodox Judaism, some public figures, newspaper editorials, etc. ridiculed the fascination of so many people, young and old, with the imaginary tales of Harry Potter and his friends, arguing that it is indicative of the ‘... emptiness of the Western cultures’ (Source: Meir Shalev, ‘Holy Harry Potter,’ Yedioth Ahronoth (daily news- paper published in Israel in Hebrew), August 4, 2000). Some Rabbinical authorities, however, were more open minded. See, for example, Yvette Alt Miller, ‘Harry Potter and Jewish Values,’ December 21, 2014 (Source: https:// aish.com/harry-potter-and-jewish-values/, accessed June 24, 2022), ‘5 Quintessentially Jewish Concepts in the Harry Potter Saga,’ Forward, (Source: https:// forward.com/opinion/375984/5-quintessentially-jewish- concepts-in-the-harry-potter-saga/, accessed June 24, 2022), and Judy Siegel-Itzkovich, ‘Researchers: Harry Potter’s Wizardry OK with Rambam,’ Jerusalem Post, May 17, 2006 (Source: https://www.jpost.com/jewish-world/ judaism/researchers-harry-potters-wizardry-ok-with- rambam, accessed June 24, 2022). Even in liberal-minded countries such as Sweden and Norway, which boasts the earliest press freedom laws, surveillance of public and school libraries remained a concern to authors and publishers even through the latter part of the 20th century. HOW FICTION REFLECTS AND AFFECTS POPULAR VIEWS AND PERCEPTIONS Literary works serve as a mirror of society. According to Bloch (1961, p. 102), for example, ‘In every litera- ture, a society contemplates its own image.’ Similarly, Tiemensma (2010, p. 3) states that ‘Stories are the structural coding of social values, beliefs, and goals that underlie human interaction.’ Literature, even fictional, reflects the life, the views, the norms and the beliefs of the society. Indeed, texts are often used in social sciences to learn how people of different societies and different times view the world (Dickstein, 2005). 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 . . Most of us believe that we know how to separate fantasy and reality . . . this is not always the case. In the same mental bin, we mix information gleaned from both fiction and nonfiction.’ Vezzali et al. (2012) report that after reading books where characters with different cultural backgrounds had positive interactions with one another, Italian teenagers displayed more positive and less stereotypical attitudes toward immigrants. Peter Smith of the UK Teachers Association warns against the supernatural: ‘Children who had enjoyed the magic and wizardry of the stories should be careful about extending their interest in the occult.’ (Source news. bbc.co.uk/2/hi/uk_news/education/1638887.stm, June 6, 2022.) Some schools have banned the books because ‘ . . . they go against the Bible’s teaching.’ (Source: news. bbc.co.uk/2/hi/uk_news/education/693779.stm, June 6, 2022.) Some British toy shops have even refused to stock Potterian merchandise fearing it will attract children to the occult (Source: news.bbc.co.uk/2/hi/ entertainment/1560335.stm, accessed June 6, 2022.). According to the American Library Association, in 2005 ‘ . . . there were 26 challenges to remove the Harry Potter books from bookshelves in 16 states.’ (Source: www. educationworld.com/a_admin/admin/admin157.shtml, accessed June 6, 2022.) Mar (2004, p. 1414), based on evidence from neu- roimaging, argues that ‘ . . . reader attitudes shift to become more congruent with the ideas expressed in a narrative after exposure to fiction.’ Appel & Richter (2007) and Appel (2008) find that fiction shapes readers’ views on fairness and justice (Note: The July 29, 2014 edition of ‘Room for Debate’ of the NY Times asked, ‘Will Fiction Influence How We React to Climate Change?’) Vezzali et al. (2015) survey psychology literature, which shows that novels can have positive social impact. Green et al. (2004) find that fictional worlds alter the way we process information, and that the deeper we are immersed in a story, the more influential the story is. According to Gottschall (2012, p. 135), ‘Fiction readers who reported a high level of absorption tended to have their beliefs changed in a more “story-consistent” way... [and] detected significantly fewer “false notes” in stories—inaccuracies, infelicities . . . When we read nonfiction, we read with our shields up. We are critical and skeptical. But when we are absorbed in a story, we drop our intellectual guard. We are moved emotionally, and this seems to leave us defenseless’. See also Sklar (2009). 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 Not less surprising is the die-hard tradition of surveillance of books in schools and libraries in the United States. For example, the Library of Congress is holding a multi-year exhibition featuring The Books that Shaped America (https://www.loc.gov/exhibits/ books-that-shaped-america/overview.html, accessed June 6, 2022.). According to the curators, ‘Some of the titles on display have been the source of great controversy, even derision, yet they nevertheless shaped Americans’ views of their world and often the world’s view of the United States.’ Many of the books displayed at the exhibit were banned in the US (Haight, 1978). The American Library Association lists on its page the top 100 banned books during the years 2000–2009. Harry Potter books top the list, which were banned in the US by some religious authorities as well as by some schools (Scheffer, 2010) (Source: http://www.ala.org/advocacy/ bbooks/top-100-bannedchallenged-books-2000-2009, accessed June 6, 2022.). This, however, was not limited Similarly, Harry Potter books have been banned in some Islamic countries, but not everywhere. For example, the books were banned in the United Arab Emirates (Source: ‘Emirates Ban Potter Book,’ BBC News, February 12, 2002, http://news.bbc.co.uk/2/hi/ Similarly, Harry Potter books have been banned in some Islamic countries, but not everywhere. For example, the books were banned in the United Arab Emirates (Source: ‘Emirates Ban Potter Book,’ BBC News, February 12, 2002, http://news.bbc.co.uk/2/hi/ \| 7 Levy and Snir 7 entertainment/1816012.stm, accessed June 24, 2022). According to some Islamic scholars, the books themes conflict with Islamic teaching because ‘Islam pro- hibits both pointless entertainment (lahw) and sorcery’ (Source: Khalid Baig, ‘Harry Potter: Facts about Fiction,’ June 21, 2003, http://www.albalagh.net/current_affairs/ harry_potter.shtml, accessed June 24, 2022). In Iran, in contrast, the books were published by the Ministry of Culture and Islamic Guidance (Source: https://www. hogwartsprofessor.com/ayatollah-condemns-harry- potter/, accessed June 24, 2022). stereotypes. We use the term similarly to describe the way people perceive and think about the world in which they live, operate and make decisions.). The story maker penetrates our skulls and seizes control of our brains . . . fiction subtly shapes our beliefs, behaviors, ethics . . . ’ (Gottschall, 2012, pp. xvi–xvii). Hakemulder (2000) reviews dozens of studies in psychology that demonstrate that fiction can have a profound effect on the readers’ thinking. Gottschall (2012, p. 133–134) argues that ‘Fiction does mold our minds . . . influences our moral logic . . . alters our behav- ior...shaping our minds without our knowledge . 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 (2005), for example, find a drop in the number of chil- dren visiting the hospital emergency departments on the weekends that Harry Potter books are released, whereas Bennett (2003) reports that some of the young readers of the Harry Potter series have suffered from headaches because of their insistence to read the books cover-to- cover without taking a break. Neuroscientists offer further evidence on the effect of the Potterian adventures on the human mind. Recently, for example, in a series of neuroimaging studies on the influence of Harry Potter books on their readers, Hsu et al. (2014, 2015a, 2015b) provide fMRI evidence for the fiction feeling hypothesis, which states that narratives with emotional content, in contrast to stories with neu- tral content, cause readers to empathize to a stronger degree with the protagonists, thus engaging the affective empathy network of the brain. See also Wehbe et al. (2014) and Lehne et al. (2015). ( ) ( ) In sum, the existing evidence in psychology, sociology and neuroscience suggests that fictional literature in general, and Harry Potter books in particular, can have a subtle yet powerful influence on the readers’ views, opinions and attitudes. Similar to these influences, and consistent with Krueger (2002) and Cowen (2008), we propose that the readers of the Harry Potter books, con- sciously and/or subconsciously, absorb the ‘economic’ ideas the books implicitly or explicitly convey. These ideas, we argue, can potentially shape the public’s opin- ion on economics and economic issues. This is consistent with the argument of Cowen (2008) who describes this as a ‘knowledge-generation process.’ Novels and mod- els, he argues, both are mechanisms for learning that complement each other. ‘We should recognize the power of stories. Many models, especially the most relevant models, are embedded in stories, further illustrating the complementarities between novels and models . . . . Both novelists and model builders have tacit knowledge about how the real world works, and they try to articulate that knowledge in the form of either a story or equations’ (pp. 333–334). ( ) Consider, for example, Cowen’s (2008, pp. 325–326) description of the similarities between fictional stories and economic models: ‘Science fiction stories . . . embody model-like thinking. The author writes down a descrip- tion of some new technologies . . . 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 more positive and sympa- Oxford Open Economics, 2022, Vol. 1, No. 1 8 thetic. Gierzynski & Seger (2011) find that the books influence the readers’ acceptance of difference, toler- ance, equality and opposition to violence and corrup- tion. Salonikov et al. (2022) offer evidence suggesting that the Harry Potter books affect the readers’ attitudes towards economic issues, including banks, the public sector, money and monopolies. Thomas and Snyder (2010, p. vii) state along these lines: ‘Part of the appeal of [Harry Potter] is that the depic- tions resonate with readers . . . This may suggest that the depictions are consistent with readers’ and viewers’ val- ues or opinions. Alternatively, if the depictions are not reflective, they may influence the development of values or opinions . . . any influence would be . . . subtle.’ Downloaded from https://academic.oup.com/ooec/article/doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 A comparison between economic models and literary texts is possible because they share several key charac- teristics (Cowen, 2008, McCloskey, 1998, 2000, Thomson, 2001, Rubinstein 2006) (Note: According to McCloskey (1998, p. xiv): ‘Economists are poets, but don’t know it. Economists are storytellers.’ Similarly, Cowen (2008, p. 15) states that ‘Novels are more like models than is commonly believed’ and recommends studying them to better understand how people think about economics and economic matters.). Both offer imaginary tales that are abstractions of reality. Both are composed of a set of actors—characters in stories, decision-makers in models and a set of assumptions—rules in stories and con- straints and assumptions in models. In addition, the characters in a story are linked by initial relationships, the same way as decision-makers and variables in a model are linked by initial conditions. In both stories and models, the initial relationships evolve. In models, these follow the assumptions and optimal decisions and strate- gies. In stories, they follow the characters’ attributes and the actions they can and cannot take. Eventually, both economic models and stories conclude in a final state (Rockoff, 1990; Watts, 2002). Some studies have even documented that the Harry Potter books affected the readers’ health. Gwilym et al. 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 A recent episode in Israel suggests that even People of the Book might be afraid of books and find them dangerous. In 2015, the Israeli Education Ministry has decided not to include ‘Border Life,’ a novel by Dorit Rabinyan, in its list of required readings for the matric- ulation exams, because it includes a love story between a Jewish Israeli woman and a Muslim Palestinian man. Many Israeli school directors and educators fought the Ministry over this decision and insisted on including the book in their school’s required readings lists. Authors also believe that their work can affect the readers. According to Gottschall (2012), Tolstoy believed that an artist’s job is to ‘infect’ his audience with his own ideas and emotions—‘the stronger the infection, the better is the art as art’ (p. 134). Similarly, ‘In his book “The Act of Reading,” Wolfgang Iser writes that ideally, a book should transform a reader by “disconfirming” his habitual notions and perceptions and thus forcing him or her to a new understanding of them’ (Tuck, 2015). Indeed, there are many fictional works with a long-lasting impact on popular views and opinions. These include Uncle Tom’s Cabin, Black Beauty, The Birth of a Nation, Jaws, 1984, Dark- ness at Noon, Roots, etc. We focus on the Harry Potter books because of the evi- dence that these particular books affect popular views. Indeed, studies suggest that the attitudes of the readers of the Harry Potter books are influenced by the messages these books convey. Hallett (2005) notes that Harry Potter books influence culture. Brown & Patterson (2009) study brand assessment by focusing on ‘Pottermania.’ Brown & Patterson (2010) study consumers’ treatment of the Harry Potter brand. See also the opening quote from Barton (2006). The belief that books influence the readers has been corroborated by recent studies in psychology that offer systematic evidence of fictional stories’ effects on the human mind and attitudes. These studies find that when we read fiction, ‘ . . . we allow ourselves to be invaded by the teller (Note: In social psychology, the term ‘attitude’ refers to personal and social norms, prejudices, and Some studies offer even more direct evidence on the effect of Harry Potter books on the readers. Vezzali et al. (2015) report that Harry Potter books make the readers’ attitudes toward stigmatized groups such as immigrants, homosexuals, refugees, etc. 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 The author then traces through the effects of these technologies and outlines how things would work or outlines an equilibrium in economic terminology. That equilibrium is then “dis- turbed” by some new change, such as an alien invasion or a new technology. The bulk of the novel then traces through the effects of the change, performing a kind of comparative statics exercise . . . these novels . . . use a stylized setting to show how one set of causes leads to particular effects, working through a mechanism of some generality. The mechanism is not always spelled out explicitly...They are like the models from earlier in the history of economics. Before the mathematization of the economics profession . . . models without explicit math- ematical forms... It is no accident that contemporary model builders sometimes refer to earlier, non-formal economists as “telling stories”.’ The success of Harry Potter books suggests that in addition to telling a story that appeals to a broad and diverse audience, the author may have also been able to capture the readers’ popular beliefs. Given the books’ universal appeal, therefore, studying the Potterian econ- omy and comparing it to standard economic models can shed light on the beliefs and views of millions of people with diverse cultures and norms about the economy and economic ideas. Another similarity between models and stories is that stories must maintain face validity, the same way as models need to maintain internal validity. Well-defined rules of math and logic ensure models’ internal valid- ity. Maintaining face validity in literary texts depends on the genre, on adhering to the readers’ norms/ex- pectations and on preserving the story’s internal logic Levy and Snir 9 secrets about Voldemort. The book ends with Harry and his friends saving Ginny while fighting the monster. In the process, they inadvertently destroy a part of Voldemort’s soul (‘Horcrux’). (Derrida, 1993). For example, if magic can solve all prob- lems, then the resulting story, even fictional, is unlikely to be interesting. The Harry Potter books’ success and near-universal popularity suggest that they satisfy this condition, and therefore, we believe that Potterian eco- nomics can offer lessons about the economic principles the books teach and convey. In the third volume, Harry Potter and the Prisoner of Azkaban, Harry learns about Remus Lupin and Sirius Black, who were his father’s friends. GENERAL BACKGROUND (SPOILER WARNING: SKIP IF YOU HAVE NOT READ THE HARRY POTTER BOOKS, BUT PLAN TO READ THEM!) The Harry Potter story, an imaginary tale of a boy with extraordinary wizardry powers, is a series of seven books (Note: See Rowling (1998, 1999a, 1999b, 2000, 2003, 2005 and 2007). The discussion in this section is based on Anandane’s (2011) descriptive summary of the seven volumes.). Most of Harry’s adventures take place in and around Hogwarts, a boarding school of witchcraft and wizardry located in northern England, where Harry spends 7 years. The books follow Harry and his friends, as they grow up and mature, and the 7 volumes describe correspondingly the 7 years Harry spends at Hogwarts. In volume 5, Harry Potter and the Order of the Phoenix, Harry learns about a secret society called the ‘Order of the Phoenix’, which is re-activated to protect Harry and his friends from Voldemort and his supporters. The school’s new Headmaster Dolores Umbridge (‘the High Inquisitor of Hogwarts’) does not permit the students to learn defense against dark magic. Harry, therefore, forms a secret study group called ‘Dumbledore’s Army’, where he teaches his friends how to fight and defeat dark arts. Eventually, Harry foresees Voldemort’s actions, and thus manages to save Hogwarts from Voldemort’s supporters, the Death Eaters. The story begins in 1991 with Harry Potter and the Philosopher’s Stone, where Harry is a shy orphan living with his aunt in a suburb of London. At age 11, Harry learns that his parents were wizards and that they were killed by the most powerful wizard of the era, ‘Lord Voldemort’, whose reputation is so fearsome that his name is not mentioned and instead is referred to as ‘You-Know-Who’ or ‘He Who Must Not Be Named.’ Harry also learns that although he grew up among non-wizards (‘muggles’), he has the power to become a wizard (Note: According to the Oxford dictionary, ‘muggle’ is now an official word, and it means ‘a person who is not conversant with a particular activity or skill.’). Furthermore, he discovers that Lord Voldemort tried to kill him after killing his parents, which left a scar on his forehead. Harry survived the attack, which gives him a special role in the wizards’ society. The school headmaster, Dumbledore, who knows about this incident, leaves Harry with his aunt until he is 11, when he is ready to enroll in Hogwarts. 6 \| Oxford Open Economics, 2022, Vol. 1, No. 1 Lupin is a teacher of defensive measures against dark magic. Black, it turns out, is a murderer believed to have helped Voldemort in killing Harry’s parents. In volume 4, Harry Potter and the Goblet of Fire, Harry is pressed by a Voldemort supporter, Barty Crouch (disguised as Professor Alastor Moody), to participate in a dangerous Tri-Wizard Tournament. Harry luckily escapes Crouch’s plans, and at the same time he forces Voldemort to reenter the wizarding world as a mortal. GENERAL BACKGROUND (SPOILER WARNING: SKIP IF YOU HAVE NOT READ THE HARRY POTTER BOOKS, BUT PLAN TO READ THEM!) Following the Hogwarts’ invitation, which is delivered by an owl, Harry enters the school, where he and his new friends, Hermione Granger and Ron Weasley, explore the world of magic, and slowly discover the powers of witchcraft and Wizardry. The first year ends when they recover the Philosopher stone, which can be used to brew an elixir that can make the drinker immortal. By the sixth year, in Harry Potter and the Half-Blood Prince, the 17-year-old Harry, who is dating Ginny Weasley, incidentally, comes across an old potions textbook filled with annotations signed by an anonymous individual, the Half-Blood Prince. He also discovers that Voldemort’s soul is split into a series of horcruxes (evil-enchanted items hidden in various locations). Draco Malfoy, Harry’s foe, attempts to attack Dumbledore several times. Even- tually, Dumbledore is killed by Professor Snape, another Harry adversary. In the last volume, Harry Potter and the Deathly Hallows, Lord Voldemort takes control of the Ministry of Magic. Harry and his friends quit school and go on a mis- sion of finding and destroying the remaining horcruxes of Voldemort. Harry discovers, however, that he is one of the horcruxes, and thus surrenders to Voldemort. It turns out, however, that the horcrux inside Harry has been destroyed when Voldemort tried to regain his full strength. In the end, the Order of the Phoenix, along with Harry and Harry’s friends, defeat Voldemort and his supporters, and thus save the world of Witchcraft and Wizardry. In the second year at Hogwarts, Harry Potter and the Chamber of Secrets, Ron’s younger sister Ginny, discovers Voldemort’s old notebook, in which she reads about a ‘Chamber of Secrets,’ which, it turns out, leads to a mon- ster. In this volume, Harry and his friends learn about the history of Hogwarts. Harry also discovers that he has some special skills, such as the ability to communicate with snakes (rather rare dark art). He also discovers some Money Wizards use commodity money. They have three types of coins: gold Galleons, silver Sickles and bronze Knuts, where one Galleon equals 17 Sickles, and one Sickle Oxford Open Economics, 2022, Vol. 1, No. 1 10 equals 29 Knuts (Rowling, 1998, p. 49). The wizards’ mon- etary system is therefore similar to the old English mone- tary system that existed from medieval times until 1971. In that system, one pound was worth 20 shillings and one shilling was worth 12 pennies. The similarity is also in the types of metals used in minting the coins. The pound (also known as Guinea) was originally made of gold, the Shilling (originally Scilling) was made of silver, and the penny (after 1796) was made of copper. modern decimal system is in the number of ways a pound could be divided into combinations of shillings and pen- nies. This divisibility allowed the minting of many coins that were combinations of pennies, shillings and pounds. For example, common English coins included 2 shillings, 2 shillings 6 pence, 5 shillings and many more. In the case of Potterian money, however, the exchange rate between the Galleons and the Sickles and between the Sickles and the Knuts, are both prime numbers. It is, therefore, less useful to mint coins that are multiples of these basic coins. However, despite their superficial similarity, the wizards’ monetary system differs greatly from its real- world counterpart. First, whereas in the old English system the value of the pound relative to the shilling and the penny fluctuated freely as a result of changes in the relative prices of gold, silver and copper, the relative values of the Galleons, Sickles and Knuts are fixed. Furthermore, whereas the values of the English coins depended on the amount of the precious metal minted in the coins, the values of the Potterian coins are independent of their physical size and weight. For example, when wizards from several countries gather, it seems that the value of all gold coins is the same, even though the foreign Galleons are ‘the size of hubcaps’ (Rowling, 2000, p. 50), suggesting that the Potterian money is not homogenous, a key property that money should have (Levy and Bergen, 1993). The Potterians however, seem not to care about this. Money Medieval merchants and bankers took advantage of this benefit of paper checks (Quinn and Roberds, 2008), yet it seems that Potterians have yet to discover this technol- ogy. It is possible that the Potterians use precious metals as money because it is harder to counterfeit money that is minted of precious metals than of other materials. However, it is possible to counterfeit the wizard’s money. It is also possible that the Potterians use commodity money because they believe that pegging the value of money to commodities might be an effective way of controlling inflation (Woodford, 2003). However, it turns out that when the supply chain is disrupted, the outcome is inflation, as in an economy that uses fiduciary money. Indeed, just like fiduciary money, the values of all types of Potterian coins decrease simultaneously when there is inflation as a result of a war that disrupts the supply (Rowling, 2005, p. 43). The values of the three types of coin denominations, however, do not change relative to each other, although we are told explicitly that at the same time as the inflation bout, the demand for silver increases because silver is used for making charms and apparatus that are in demand (Rowling, 2005, p. 73). Another important shortcoming of the Potterian mon- etary system is the lack of paper checks that could be used to transfer funds without the need to carry cum- bersome coins, making transactions far more efficient. Medieval merchants and bankers took advantage of this benefit of paper checks (Quinn and Roberds, 2008), yet it seems that Potterians have yet to discover this technol- ogy. Thus, in the Potterian economy, commodity value of the money is distinct from the exchange value of money. This is counter to the models of commodity money where the value of the money is pegged to the value of the commodity it is made of (Sargent and Wallace, 1983; Rockoff, 1990) (Note: In The Lord of the Rings, for example, the value of gold as a medium of exchange is determined by weighing it. Thus, Tolkien seems to have understood this aspect of the difference between commodity money and fiduciary money.). Money Whatever the reason for using commodity money, its use has real effects beyond the opportunity cost of using the precious metals for making jewelry or silver dag- gers (Rowling, 1998, p. 8, Rowling, 2000, p. 413, Rowling, 2003, p. 45). Carrying too many coins is risky and cum- bersome. Therefore, Wizards store most of their money in bank vaults. Withdrawals and deposits, however, are time-consuming, as they require a lengthy bureaucratic procedure, implying a high transaction cost (Rowling, 1998, pp. 47–49). Consequently, wizards make infrequent withdrawals from the bank (Rowling, 2000, pp. 61, 352). In sum, the Potterian money lacks some of the basic properties that economists believe money should have, for it to serve its functions efficiently: homogeneity, portability, divisibility and storability (Levy and Bergen, 1993). The Potterian money is not homogenous, it is not easy to transport, it is not easily divisible and it is difficult to store. In any efficient economy, the Potterian money would have therefore been replaced by more efficient money, which is more divisible and less cumbersome. Thus, although the wizards’ money is a type of com- modity money, it behaves as if it was fiduciary money. For example, in the muggle (non-wizard) world, the ratio of the price of gold to the price of silver, over the period 1991–1998, when Harry Potter’s adventures take place, varied between 48.2 to 91.6 (Source: https://sdbullion. com/, accessed June 6, 2022.). Yet the conversion rates between the Galleon, Sickle and Knut remained fixed. y It is possible that the Potterians use precious metals as money because it is harder to counterfeit money that is minted of precious metals than of other materials. However, it is possible to counterfeit the wizard’s money. It is also possible that the Potterians use commodity money because they believe that pegging the value of money to commodities might be an effective way of controlling inflation (Woodford, 2003). However, it turns out that when the supply chain is disrupted, the outcome is inflation, as in an economy that uses fiduciary money. Another important shortcoming of the Potterian mon- etary system is the lack of paper checks that could be used to transfer funds without the need to carry cum- bersome coins, making transactions far more efficient. Banks, interest and credit Muggles also have limited opportunities to exchange muggle money for wizards money because most muggles are not aware of the wizards’ existence (Rowling, 1998, p. 42, Rowling, 1999b, p. 80, Rowling, 2000, p. 50). The books do not explain the reasons for the lack of financial intermediaries. However, it seems that the Potterians view financial service providers as immoral, the same way as prisoners of war were suspicious of those who offered financial services at the POW camp according to the account of Radford (1945) (Notes: See Rowling (1998, p. 41), Rowling (2000, pp. 81, 287, 471), Carlton (1995), and Hillman (2010). Portraying bankers in fictional works as evil has been common throughout history. Examples include Shylock in Merchant of Venice, Harpagon in Miser, Danglars in The Count of Monte Christo, Mr. Merdle in Little Dorrit, Mr. Banks in Mary Poppins, the hero in American Psycho, and Le Chiffre in Casino Royale.). The negative image of bankers is so strong that wizards shy away from such jobs. Consequently, the banking jobs are taken by Goblins, an inferior social group. (Note: Gringotts apparently employs some humans. Fleur Dela- cour (Rowling, 2003, p. 53) and Bill Weasley (Rowling, 2007, p. 196) are two such examples.). Gringotts, however, is not an ordinary bank, as it does not offer lending and borrowing services. In fact, Pot- terians do not have a financial institution that offers such services. That is, unlike modern banks, the Potte- rian bank does not operate based on fractional reserves. For example, we do not find a single case of someone borrowing money from Gringotts, which suggests that Gringotts is not offering loans. This is the interpreta- tion of other Harry Potter scholars as well. For exam- ple, consider Schooner’s (2010, p. 265) interpretation: ‘Most important, however, is the question of lending. If Gringotts is a true bank, then it not only takes deposits, but it also lends out a portion of those deposits to other customers. The image of Harry and his vault full of money suggests that all of Harry’s money remains in the vault at all times. This would mean that Gringotts does not operate on the basis of fractional reserves, i.e. it does not lend out a percentage of the money deposited by its customers.’ Wizards that want to borrow, must therefore borrow from a friend or from illegal usurers (Rowling, 2005, p. 78, Rowling, 2000, p. Banks, interest and credit Potterians have only one bank, Gringotts, making it a textbook version of a perfect monopoly. The bank serves mostly wizards, although the bank’s owners and employ- ees are Goblins (Rowling, 1998, p. 41)—greedy, gold loving, selfish and unfriendly humanoids (Rowling, 2000, p. 81). Gringotts offers several services. First, it is in charge of minting money and preventing its counterfeiting. There Potterians have only one bank, Gringotts, making it a textbook version of a perfect monopoly. The bank serves mostly wizards, although the bank’s owners and employ- ees are Goblins (Rowling, 1998, p. 41)—greedy, gold loving, selfish and unfriendly humanoids (Rowling, 2000, p. 81). Another shortcoming of the Potterian monetary sys- tem is that, unlike monetary economy models, Potterian money lacks some key features that would facilitate trade. One advantage the old English system had over the Gringotts offers several services. First, it is in charge of minting money and preventing its counterfeiting. There Levy and Snir 11 government also cannot issue debt and, therefore, it often depends on donations from wealthy individuals for funding public goods (Rowling, 2000, p. 66, 456). are, however, several ways to counterfeit money, and even schoolchildren can do it (Rowling, 2003, p. 297). The amount of counterfeit money in circulation is low, nevertheless. It appears that it is not because of a lack of willingness that Gringotts does not lend money. Indeed, Gringotts’ employees sometimes offer private usury services (Rowling, 2000, p. 471). There is no shortage of demand for loans either. On the contrary, the lack of borrowing options is a significant constraint. For example, wizards that make windfall gains spend them immediately, and entrepreneurs without capital cannot open businesses, suggesting that both consumers and businessmen face credit constraints (Rowling, 1999b, p. 5). Second, wizards take advantage of the Gringotts’ money-storage and safekeeping services for storing their gold and other valuables (Rowling, 1998, pp. 48–49). Third, the bank offers currency exchange services, which include exchanging wizard money for precious stones, pieces of art and for muggle money (Rowling, 1998, p. 47, Rowling, 1999a, p. 37). The amount of money exchanged, however, is limited. Wizards use muggle money only when they run errands in the non-wizard parts of England. This happens rarely, and when it does, they face significant difficulties in using muggle banknotes (Rowling, 1998, p. 43, Rowling, 2000, p. 50). Banks, interest and credit 471). doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 p ) p ) Thus, in the Potterian economy, economic incentives are overridden by the power of socially stigmatizing financial service providers. In Europe, such processes led to the prosecution and stigmatization of minority groups (Carlton, 1995; Hillman, 2013a). Some have noted that in the movie adaptation of the Harry Potter books, the goblin bankers are depicted with aquiline noses and greedy-looking faces, similar to the cartoons that were used to depict stereotypical bankers and financiers in Europe in the late 19th century (Source: momentmaga zine.wordpress.com/2011/07/14/debunking-the-harry- potter-anti-semitism-myth/, accessed June 6, 2022.) A reader has suggested that the goblins are merely a joke on the theme of Gnomes of Zurich. That, however, does not explain why they are depicted in the movies with aquiline noses. On the other hand, it could be argued that even if goblin characters were historically used to depict financial service providers negatively, currently they are a part of Western literary characters. (Note: A eugenics view could also be suggested. See Peart & Levy (2005) on eugenics in the post-classical 19th century economics.). Similarly, in the Potterian economy, the stigmatization of financial intermediation leads to a tension between wizards and Goblins, with Goblins being treated as inferiors even though they provide cheap, efficient, and economically beneficial services. It also Indeed, we find numerous episodes where wizards bor- row money from friends, private usurers, or some other wealthy individuals, but not from Gringotts. For example, to open their joke shop, the ‘Weasleys’ Wizard Wheezes,’ Fred and George Weasley borrow the necessary start-up money from Harry Potter (Rowling, 2003, p. 79, Rowling, 2005, p. 78). As another example, Ludo Bagman, a senior public official who is in debt after running a failing private enterprise, borrows ‘loads of gold’ from Goblins (Rowling, 2000, p. 471). Moreover, we find cases where wizards in need of money resort to gambling with the hope of winning the necessary funds. For example, the Weasley twins, before discovering that they can borrow money from Harry, consider gambling as a means of obtaining the funds needed to open their shop (Rowling, 2000, p. 57, Rowling, 2005, p. 78). Because there are no financial markets, the Oxford Open Economics, 2022, Vol. 1, No. 1 12 inhibits most forms of interaction between wizards and Goblins (Rowling, 2007, pp. 323, 342). Potterian economy, leads to wizards facing a cash (i.e. Clower) constraint. Downloaded from https://academic.oup.com/ooec/article/doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 However, beyond their effect on the price level, the cumbersomeness of the Potterian money also affects the pricing strategies and possibly even the extent of price rigidity (Dutta et al., 2002). Many prices are set in round numbers, such as 5 Knuts, 10 Sickles, 30 Galleons, etc. Examples include Goblin-made armor for 500 Galleons (Rowling, 2005, p. 286), Acromantula venom for 100 Galleons/pint (Rowling, 2005, p. 316), Deflagration Deluxe for 20 Galleons (Rowling, 2003, p. 472), Omnioculars for 10 Galleons (Rowling, 2000, p. 60), weekly pay of 10 Galleons to house-elf (Rowling, 2000, p. 244), Metamorph-Medals for changing appearance for 10 Galleons (Rowling, 2005, p. 58), Basic Blaze box for 5 Galleons (Rowling, 2003, p. 472), Glittery-Black Beetle Eyes for 5 Knuts/scoop (Rowling, 1998, p. 52), etc. In cases where prices are not round, then they are still denominated in units of a single type of coin, and thus they can be paid using one type of coin. Examples of such convenient prices include a new copy of Advanced Potion-Making for 9 Galleons (Rowling, 2005, p. 144), a ticket for a night bus to London for 11 Sickles (Rowling, 1999b, p. 22), hot chocolate on a night bus for 2 Sickles (Rowling, 1999b, p. 22), membership in the Society for the Promotion of Elfish Welfare for 2 Sickles (Rowling, 2000, pp. 144–145), etc. Such price-setting rules, however, have a disadvantage because they lead to a type of grid pricing, which is inefficient. For example, a Galleon is composed of 17×29 = 493 Knuts, but it seems that prices that are set in Galleons only change by Galleons, and, consequently, retailers cannot change them by smaller amounts in response to small shocks. It is not surprising, therefore, that once there are disturbances to supply, prices go ‘sky high’ (Rowling, 2005, p. 65). Thus, the Potterians’ ‘ban’ on lending, leads to finan- cial transactions being handled by goblins, which further strengthens their stereotypical image as greedy. These observations are striking given that the books are often viewed and interpreted as opposing all types of stereo- typical biases (Gierzynski and Seger, 2011; Vezzali et al., 2015). In summary, the ban on lending leads to an outcome that is opposite to the values that the books are believed to promote. Downloaded from https://academic.oup.com/ooec/article/doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 Further, these credit constraints reduce wel- fare, increase corruption because it limits the ability of would-be-entrepreneurs to open new businesses and thus increase the influence of wealthy wizards (Paldam, 2002; Fudenberg and Levine, 2006). In sum, in the Potterian economy, the role of financial intermediation is completely orthogonal to its role in eco- nomic models. Economists view financial intermediation as a key mechanism for promoting investment through efficient channeling of funds from savers to investors. The Potterians, whose bank enjoys monopoly power, in contrast, view financial intermediation as means to impoverish the borrowers and enrich the lenders. Thus, whereas retailers in real economies tend to set many prices at psychological price points such as 9- ending prices (Levy et al., 2011, Levy et al., 2020), which studies find affect the quantity demanded (Snir and Levy, 2021; Chen et al., 2022), Potterian retailers stick to conve- nient prices. The only product with a 9-ending price we were able to find is a new copy of Advanced Potion-Making from Flourish and Blotts, with a price tag of exactly 9 Galleons (Rowling, 2005, p. 144). Since the Potterian currency system is not decimal, it could be argued that the Potterian parallels of the Muggles’ 9-ending prices are the prices that end with either 16 Sickles or 28 Knuts (i.e. ‘just below prices’), which would be equivalent to prices like 11 pennies or 19 shillings that were documented by Gabor and Granger (1966) in the UK under the pre- decimal currency system. However, we find only one such good, dragon liver, with a price of 16 Sickles per ounce doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 ( g pp ) Such negative attitudes still prevail. For example, in a 2005 Roper poll, ‘Only 9% . . . had full trust in financial ser- vices institutions, down from 14% last year’ (Source: ‘New Surveys Show that Big Business Has a P.R. Problem’, by C. Deutsch, New York Times, December 9, 2005, Late Edition- Final, Sec. C, Column 2). The 2008 financial markets’ troubles have likely deepened the distrust between the public and bankers even further. For example, in July 2015, Argentines’ President, Cristina Fernández de Kirch- ner, according to Wall Street Journal, told Buenos Aires schoolchildren, after learning that they were reading Romeo and Juliet, that they should ‘ . . . read “The Merchant of Venice” to understand the vulture funds’, alluding to a decade-long financial battle to collect $1.5 billion from Argentina on defaulted foreign bonds held by a US hedge fund. In a follow up tweet, Kirchner added: ‘No, don’t laugh. Usury and bloodsuckers have been immortalized in the greatest literature for centuries.’ As the author notes, ‘The persistence of anti-Semitism over time and across cultures is one of mankind’s darkest puzzles. So is the hatred of capitalism, property rights and freedom.’ (Source: ‘The Bigot Defense: The Oldest Prejudice Reap- pears in Attacks on American Capitalists’, Wall Street Journal, July 10, 2015. The cash constraint forces retailers to set low prices for basic goods and services, as otherwise wizards would be caught cashless (Rowling, 1998, p. 41). For example, Harry Potter withdraws money only once every year, before going to Hogwarts, and the sum he withdraws has to last him through the entire school year (Rowling, 1999a, 50). PRICES In sum, the price rigidity in the Potterian economy seems to be due to a combination of three factors. First, thanks to the monopolistic structure of the market, the Potterian retailers have relatively high profit margins, which allows them not to change the prices in response to small cost changes, and still remain profitable. Second, the lack of growth of the Potterian economy makes large changes in the cost and demand rare. Third, the prevalent use of convenient prices suggests that retailers believe that such prices increase the quantities sold. Retailers therefore change prices only when the cost or demand shock is large enough to merit a change to another convenient price. Given the low price level, however, this means a large price change (in percentage terms), which is unlikely because the Potterian economy is not growing (Snir and Levy, 2010). Moreover, the Potterian economy is not growing, im- plying that there is no upward trend in the aggregate demand (Snir and Levy, 2010). Nor do the Potterian retail- ers experience major changes in their cost structure. These factors, along with the currency structure decrease the need and the willingness of the Potterian retailers to adjust prices. Simply, there are not sufficient changes in the market conditions that would warrant such price ad- justments, except when a war breaks out (see section 13). justments, except when a war breaks out (see section 13). In sum, the price rigidity in the Potterian economy seems to be due to a combination of three factors. First, thanks to the monopolistic structure of the market, the Potterian retailers have relatively high profit margins, which allows them not to change the prices in response to small cost changes, and still remain profitable. Second, the lack of growth of the Potterian economy makes large changes in the cost and demand rare. Third, the prevalent use of convenient prices suggests that retailers believe that such prices increase the quantities sold. Retailers therefore change prices only when the cost or demand shock is large enough to merit a change to another convenient price. Given the low price level, however, this means a large price change (in percentage terms), which is unlikely because the Potterian economy is not growing (Snir and Levy, 2010). The abundance of convenient prices seems to affect price rigidity (Snir et al., 2021). Prices in the Potterian economy change only infrequently. PRICES The difficulties in carrying and transporting money, together with the lack of credit market, have real effects on the price level and on pricing strategies. Since bank withdrawals and deposits are time-consuming, wizards make infrequent cash withdrawals, as predicted by the Baumol–Tobin inventory model of money holding. This, in addition to the limited (or no) supply of credit in the 13 Levy and Snir menu cost (Levy et al., 1997 and 1998, Dutta et al., 1999, Zbaracki et al., 2004, Fisher & Konieczny, 2006) because most prices in the Potterian economy are not posted.). In that case, round prices will be adjusted only if the round endings can be preserved. Coca-Cola faced a similar con- straint in 1950s. It was unable to adjust the price because increasing it while allowing people to use a single coin to buy it, meant doubling the price from 5c (a nickel) to 10c (a dime) (See Levy & Young (2004, 2021) and Young & Levy (2014).). In modern markets, unlike the Potterian markets, however, round prices are usually limited to particular types of goods and settings (Snir et al., 2022). Thus, the Potterians’ currency structure and the resulting pricing practices lead to prices that are set at a few convenient price points, which hinder price changes. (Rowling, 1998, pp. 55–56). Thus, there are almost no ‘just below’ prices, despite the widespread recognition that they increase sales (Anderson and Simester 2003) and despite the fact that they were common in the old English monetary system (Gabor and Granger 1966). Thus, the inefficiency of the monetary system leads to inefficien- cies in sellers’ price setting practices. (Rowling, 1998, pp. 55–56). Thus, there are almost no ‘just below’ prices, despite the widespread recognition that they increase sales (Anderson and Simester 2003) and despite the fact that they were common in the old English monetary system (Gabor and Granger 1966). Thus, the inefficiency of the monetary system leads to inefficien- cies in sellers’ price setting practices. Thus, unlike modern marketing practitioners and scholars, the Potterian retailers do not use psychological price points. It might be that unlike Muggle retailers (Anderson and Simester, 2003), Potterian sellers do not believe that psychological price points affect consumer demand. PRICES For example, the price of Floo-powder remained the same for over 100 years, two Sickles a scoop (Source: pottermorehead. tumblr.com/post/102175802190/floo-powder, accessed June 6, 2022.). For comparison, the longest spell of price rigidity that economists have documented belongs to the 6.5 ounces Coca-Cola, whose price remained 5 cents between 1886 and 1960, over 74 years (Levy and Young, 2004). Thus, price rigidity in the Potterian economy is at least as prevalent as in the US, in line with the findings reported in some recent New Keynesian studies. One reason for the price rigidity might be that retail- ers that set prices to maximize transaction convenience are reluctant to change them, as changing the price might, like in modern markets, make a transaction less convenient because of the increase in the number of coins used in the transaction, or because round prices are cognitively more accessible (Dehaene, 1997; Knotek 2008 and 2011; Snir et al., 2022, Fisher & Konieczny, 2006) (Note: This price rigidity cannot be explained by PRICES It might also be that the computational efforts needed in the triple coinage system are cognitively demanding and, consequently, the retailers use prices that reduce the cognitive load (Dehaene, 1997; Knotek II, 2008, 2011; Snir et al., 2022). This seems likely because it is clear that many wizards find it difficult to calculate the value of goods denominated in foreign currency units (Rowling, 1998, p. 43, Rowling, 2000, p. 50), similar to the difficulties reported in the EU during the transition to the Euro (Ehrmann, 2011). Thus, it is possible that wizards also find that converting prices between Galleons, Sickles and Knuts is a cognitively difficult task especially given that the exchange rates are prime numbers. Another aspect of the behavior of prices that we should note is that there is very little price dispersion, even across highly heterogeneous goods, suggesting that the prices in the Potterian economy are not set in perfectly competitive markets. For example, the Omnioculars (Rowling, 2000, p. 60), Metamorph-Medals for changing one’s appearance, one week’s work of a house-elf (Rowling, 2005, p. 58), Slytherin’s Locket (Rowling, 2005, p. 171) and Unicorn hair (Rowling, 2005, p. 320) all cost 10 Galleons. This type of pricing resembles somewhat the pricing of modern dollar stores, where highly heterogeneous products are all sold for $1. It could be that the complicated exchange rate between the Knut, Sickle and Galleon may have confused even the author and/or her book’s editor. In the first edition of Rowling (1998), the price of one ounce of dragon liver is 17 sickles, that is, one galleon. But that would be like saying that an item costs 100c, instead of $1. After the mistake was noticed, the price in the later editions was changed to 16 sickles (Source: http://harrypotter.wikia. com/wiki/Dragon_liver, accessed June 6, 2022, and www. hp-lexicon.org/edits-changes-text-ps/, accessed June 6, 2022.). Moreover, the Potterian economy is not growing, im- plying that there is no upward trend in the aggregate demand (Snir and Levy, 2010). Nor do the Potterian retail- ers experience major changes in their cost structure. These factors, along with the currency structure decrease the need and the willingness of the Potterian retailers to adjust prices. Simply, there are not sufficient changes in the market conditions that would warrant such price ad- justments, except when a war breaks out (see section 13). FOREIGN EXCHANGE RATE AND THE COMMODITY VALUE OF THE GALLEON The Potterian monetary system has another important flaw: there is an unreasonably large gap between the Oxford Open Economics, 2022, Vol. 1, No. 1 14 reasonable. As another example, Arthur Weasley, the Head the Office for the Detection and Confiscation of Counterfeit Defensive Spells and Protective Objects, is fined 50 Galleons for bewitching a Muggle car (Rowling, 1999a, p. 142), which is equivalent to about $375.00. This is comparable to the monetary fines assessed for various types of traffic violations in the real Muggle world, such as in California (Source: catrafficticket.com/ ca-traffic-ticket-fines/, accessed on June 6, 2022.). Other examples include the cost of traveling on a night bus to London, 11 Sickles or about $4.95 (Rowling, 1999b, p. 22), birthday present of 10 Galleons, which is equivalent to about $73 (Rowling, 1999b, p. 36), etc. All these prices seem reasonable (Note: Using the above exchange rate, $7.5/Galleon, CNN has even published the Wizard Calculator, offering conversion services to/from the US dollars to/from the Potterian currency units. Source: money.cnn.com/2001/10/23/saving/wizard_calc/index. htm, accessed on June 6, 2022.). commodity value of a gold Galleon and its exchange value. We draw this conclusion based on the Galleon– Dollar exchange rate on the one hand, and the estimated weight of the Gold Galleon, on the other. Consider first the Galleon–Dollar exchange rate (i.e. the exchange rate between the muggles’ money and the wizards’ money), which is not mentioned explicitly in the original 7-volume Harry Potter books, but we know from the books that the Gringotts handles such exchanges (Rowling, 1999a, p. 50). Based on information from three sources, we estimate that the Galleon–Dollar exchange rate is about $7.30/Galleon. Downloaded from https://academic.oup.com/ooec/article/doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 $ First, J.K. Rowling authored two additional, although less known, Harry Potter books as a charity for the UK Comic Relief: (a) Fantastic Beasts & Where to Find Them, and (b) Quidditch through the Ages. Fantastic Beasts & Where to Find Them is included in the list of the things the first-year students are required to have at the Hogwarts School of Witchcraft and Wizardry (Rowling, 1998, p. 43). Quidditch through the Ages is mentioned in Rowling (1998, p. FOREIGN EXCHANGE RATE AND THE COMMODITY VALUE OF THE GALLEON 93), where we are told that Hermione Granger borrows the book from the Hogwarts library, and then later we are told that she lends the book to Harry to help him better prepare for Quidditch practice (Rowling, 1998, p. 117). The foreword to both books is ‘written’ by Albus Dumbledore himself. In the foreword to Fantastic Beasts & Where to Find Them, Dumbledore writes that ‘Comic Relief U.K. has raised over 250 million dollars since 1985 (that’s also 174 million pounds, or thirty-four million, eight hundred and seventy-two Galleons, fourteen Sickles and seven Knuts)’ (Rowling, 2001a, pp. vii–viii). In the foreword to Quidditch through the Ages, Dumbledore writes that ‘Comic Relief U.K. uses laughter to fight poverty, injustice, and disaster. Widespread amusement is converted into large quantities of money (over 250 million dollars since they started in 1985—which is the equivalent of over 174 million pounds or thirty-four million Galleons)’ (Rowling, 2001b, p. vii). These figures imply that one Galleon is equivalent to £4.95 or $7.30, and one Sickle is equivalent to £0.30 or $0.45. Third, in an interview on March 12, 2001, when asked by Rebecca Boswell, ‘What is the approximate value of a galleon?’ J.K. Rowling’s reply was ‘About five pounds, though the exchange rate varies!’ (Source: https://www.hp-lexicon.org/2007/02/04/wizard-money/, accessed June 6, 2022.) We conclude therefore that the Galleon–Dollar exchange rate is about $7.30/Galleon. Next, consider the commodity value of a gold Galleon. To assess it, we need to know the weight of the gold it contains. Galleon’s weight, however, is not mentioned in the Harry Potter books, although we know that it is not trivial. For example, in Rowling (2000, p. 61), Harry Potter finds that his money bag is ‘considerably lighter’ after paying 20–30 Galleons for some goods he bought. To estimate the Galleon’s weight, we take the aver- age weight of three gold coins that were issued in the Kingdom of England. These are (1) the Noble, weighing 7.74 gr, which was issued during King Edward III, (2) the Ryal, weighing 7.58 gr, which was issued during King Edward IV and (3) the Angel, weighing 5.20 gr, which was issued during King Henry VII (Deutsche Bundes- bank, 1983). These figures yield an average weight of 6.84 gr. Alternatively, we could consider the gold Guinea, which was used in England until 1814, and weighed up to 8.50 gr. (Source: https://www.royalmint.com/our-coins/ ranges/guinea/, accessed June 6, 2022.). FOREIGN EXCHANGE RATE AND THE COMMODITY VALUE OF THE GALLEON We thus estimate the weight of a gold Galleon to be in the range of 6.84–8.50 gr., which we consider an upper bound of the Galleon’s true weight. Second, we compare the prices of the products consumed by wizards to the prices of similar products consumed by ordinary muggles in the same period, using the above exchange rate, i.e. one Galleon equals £4.95 or $7.30. For example, Hermione, Ron and Harry pay six Sickles for three butter beers (Rowling, 2003, p. 251), i.e. two Sickles per butter beer. We can reasonably assume that butter beer is not a real beer with significant alcoholic content, because otherwise, it would imply that Harry Potter and his friends, all of them underage wizards, are consuming alcoholic beverages, which is unlikely. In other words, we assume that butter beer is more like Muggles’ root beer or cream soda. In that case, the price of a single bottle of butter beer, two Sickles, which is equivalent to about £0.60 or $0.90, seems reasonable for a price paid by school kids for a soft drink. Hot chocolate on a night bus also costs two Sickles (Rowling, 1999b, p. 22), i.e. £0.60 or $0.90, also The actual price of 1 gr. gold in the years the Harry Potter story takes place, 1991–1998, varied between $10.14–$13.80 (Source: www.kitco.com/charts/historical gold.html, accessed June 6, 2022.), which implies that the commodity value of a gold Galleon is in the range of $69.36–$117.30. Thus, the commodity value of a gold Galleon is between 9 and 16 times the Galleon– Dollar exchange rate. As a lower bound of the Galleon’s weight, we consider the estimate of Generalov (2006), who assess the weight of a gold Galleon and reaches a 15 Levy and Snir ones. Also, it seems that wizards can fake Galleons relatively easily—Hermione does that when she was 16, in Rowling (2003) (Note: One of the readers of this paper, when he was young, was bewildered why wizards and witches did not fake 10 Euro bills from the muggle world with the appropriate charm so that they could at least live well in the muggle world.). Bank officials can probably identify counterfeit coins. Most wizards, how- ever, cannot. Therefore, the only way to prevent wizards from counterfeiting Galleons on a large scale is if its cost is prohibitively high—i.e. FOREIGN EXCHANGE RATE AND THE COMMODITY VALUE OF THE GALLEON Indeed, if gold in the Potterian economy is so cheap for the bank officials that they value it at $7.30 while muggles’ valuation is as much as $69.36–$117.30, wizards could melt the gold and sell it to muggles in return for other things they value, such as other precious metals, gems, etc. They could buy, for example, silver and copper, because the commodity values of silver and copper in the Potterian economy, as can be deduced from the value of the Sickle and the Knut, are closer to the values of silver and copper in the muggle economy, in comparison to the value of gold. If they were to do so, the price differ- ences between the two economies should have become smaller, or disappeared altogether, following the law of one price. Third, it could be that gold Galleons are not made of pure, 100% gold. Indeed, gold is a soft substance and therefore 24 k gold is rarely used for making coins or jewelry. Historically, actual gold coins in circulation in the UK were made from 22 k (91.6%, known as ‘Crown Gold’) or 23 k (95.83%) gold. For example, the gold Pound Sterling coin that was issued in 1489 under King Henry VII was made of 23 k gold. In 1526, under King Henry VIII, and in 1549, under King Edward VI, lower grade gold, 22 k, was used to make the coins. The Crown gold became the standard for making English gold coins from 1526 onward. In the US, 21.6 k (90% gold) became the standard for making American coins for circulation from 1837 onward. p Some possible arguments could be made to confront the arbitrage opportunity puzzle (See: Generalov (2006), gilletts.com.au/jewellery-metal-information-i-39.html, www.goldpriceoz.com/gold-karat.html, taxfreegold.co. uk/goldcoinsbriefhistory.html, en.wikipedia.org/wiki/ Gold_coin, and https://harrypotter.fandom.com/wiki/ Wizarding_currency, all accessed June 6, 2022.). First, it could be that gold Galleons are not made of real gold but rather they are only gold colored, although there is nothing in the books that indicates this. Also, if wizards were to use coins that do not contain precious metals, then they could just as well use paper money, whose commodity value would be zero, or close to zero, like colored coins, but more convenient. Further, there are fake Galleons, made of Leprechaun gold, which look like the real gold Galleons, but they vanish within hours (Rowling, 2000, p. 350). FOREIGN EXCHANGE RATE AND THE COMMODITY VALUE OF THE GALLEON if the coins are made of gold, because the only magical way to produce gold is by using the extremely rare philosopher’s stone. This is essential for the stability of the Potterian currency as otherwise the Potterian economy would be flooded with fake gold coins, which would quickly make the Potterian money worthless. conclusion that a Galleon weighs about 2 gr. This is a more conservative estimate, yielding a commodity value of gold Galleon in the range of $20.28−$27.60, which is 3−4 times the Galleon–Dollar exchange rate. Because we know that wizards value gold, using gold for making coins with such a low purchasing value relative to its commodity value seems to be a misallocation: instead of using gold as a commodity, it is used as a medium of exchange. In this setting, gold is not directed to its most beneficial use, which is inefficient (Friedman, 2002). Furthermore,the ridiculously low exchange rate points at an arbitrage opportunity. Wizards could melt the gold, sell it to muggles as a commodity and then exchange the muggle money for Galleons at Gringotts. However, wizards do not seem to take advantage of this arbitrage opportunity, although it promises immense profits and no risk. Not even the bankers, including the greediest ones, seem to notice it. Nor do rich businessmen who have interest in undermining the Potterians’ political stability. Clearly, in any commodity money model, at least some of those who stand to make a profit would have exploited such profitable arbitrage opportunities lead- ing to their eventual disappearance. Thus, the foreign exchange valuation of the gold Galleon is a textbook example of an inefficient market outcome. Second, it could be that Galleons are made of real gold, but there are some limitations on the wizards’ ability to take advantage of it for arbitrage purposes. For exam- ple, there may be some spell, prohibition or some other kind of restriction that prevents the wizards, even the greediest ones, from engaging in arbitrage. We, however, do not find any explicit mention of such restrictions in the books. Even if wizards are prohibited from melting the gold and selling it to muggles, the goblins that mint the coins are not. The goblins, however, also do not take advantage of this opportunity, although they trade with muggles regularly. FOREIGN EXCHANGE RATE AND THE COMMODITY VALUE OF THE GALLEON The mere existence of ‘fake’ Galleons, however, suggests that the ‘real’ Galleons are ‘real,’ i.e. they are made of real gold, unlike the ‘fake’ However, even if the Galleon is made of 21 k–23 k gold like the Muggle gold coins, the puzzle remains because the price difference between 21 k gold and 24 k gold is not that significant and thus the Galleon’s commodity value as gold is still high. POTTERIAN PUBLIC SECTOR: THE GOVERNMENT The Potterian government is large, corrupt and ineffi- cient. The government controls and regulates the pro- duction of every major good and service produced in the economy, including health, law, education, etc. Thus, to a large extent, the Potterian economy is planned. The government determines which products will be produced Oxford Open Economics, 2022, Vol. 1, No. 1 16 of his sons, Percy and Ron, are all employed by the Min- istry of Magic. Arthur Weasley works as the head of the Office for the Detection and Confiscation of Counterfeit Defensive Spells and Protective Objects (Rowling, 2005, p. 56), after promotion from his former position as the Head of the Office for the Misuse of Muggle Artifacts. Percy Weasley worked in the Department of Interna- tional Magical Cooperation under Barty Crouch, Sr., until he became a Junior Assistant to the Minister of Magic (Rowling, 2000, p. 268, Rowling, 2003, p. 229). In Rowling (2003), Ron Weasley is about to become an Auror in the Ministry of Magic (Rowling, 2003, p. 170, Rowling, 2000, p. 392). or imported and which will not. The government policies also ensure economic stability, as in the Marxian model, in the sense that Potterians do not experience busi- ness cycle fluctuations and, consequently, all workers are employed at all times. Downloaded from https://academic.oup.com/ooec/article/doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 Yet there is a private bank, and there is some private enterprise. The regulations, however, restrict competition in the private sector (Shughart II and Thomas, 2015). Firms in the private sector, therefore, do not have to fire workers or reorganize. Thus, Potterians have both stable prices and stable output in an economy that is not growing. The public, however, knows very little about their government’s decision-making process because of its lack of transparency, which makes it easier for corrupt public officials and interest groups to engage in rent- seeking activities (Tullock, 1967, 1989). Junior public officials engage in rent-seeking (Hillman, 2013b) by putting effort into pleasing their superiors rather than doing their jobs, thereby increasing their prospects for promotion and for gaining higher status (Kahana and Liu, 2010). This, however, further deepens the inefficiency and corruption (Rowling, 2000, pp. 56, 58–59, 273). The tenure system makes it impossible to fire even the most inefficient workers, leading to hidden unemployment. In addition, undeserving promotions create underemployment. POTTERIAN PUBLIC SECTOR: THE GOVERNMENT As a result, many offices are overstaffed with low productivity workers. For example, Bertha Jorkins’ disappearance from her office is unnoticed for several weeks (Rowling, 2000, p. 40). These inefficiencies persist because the budget of each department depends primarily on the influence its head has in the Ministry or with the media. The resulting contest for contacts with powerful politicians and influence on their policy decisions over budgetary allocations leads to wasteful social welfare loss (Katz and Rosenberg, 1989). The lack of transparency is partly because in the Pot- terian economy there is a monopoly on information. The only important information source is a daily newspaper, the Daily Prophet, and its editors are on good terms with senior public officials and wealthy individuals. The latter often fund public goods and thus have a significant influ- ence on the officials and their policy (Rowling, 2003, p. 116). The newspaper’s reporters, therefore, publish infor- mation that favors the officials, who reciprocate by mak- ing decisions favoring the reporters and wealthy wizards (Rowling, 2003, p. 423, Strömberg, 2004, Gentzkow and Shapiro, 2006). Power concentration and lack of transparency help senior officials in seeking rent and in obtaining other ben- efits such as bribes (Rowling, 2007, pp. 144, 172). Wealthy wizards that fund their office expenditures are recip- rocated by getting access to the officials, and swaying their decisions (Rowling, 1999a, pp. 33–34, Rowling, 2003, p. 116). Although not identical, this process resembles political benefits gained through campaign donations (Ursprung, 1990). However, junior public employees seem to be doing their best at carrying out their jobs, which contradicts the internal, bureaucratic rent-seeking noted above. Even extremely inefficient workers like Bertha Jorkins seem to be doing their best, and it is only their lack of talent that makes them inefficient, not their lack of effort (Rowling, 2000, p. 40). Thus, as in the Marxian model, the work- ers contribute according to their ability, and it is their work efforts, more than the pecuniary compensation that gives them satisfaction. Consider the following exchange that reads like a text- book case of rent-seeking, as surveyed for example, in Hillman (2013b): ‘What are you doing here, anyway?’ Harry asked Lucius Malfoy. ‘I don’t think private mat- ters between myself and the Minister [of Magic, a posi- tion that parallels the position of Prime Minister in the muggle-world] are any concern of yours, Potter,’ said Malfoy . . . POTTERIAN PUBLIC SECTOR: THE GOVERNMENT Thus, consistent with public choice theory, when pub- lic officials amass power over the entire economy and transparency is low, they use their power to advance their private goals and procure more power. The officials that comprise the upper layers of the public sector share sim- ilar values because only those with similar backgrounds are promoted. The Potterians’ legal institutions are rather limited in both breadth and scope. There are no lawyers, no independent court system or other muggle-type judicial entities. The Ministry of Magic seems to have all the regulatory, legislative and judicial powers, implying that the Potterians are not familiar with the notion of separa- tion of powers. For example, the Ministry of Magic is not subject to any practical limits on its powers since there are no laws or regulations that impose any restrictions on what it can or cannot do. The Ministry operates the court, and the Minister of Magic himself serves as one of the ‘inquisitors,’ the Potterian equivalent of a prosecutor, as well as one of the judges. Furthermore, it seems that one of the incentives to climb the ranks of the civil service is ego rents provided by status and power (Olson, 1965). The school headmaster, Dumbledore, for example, does not want to become a minister of magic because he is not as power hungry as those who apply for the job (Rowling, 2003, p. 89). The urge to satisfy his ego rent ambition for power even drives the Minister of Magic Pius Thicknesse to fight on the side of He-Who-Must-Not-Be-Named, the evil Voldemort (Rowling, 2007, p. 636). Hayek (2006) predicts that in a society in which power is concentrated in the hands of a few, the ones to get to the top are those that are the most power and status hungry, not the most competent or the most benevolent. Many characters that hold high positions in the Potterian economy fit this characterization. Even the laws that are in place cannot be trusted because according to the Minister of Magic, there is no prohibition on their retroactive revision. On the contrary, the Ministry changes and manipulates the laws at will depending on its interests and circumstances. For example, the Ministry decides to change the time of Harry’s court hearing but informs him of this change at the last minute, causing Harry to be late for the hearing (Rowling, 2003, p. 103). POTTERIAN PUBLIC SECTOR: THE GOVERNMENT Harry distinctly heard the gentle clinking of what sounded like a full pocket of gold. ‘ . . . shall we go up to your office, then, Minister?’ ‘Certainly,’ said Fudge . . . ‘This way, Lucius’ . . . ‘What private business have they got together, anyway?’ ‘Gold, I expect,’ said Mr. [Arthur] Weasley angrily. ‘Malfoy’s been giving gen- erously to all sorts of things for years . . . gets him in with the right people . . . then he can ask favors . . . delay laws he doesn’t want passed . . . oh, he’s very well-connected, Lucius Malfoy’ (Rowling, 2003, pp. 115–116). However, unlike the workers, those high in the social hierarchy behave as predicted by public choice theory. Senior public officials use their powers and the lack of transparency to advance their own goals, by taking advantage of the rent-seeking opportunities the Potterian government offers, from promoting their associates to releasing their relatives from jail. They also reciprocate by benefiting wealthy landowners, granting special ben- efits to those who donate to causes of their interest. Senior officials also do not hesitate to use fear and violence, if needed, to achieve their goals or hide their mistakes (Levin and Satarov, 2015). When Harry and his colleagues express doubts about the threat of war, the press is used to intimidate and silence them, and some of those that raise doubts like the headmaster of the Nepotism is common and family members and asso- ciates of senior officials receive perks and benefits not offered to others. For example, Arthur Weasley and two Levy and Snir 17 trumped-up charges); conducted criminal trials with- out defense counsel; used truth serum to force confes- sions; maintained constant surveillance over all citizens; offered no elections and no democratic law-making pro- cess, and controlled the press? You might assume that the above list is the work of some despotic central African nation, but it is actually the product of the Ministry of Magic’ (Barton, 2006, pp. 1523–1524). school lose their jobs. Others, including Harry Potter, are threatened (Rowling, 2005, pp. 341–348). At least on one occasion, a senior public official orders a ‘neutralization’ of a man to silence him (Rowling, 2000, pp. 453–454). POTTERIAN PUBLIC SECTOR: THE GOVERNMENT Moreover, the presiding judge (the ‘Chief Inquisitor’) in the hearing, the Minister of Magic himself, states during the court proceedings that ‘laws can be changed.’ For example, During Harry’s trial for using underage magic,which the Ministry’s laws prohibit, Dumbledore (who attends the court hearing as a witness) debates the Minister of Magic Cornelius Fudge about the applicability of the relevant laws (Rowling, 2003, p. 112): ‘The Ministry does not have the power to expel Hogwarts students, Cornelius, as I reminded you on the night of the second of August,’ said Dumbledore. ‘Nor does it have the right to confiscate wands until charges have been successfully proven . . . In your admirable haste to ensure that the law is upheld, you appear, inadvertently I am sure, to have overlooked a few laws yourself.’ ‘Laws can be changed,’ said Fudge savagely. ‘Of course, they can,’ said Dumbledore, inclining his head. ‘And you certainly seem to be making many changes, Cornelius. Why, in the few short weeks since I was asked to leave the Wizengamot [the court of the Ministry of Magic], it has already become the practice to hold a full criminal trial to deal with a simple matter of underage magic!’ Despite these inefficiencies, the government has con- siderable public support. Some of this support might be due to the public being unaware of the inefficiencies, due to the low transparency. It might also be because of the government’s size (Alesina & Fuchs-Schuendeln, 2007). The public sector is the largest employer, and wizards depend on it for both work and services. Furthermore, the size of the government allows for full employment, with even the most unproductive workers finding a job in the public sector. Indeed, it seems that for most wizards, a job in the public sector is the default, and many of them do not even consider a job in the private sector. Therefore, further rent-seeking may be taking place through the benefits of a majority from the government (Paldam, 2015). Thus, the support for the government seems to be partly because the public is unaware of much of the inefficiencies, and partly because those that are aware ignore them and look the other way, to protect their private benefits such as job security. LAW AND ORDER Consider the following: ‘What would you think of a gov- ernment that engaged in this list of tyrannical activities: tortured children for lying; designed its prison specifi- cally to suck all life and hope out of the inmates; placed citizens in that prison without a hearing; ordered the death penalty without a trial; allowed the powerful, rich or famous to control policy; selectively prosecuted crimes (the powerful go unpunished and the unpopular face There are not even rules that govern the process of elections. There is no evidence of elections taking place for any key public office position. For example, we are told that Rufus Scrimgeour is appointed (not elected) as a Minister of Magic, but we do not know by who (Rowl- ing, 2005, p. 27). Ministry officials often take advantage Oxford Open Economics, 2022, Vol. 1, No. 1 18 of their powers by drafting laws and regulations that promote their and/or their friends’ interests. Further, the implementation and enforcement of the laws are often done selectively. For example, Harry has committed in the past a worse crime, blowing up Ms. Marjorie Dursley, but no charges were filed against him because, in that period, Harry was still the darling of the Minister of Magic (Rowling, 1999b, p. 28). There are even cases where wiz- ards are accused and sentenced without any trial or court hearing. Since there is no constitution, the authoritarian Ministry can arbitrarily manipulate the existing laws and regulations to fit its needs. For example, at a certain point, the Ministry decides that all halfblood and mudblood wizards must be questioned to make sure that they did not ‘steal’ magic powers (Rowling, 2007, p. 136) (Note: Wizards with one muggle-parent are called ‘halfblood’ (Rowling, 2003, p. 584). Wizards with two muggle parents are called ‘mudbloods’ (Rowling, 1999a, p.96).). The wiz- ards that fail the test are imprisoned. existence secret by adding their name to a list of the attendees: ‘So if you sign, you’re agreeing not to tell Umbridge or anybody else what we’re up to.’ ‘There was an odd feeling in the group now. It was as though they had just signed some kind of contract’ (Rowling, 2003, p. 259). LAW AND ORDER The third time the term ‘contract’ is mentioned when we are told that the house-elf Hokey’s contract prohibits her from saying what she thinks about her mistress Hepzibah Smith’s looks (Rowling, 2005, 285–286): ‘“How do I look?” Said Hepzibah, turning her head to admire the various angles of her face in the mirror. ‘Lovely, madam,’ squeaked Hokey. Harry could only assume that it was down in Hokey’s contract that she must lie through her teeth when asked this question because Hepzibah Smith looked a long way from lovely in his opinion’. As Gava & Paterson (2010) note, however, this does not consti- tute a contract in a legal sense because it is not signed voluntarily: house elves are enslaved by their masters and thus they must follow their orders. In addition, there is a mention of an ‘unbreakable vow’ that Snape made with Draco Malfoy’s mom that he would protect Draco (Rowling, 2005, p. 214). The vow, however, more resembles a promise than a contract. p Day-to-day policing is done by the wizards and witches that work at the Ministry. Some of them are aurors— wizards whose job is to locate and apprehend ‘dark wizards’—wizards that practice various types of ‘dark arts’ and are known for their dislike of muggles. There are also divisions in the Ministry that handle specific types of offenses and crimes. Examples include the Office of the Improper Use of Magic, the Office of the Regulation and Control of Magical Creatures, the Office of the Magical Law Enforcement, etc. Some of the offenses they deal with include buying and selling stolen caldrons (Rowling, 2003, pp. 18, 80), car theft (Rowling, 2003, p. 377), sale of fake amulets that supposedly protect against werewolves (Rowling, 2005, p. 73) and various kinds of fake protections that supposedly guard against You- Know-Who and Death Eaters (Rowling, 2005, p. 56), etc. Thus, like muggles, the Potterians seem to have their share of thieves, crooks and other common criminals. ‘Private property’ is mentioned in the story only once, when Mr. Gaunt tells a ministry official that he cannot just show up unannounced on his private property (Rowl- ing, 2005, p. 132, Demsetz, 1967). But there is indirect evidence that Potterian wizards respect private property, at least in the context of inheritance, which seems to be rather common in their economy. LAW AND ORDER For example, Harry inherits a large amount of money from his parents (Rowl- ing, 1998, p. 48, Rowling, 1999a, p. 30). He also inherits Sirius Black’s house along with everything in the house, including the house-elf Kreacher (Rowling, 2005, pp. 33– 34). There are numerous other cases of inheritance trans- fer from generation to generation. The government, however, does not seem to be com- mitted to respecting the Potterians’ inheritance wills. The Decree for Justifiable Confiscation gives the Ministry the power to confiscate ‘within 31 days’ anything that is inherited. This particular law was created to prevent wiz- ards from passing on Dark Artifacts, and it can be applied only if there is ‘powerful evidence that the deceased’s possessions are illegal’ (Rowling, 2007, p. 80). However, the Ministry of Magic applies to the law its own inter- pretation, by arbitrarily and selectively using it to con- fiscate valuable artifacts. For example, the Minister of Magic Rufus Scrimgeour relies on this law to harass and interrogate Harry and his friends when he informs them about the inheritance Dumbledore has left for them (Rowling, 2007, pp. 80–85). Moreover, the Minister refuses to hand the sword of Godric Gryffindor to Harry, against the will that Dumbledore has left, arguing that ‘the sword of Godric Gryffindor is an important historical artifact’ (Rowling, 2007, p. 85). As another example, the High Inquisitor and Head Mistress of Hogwarts, Ms. Dolores Umbridge uses her absolute powers and authority to Further, in the Potterian legal system, we see very little of what might resemble private laws such as tort laws or contract laws. For example, although the Hogwarts students are often injured (e.g. during the Quidditch games), there is no mention of any kind of lawsuit in this context. The word ‘contract’ is mentioned only three times in the 7-volume set. The first time it is mentioned when Dumbledore explains the Triwizard Tournament rules and warns the students: ‘Once a champion has been selected by the Goblet of Fire, he or she is obliged to see the tournament through to the end. The placing of your name in the goblet constitutes a binding, magical contract’ (Rowling, 2000, pp. 166, 179). However, we are not told how this contract is enforced. LAW AND ORDER 75–76, 82). In another instance, a public official, Percy Weasley, is asked to formulate and pass a law that will prohibit the importation of caldrons (Rowling, 2000, pp. 36–37), and thus protect the interests of a local producer. The public ends up paying higher prices because of these import restrictions. Clearly, Potterians’ legal system is far from a rule of law, as characterized by Hayek (2006). Rather, it seems closer to a rule of government in Nietzschean spirit (Niet- zsche 1997, Hillman, 2009). Since there are no lawyers, no independent judiciary and no court system, rule of law is doubtful. Moreover, given the low ethical and moral stan- dards of many of the Ministry’s officials, the Potterians’ fate, freedom and welfare seem to depend entirely on the goodwill of the government’s officials. The Potterian wiz- ards are not equal before the law. The Ministry of Magic has no regard for the rule of law, has no ethical restraints, uses its monopoly power selectively by applying its own interpretation to the laws and by denying the rights of the wizards that it dislikes. Since the government does not treat or view them equally, the wizards cannot trust their government or its intentions. They are terrified by the government’s powerful bureaucrats and officials, and by the ease at which they selectively interpret, manipulate or even retroactively change the laws and regulations to harass citizens they dislike, while protecting and helping their, usually wealthy and well-connected, friends. Second, the lack of financial markets further reduces business turnover. Most wizards do not have the capital needed to open a business and are therefore forced to solicit the help of wealthy wizards. That is how Borgin and Burkes opened a shop in the 19th century, and that is how the Weasley twins managed to open a shop about 100 years afterward. There is no mention in the books of any other shop opening in Diagon Alley during that period. A third factor contributing to the low business turnover is the monopoly on information. The Daily Prophet has a monopoly, and its reporting is system- atically biased in favor of wealthy wizards (Rowling, 2000, p. 213, Rowling, 2003, pp. 72, 423) (There is another publication, The Quibbler (Rowling, 2003, p. LAW AND ORDER 144), which is as reliable as the modern-day tabloids that publish articles such as ‘I Was Kidnapped by Aliens!’ ‘I Saw Bigfoot in My Back Yard!’ etc.). Also, it selectively prevents information about new products from reaching the public. When the Weasley twins open their shop, the paper does not publish their advertisements, nor share with the public information about the shop, despite the shop’s success. (For example, it seems that the wizards that read the Daily Prophet, including Harry Potter—a devoted reader of the newspaper, know nothing about the twins’ joke shop (Rowling, 2000, p. 36). LAW AND ORDER The second time contract is mentioned is when Hermione Granger organizes a study group of students that take lessons (without the school’s permission) from Harry on how to defend against Voldemort’s Death Eaters. At the meeting where they decide on the formation of the group, they agree to keep the group’s 19 Levy and Snir arbitrarily confiscate Harry Potter’s and Ron Weasley’s broomsticks without any hearing or other due process (Rowling, 2003, p. 311). Most businesses operating at these shopping centers have been around for a long period. The low business turnover is the result of several factors. First, the Potterian government’s regulations give few existing well-connected businesses monopoly power. By applying these regulations selectively, public officials block com- petition. Consequently, new businesses open rarely. For example, at one point an entrepreneur, Ali Bashir, wants to import flying carpets that would substitute for flying brooms. The proposal is supported by a senior official, Mr. Barty Crouch, who has a personal interest in the importa- tion of carpets. However, his initiative is blocked because the Ministry defines carpets as Muggle Artifacts, i.e. as objects that are ‘too similar to objects used by muggles’ (Rowling, 2000, p. 59). Although the argument is weak, the regulations cannot be changed due to technicalities. The same official, however, does not prohibit his sons from selling muggle products (Rowling, 2005, p.118). The Potterian government also interferes at will in the operation of private businesses. Goblins, for example, complain at one point that the Ministry interferes with Gringotts’ affairs, making it unsafe for its employees (Rowling, 2007, p. 296). According to Hayek (2006), rule of law ‘ . . . means that government in all its actions is bound by rules fixed and announced beforehand—rules which make it possible to foresee with fair certainty how the authority will use its coercive powers in given circumstances . . . the essential point, that the discretion left to the executive organs wielding coercive power should be reduced as much as possible, is clear enough . . . under the Rule of Law, the government is prevented from stultifying individual efforts by ad hoc action . . . It is the Rule of Law, in the sense of the rule of formal law, the absence of legal privileges of particular people designated by authority, which safeguards that equality before the law which is the opposite of arbitrary government’ (pp. MARKET STRUCTURE: MONOPOLIES, OLIGARCHIES, AND OTHER PATHOLOGIES The Weasleys’ mother does not encourage her children’s business aspi- rations because she believes that public sector jobs bring a better reputation (Rowling, 2000, p. 36, Rowling, 2003, p. 79). Thus, the Potterian economy is a small, monopoly- dominated economy with a large corrupt public sector and an inefficient credit market. Potterian markets lack competition because of the government’s selective use of its regulatory powers in many spheres of life. This leads to low business turnover, a limited variety of goods and services, and thus to a limited choice, welfare loss and inefficient outcomes. The monopoly on information worsens the state of affairs further because the wizards have no access to useful information that could help them improve their private lives (Note: Compare this with the effect of the internet, which allows consumers to compare the prices of similar goods from thousands of sellers almost effortlessly, on the demand for goods at shopping malls.). ) Another reason for the low social mobility is that wealthy wizards view themselves as superior, especially to wizards with muggle predecessors (Rowling, 2003, pp. 84, 584, Rowling, 2005, p. 417), who are considered a threat because of their different culture (Rowling, 2000, p. 66). Muggle-born wizards are subject to constant harassment, ridicule, abuse and discrimination, and are derogatorily called ‘mudbloods’ (Rowling, 1999a, pp. 72– 74, Rowling, 2005, p. 74). Wealthy wizards favor policies that limit the influence of middle-class wizards. Most of the supporters of Voldemort, for example, come from wealthy families, and their goal is to enslave the middle class, especially the mudbloods. The middle-class wizards are unlikely to move up the social ladder through marriage as they rarely socialize with wealthy wizards. Wealthy wizards are often dis- inherited by their families if they associate themselves with middle-class wizards (Rowling, 1999a, p. 96, Rowling, 2003, p. 103), which along with intermarriages further block upward mobility (Rowling, 2003, p. 85). Thus, the biases of the elite against lower-class wizards minimize the opportunities for upward social mobility, resulting in social mobility being mostly downward (Dearden et al., 1997) and is expected to persist (Mulligan, 1999). The inefficient and selective regulatory interventions of the Potterian public officials suggest that the world- view the books promote is in line with the economic models of public choice. MARKET STRUCTURE: MONOPOLIES, OLIGARCHIES, AND OTHER PATHOLOGIES The number of consumers in the Potterian economy is relatively small. Moreover, the population size has remained stable for generations. Hogwarts was founded around 990 A.D. and its size has not changed since, suggesting that the number of wizard children remained stable for at least that long. In addition, most wizards live in London or near Hogsmeade, a village in north- ern England. Consequently, two shopping centers, one in Hogsmeade and the other in Diagon Alley in London, seem to satisfy the shopping needs of the English wiz- ards. These barriers and restrictions lead to high entry costs, limit the number of sellers and give the existing firms market power. For example, the UK wizards have only one wand-maker, the Ollivanders (Rowling, 1998, p. 53). While it is hard to assess the effect of the market concentration on markups, the extent of price rigidity and the frequent use of convenient prices suggest that the Potterian retail- ers have substantial profit margins. These margins would Oxford Open Economics, 2022, Vol. 1, No. 1 20 likely fall if the markets were to open to imports (Rowling, 2000, pp. 36–37). from a well-established family that was stripped of its assets (Rowling, 2005, p. 138).). Mobility between low- and middle-class wizards is more common. For example, Hermione starts as a mudblood wizard and ends as the Minister of Magic (Source: https://harrypotter.fandom. com/wiki/Hermione_Granger, accessed August 5, 2021.). Most of the Weasley brothers have also improved their economic conditions. The low business turnover also limits product variety and innovations. For example, the same textbooks have been used for over 20 years. Similarly, children eat the same candies and collect the same cards as did their parents (Rowling, 2005, p. 31). Thus, the lack of compe- tition limits the choice of the Potterians, although there is demand for new products. For example, after Zonko’s Joke Shop goes bankrupt, the Weasleys’ Wizard Wheezes has no competition, although there is a demand for the new jokes and tricks that the Weasleys sell (See Rowling (2000, p. 36), Rowling (2003, p. 403), Rowling (2005, pp. 76, 78, 158–159) and Tirole (1988).). Downloaded from https://academic.oup.com/ooec/article/doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 One reason for the limited social mobility is the insuf- ficient free enterprise due to negative social images of businesspeople in the Potterian society. MARKET STRUCTURE: MONOPOLIES, OLIGARCHIES, AND OTHER PATHOLOGIES At the same time, however, the actions of these officials and their outcomes for the Potterian economy and the Potterian public resemble the communist model. The Daily Prophet reminds us of the Soviet Pravda, which had a monopoly on information in the former Soviet Union and served as a propaganda machine for the communist government. More generally, the limited choice of the Potterians very much resembles the limited choice in the former USSR and other Soviet- bloc communist countries. The social structure of the wizard society leads to a struggle between middle- and higher-class wizards. The upper-class wizards use their influence and even vio- lence, to repress the middle class, while the middle-class wizards scorn the upper-class wizards. The upper-class wizards even try to control the thoughts and beliefs of the middle-class wizards by influencing the curriculum used in the school. INTERNATIONAL TRADE AND MIGRATION Wizards can travel long distances easily and cheaply. There are hardly any restrictions on international travel. Nevertheless, there is little international trade, which is partly due to regulations protecting local producers. Trade and travel are limited also by linguistic barriers as most wizards are monolingual. In addition, there are cultural barriers, as the wizards do not know much about other people’s customs, traditions, etc. (Rowling, 2000, pp. 163–164, 363). In sum, wizards could benefit by trading with muggles. They could also import muggle-produced goods or adapt their designs and thus increase the variety of goods at a relatively low cost. However, trade is limited by cul- tural norms and prejudices that make most wizards view muggle-made goods as inferior (Rowling, 1999a, p. 25, Rowling, 2005, p. 77). Further, cultural differences prevent most trade between wizards from different countries, and between wizards and other humanoids. It, therefore, seems that unlike most models of trade, transaction opportunities in the Potterian economy are limited by culture. Thus,much of the potential trade in the Potterian economy is blocked by protectionism (Grossman and Helpman, 1994) and cultural prejudices (Bala and Long, 2005). These barriers reduce the demand for foreign goods, which may explain the absence of foreign or ethnic restaurants in Potterian London and Hogsmeade. Wiz- ards also know very little about the quality of foreign goods. For example, when a leading English wand-maker disappears, wizards do not know where to find another wand-maker although there are several quality wand producers in other countries (Rowling, 2005, p. 70). ( g ) In addition, prejudices against foreigners and non- wizards eliminate interactions with muggles almost completely. Indeed, one of the main responsibilities of the Ministry of Magic is to ensure that muggles are not aware of wizards’ existence (Rowling, 1998, p. 42). For example, Arthur Weasley’s job in The Office of Misuse of Muggle Artifacts is to prevent any interaction between muggles and wizards (Rowling, 1999a, p. 20). Thus, although trade in muggle goods could be beneficial, it takes place only under special circumstances and only for specific goods (Rowling, 2005, p. 77). Another barrier to trade is the absence of immigrant networks (Rauch, 2001). In the Potterian economy, immigrants are rare. For example, there are only three characters with substantive roles, which have a foreign background, or their names suggest so. INCOME DISTRIBUTION, INCOME INEQUALITY, AND SOCIAL IMMOBILITY The wizards’ society is composed of a low class, a large middle class and a small elite. The middle-class wizards earn enough to live comfortably but not enough to save. They, therefore, work almost their entire lives. Wealthy wizards enjoy a luxurious lifestyle and own almost all the assets and capital (Rowling, 1999a, p. 19). Although this seems like a Marxian-style social strug- gle, it is a struggle that is fought in a society with central planning where the government owns and regulates pro- duction. In the Marxian model, free markets are usually considered the cause of social struggles because the own- ers of the capital (‘capitalists’) can collect riches only by robbing the middle class (‘proletariat’). Also in Marxian models, central planning is the solution, because it takes the power away from the upper classes and transfers it to the government officials who represent the proletariat, Social mobility between the high class and the other classes is mostly downward. There is just one promi- nent character that makes it from the bottom to the top, Voldemort, but several characters, including Sirius Black, Andromeda Tonks (Black) and the Gaunts, that moved down the social ladder (Note: Voldemort comes 21 Levy and Snir (Source: the UK Department of Education’s Statistical Frist Release, ‘Schools, pupils, and their characteristics: January 2015.’). and who make the decisions for all. Yet in the Potterian model, the government is controlled by the elite, and the elite use the government to repress the middle class (Murphy et al., 1993). These social norms and prejudices translate into formal trade barriers. For example, there is no protest when a junior official uses quality as a pretext to block the importation of considerably cheaper foreign goods, although the official himself admits that they are of almost the same quality as the locally produced goods (Rowling, 2000, pp. 36–37, 59). The outcome is that in the Potterian model, the class struggle focuses on controlling the government. The Marxian model predicts that once the middle class enjoys a relatively high standard of living and good education, there will be a class struggle that ends in the collapse of the class system and greater equality. In the Potterian economy, however, even after a class struggle that ends in the defeat of the high class, the income differences remain large. INCOME DISTRIBUTION, INCOME INEQUALITY, AND SOCIAL IMMOBILITY Thus, when it comes to inequality outcomes in a society with a large government, the Potterian model is more consistent with the public choice model than with the Marxian one. However, some types of foreign work are so profitable that they are tolerated. Wizards use ‘house elves’, a special kind of humanoids, to do manual and dirty work, perhaps analogous to foreign workers. Although the elves provide many useful services, they work in terrible con- ditions for almost no pay. They do not own even their clothing. They are nevertheless diligent, work without a break for many years, accept slavery conditions and are extremely afraid of being sacked because unemployment is almost certain death for them. These considerations make the use of elves so profitable, that despite the wizards’ prejudices against any type of humanoids, they employ elves in large numbers (Rowling, 2000 pp. 64, 80, 89). INTERNATIONAL TRADE AND MIGRATION These are Viktor Krum, a Bulgarian wizard, Fleur Delacour, who came from France and Cho Chang. Just one of them Cho Chang, studies at Hogwarts. Thus, whereas almost 20% of the UK primary school pupils are exposed at home to a language other than English, there are very few, if any, such students in the only school for wizards in the UK In summary, whereas in economic models, invest- ments depend on borrowing cost and rate of return, in the Potterian model, they are determined primarily by prejudices and social norms. This is consistent with studies that show that social norms are a barrier to trade, except that in the Potterian economy, the effect of social norms on trade is perhaps an order of magnitude higher in comparison to the effects reported in empirical studies (See McCallum (1995), Nunn & Wantchekon (2011) and Clerides et al. (2015).). WAR ECONOMICS Indeed, one of Harry Potter’s teachers, Professor Slughorn, complains that due to the war, prices are sky-high and that it is difficult to obtain even the most elementary products (Rowling, 2005, p. 43). The members of the Order of the Phoenix are therefore isolated and gain little public support. Without pub- lic support, their efforts can at best only slow down the progress of the terrorists, but they cannot prevent them from gaining almost full control over most of the institutions of the Potterian economy (Rowling, 2003, p. 88). The government also fails to prepare adequately and, consequently, it has to buy costly equipment that would be unnecessary if it had provided its staff with proper training. Additional cost born by the public because of the government inefficiency is the price paid for goods sold by swindlers that offer a false sense of security (Rowling, 2005, pp. 73, 78) (Note: Similar security needs lead some modern governments to spend millions of dollars on useless bomb detectors sold by swindlers. Source: www.guardian.co.uk/uk/2013/apr/23/ somerset-business-guilty-fake-bombs, accessed June 6, 2022. After the crash of the Russian Metrojet flight 9268 in Egypt on October 31, 2015, it was reported that many Sharm el-Sheikh hotels use fake bomb detectors. A November 10, 2015 report of CNN described them as ‘magic wand’ detectors. Source: http://edition.cnn. com/2015/11/10/middleeast/egypt-sharm-fake-bomb- detectors/, accessed June 6, 2022.). The Potterian model is therefore consistent with the economists’ insight that governments should provide basic public goods such as security because the private sector cannot provide them efficiently (Samuelson, 1954). In the Potterian model, however, the government also controls the production of many goods that are not pub- lic, suggesting public-choice type distributive outcomes (Tullock, 1959). The war underscores the Potterian government’s inef- ficiency in responding in a timely fashion to the signs of danger. Furthermore, although the war might seem like a class struggle, its outcome contradicts the Marxian interpretation of a class struggle. In the Potterian model, as in the Marxian model, the upper class struggles to secure its status. In the Marxian model, however, once the middle class becomes aware of the struggle, it fights back and ultimately all classes are eliminated and a new economic order is established. ) In addition, Terror generates fear, which influences people’s moods by making everything less enjoyable (Note: The effects of fear might be long lasting. WAR ECONOMICS In Rowling (2005, 2007), the wizards fight a war against ‘Death Eaters’, a group of wizards that either belongs to the upper class or aspire to associate themselves with the upper class. Their goal is to take control over the wizard world, thereby enslaving the middle class and driving out those wizards whose ancestors were not wizards. They Oxford Open Economics, 2022, Vol. 1, No. 1 22 are extremely committed and are willing to use any form of violence, including suicide attacks, to achieve their goal. These events demonstrate that influential events such as wars can have long-term effects on people’s prefer- ences. For example, as predicted by recent studies, the war brings about a significant drop in the demand for dining at restaurants and bars (Gould and Klor, 2010; Becker and Rubinstein, 2011). In Israel, during the Sec- ond Intifada in 2000–2002, there were Palestinian ter- ror attacks in public places. The consumers responded by eating more at home/office. Restaurants responded by offering delivery services. The share of restaurants offering delivery services, however, did not decrease after the cessation of violence, suggesting that the taste for deliveries remained. The signs of danger appear two years before the first acts of violence, but the government ignores them (Rowl- ing, 2000) (Note: Compare this to the recent criticisms of the US government concerning its slow and delayed responses to ISIS terror (www.nytimes.com/2014/09/29/ world/middleeast/president-obama.html, accessed June 6, 2022), flood water (www.msnbc.msn.com/id/9614737/, accessed June 6, 2022), bird flu (www.msnbc.msn. com/id/9661312/, accessed June 6, 2022) and Ebola outbreak (www.nytimes.com/2014/09/26/world/africa/ obama-warns-of-slow-response-to-ebola-crisis.html?_ r=0, accessed June 6, 2022).). This is consistent with Tuchman’s (1990) theory of march of folly: the govern- ment not only ignores signs of warning but also silences anybody who expresses opinions other than the official ones. When the Potterians see the inability of their govern- ment to respond, they form the ‘Order of the Phoenix,’ whose goal is to fight the terrorists. Their efforts, however, do not fully compensate for the lack of government action because of the high private costs of fighting ter- rorists. Indeed, those who fight the terrorists become primary targets. When violence breaks out, however, the government has a sudden need to obtain military equipment. As more resources are spent on supplying military needs, common people find it hard to obtain the goods they need for daily life. INNOVATIONS AND TECHNOLOGICAL PROGRESS Indeed, Harry repeatedly visits ‘Quality Quidditch Supplies’ to look at and admire the prototype Firebolt model that the store displays, and is considering spending all his savings to buy it (Rowling, 1999b, p. 32). The way the Potterian broomstick industry func- tions and develops is comparable to the modern auto industry, particularly because new broomstick models are released almost every year. The newer model broomsticks use better materials (e.g. type of wood such as Spanish Oak), have better precision, offer better balance, are faster, etc. The Firebolt, for example, can accelerate from 0 to 150 mph in 10 seconds, which is at least twice as fast as the Cleansweep. Comet-260 looks ‘ . . . like a joke next to the Firebolt’ (Rowling, 1999b, p. 162). Thus, each model is of better quality than the older one, which is indicative of technological improvements There is also evidence that over time there were non- trivial improvements in the broomsticks’ production, especially in their quality. For example, some of the early models were simple and basic, but more recent ones are more advanced. For example, the Shooting Star, the model owned by the Hogwarts School, is a basic broomstick, relatively cheap, ‘very slow and jerky’ (Rowling, 1999a, p. 30, Rowling, 1999b, p. 121), similar to the Bluebottle family series with an anti-burglar buzzer (Rowling, 2000, pp. 62–63). Nimbus-2000, on the other hand, is a top-of-the-line broomstick from the time of its release (Rowling, 1999a, p. 30), until Nimbus-2001 is released. The Firebolt, which is the latest addition, is perhaps ‘the Ferrari’ of the broomsticks. It is the best, fastest and most aerodynamically efficient model, offering a smooth action and fine control. It is a dream broomstick. Indeed, Harry repeatedly visits ‘Quality Quidditch Supplies’ to look at and admire the prototype Firebolt model that the store displays, and is considering spending all his savings to buy it (Rowling, 1999b, p. 32). Thus, in general, we do not see any evidence of the innovations in the broomstick industry spreading to the general economy. This is consistent with the finding that the Potterian economy is stagnating; there is hardly any growth in the labor force, there is no accumulation of knowledge (human capital) and we see no evidence of large-scale capital investments. WAR ECONOMICS Indeed, many wizards remember and react irrationally when they hear the name Voldemort even though for many years, he was thought to be dead (Huddy et al., 2002).). Indeed, the usually crowded ‘Leaky Cauldron’ bar is empty because even the most loyal consumers seem to have lost their appetite (Rowling, 2005, p. 72). In Rowling (2005, p. 72), the bar owner notices Hagrid, who is well known for his fondness for alcohol. From the barman’s reaction, it is clear that Hagrid is one of his last loyal customers. In addition, in the Marxist model, class struggle is an outcome of capitalistic motives, where the high class robes the middle class of its share in production. The solution in the Marxist model is central planning by the government. In the Potterian model, however, there is already central planning. The government, however, is controlled by the upper class who uses it as means to control the middle class. The Potterians’ war ends when more middle-class wizards join their comrades in the fight (Rowling, 2007, p. 511). The outcome, however, is not a new social order. Although the middle-class wins, the high-class wizards still preserve their high-class status (Rowling, 2007, 23 Levy and Snir in the broomstick industry, a process resembling a Schumpeterian mechanism of creative destruction. p. 605) and their hold over the government, which controls the economy. An interesting question that follows from these obser- vations is how much of the broomstick technological innovations translate into economy-wide innovations. Our reading of the books suggests that broomsticks are primarily used for leisure-related activities, especially for the Quidditch games. For day-to-day purposes, Potterians use other means of transportation including apparition— a spell that allows the caster to move from one spot to another instantaneously (Rowling, 2000, pp. 43–44), magic-propelled boats (Rowling, 1998, p. 42), flying horseless carriages (Rowling, 2000, p. 109), the Floo- network that allows immediate transportation to any place in the network (Rowling, 2000, p. 29), Knight-Bus (Rowling, 1999b, p. 21), a magical train, the Hogwarts Express (Rowling, 1998, p. 60), the Ministry of Magic cars (Rowling, 1999b, p. 45) and Portkeys—objects that transport to a pre-determined location when touched (Rowling, 2000, p. 46). INNOVATIONS AND TECHNOLOGICAL PROGRESS Consequently, we con- clude that very little (if any) of the broomstick technology innovations spread to other industries, or more generally, to the wider economy. The lack of technological progress is also consistent with the common image of centrally planned economies. In the Potterian economy, with the government either producing many goods or regulating their production, government officials do not benefit from the introduction of new products and therefore they have little incentive to introduce them. On the contrary, they might have incentives to erect barriers to such innovations if they see technological progress as a threat to their employment, which is a common view among labor unions. Perhaps it is not surprising, therefore, that the only industry in which technology is progressing is not a part of the public sector. The way the Potterian broomstick industry func- tions and develops is comparable to the modern auto industry, particularly because new broomstick models are released almost every year. The newer model broomsticks use better materials (e.g. type of wood such as Spanish Oak), have better precision, offer better balance, are faster, etc. The Firebolt, for example, can accelerate from 0 to 150 mph in 10 seconds, which is at least twice as fast as the Cleansweep. Comet-260 looks ‘ . . . like a joke next to the Firebolt’ (Rowling, 1999b, p. 162). Thus, each model is of better quality than the older one, which is indicative of technological improvements INNOVATIONS AND TECHNOLOGICAL PROGRESS According to Snir and Levi (2010), the Potterian economy is not growing because there is no growth in the labor force, there is no accumulation of physical or human cap- ital, and there is no evidence of any kind of technological progress. g The Potterians’ broomstick industry is an exemption. A careful reading of the Harry Potter books suggests that the Potterian broomstick industry has been experiencing significant technological progress over time. For exam- ple, we are told that there are several makes and mod- els of broomsticks. These include Cleansweep-5 (Rowl- ing, 1999a, p. 71), Cleansweep-6 (Rowling, 2003, p. 143), Cleansweep-7 (Rowling, 1998, p. 98) and Cleansweep-11 (Rowling, 2003, p. 202), Nimbus-2000 (Rowling, 1998, p. 108) and Nimbus-2001 (Rowling, 1999a, p. 71), Comet- 260 (Rowling, 1998, p. 107) and Comet-290 (Rowling, 2003, p. 128), the Shooting Star (Rowling, 1999a, p. 30), the Bluebottle (Rowling, 2000, pp. 62–63), the Silver Arrow (Rowling, 1999b, p. 162) and the top-of-the-line Firebolt (Rowling, 1999b, p. 32). In other words, the technological progress in the pro- duction of broomsticks contributes to the Potterians’ leisure-related activities, the game of Quidditch being the primary example. This conclusion is also consistent with the observation that most stores in Diagon Alley sell the same old stuff over centuries, except for the Weasley twins’ Joke Shop, which sells some new models of games and toys, which are also an example of leisure goods. There is also evidence that over time there were non- trivial improvements in the broomsticks’ production, especially in their quality. For example, some of the early models were simple and basic, but more recent ones are more advanced. For example, the Shooting Star, the model owned by the Hogwarts School, is a basic broomstick, relatively cheap, ‘very slow and jerky’ (Rowling, 1999a, p. 30, Rowling, 1999b, p. 121), similar to the Bluebottle family series with an anti-burglar buzzer (Rowling, 2000, pp. 62–63). Nimbus-2000, on the other hand, is a top-of-the-line broomstick from the time of its release (Rowling, 1999a, p. 30), until Nimbus-2001 is released. The Firebolt, which is the latest addition, is perhaps ‘the Ferrari’ of the broomsticks. It is the best, fastest and most aerodynamically efficient model, offering a smooth action and fine control. It is a dream broomstick. POTTERIAN EDUCATION SYSTEM: INVESTMENT IN HUMAN CAPITAL Harry Potter books revolve around the life at Hogwarts School of Magic. It is fitting, therefore, to end with Oxford Open Economics, 2022, Vol. 1, No. 1 24 a short discussion of the Potterian education system (Note: See Snir & Levy (2010) for a discussion of the Potterian education system in the context of Solow growth model.). The wizards’ education system is publicly financed. It gives the students basic training and ensures that all wizards graduate with knowledge that allows them to find a job. The school is also one of the only institutions where the rules treat all members of society equally. It is also the only institution where middle- and high-class wizards interact on a day-to-day basis. seemingly equal education system,there are inequalities. Upon arrival at Hogwarts, the students are divided into four ‘houses’ based, to a large extent, on their ancestral history. Most high-class wizards end up in one particular house, whereas the middle-class wizards are scattered between the other three houses. The students, school experience depends largely on the house to which they belong. They, therefore, grad- uate with a strong association with the students from their own house, and thus the high-class wizards leave the school with different values, norms and associates than the wizards that belonged to other houses. Thus, the system that should have fostered equality of values ends up instead fomenting social division, tension and struggle. Downloaded from https://academic.oup.com/ooec/article/doi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 The wizards value education highly, and thus the pub- lic image of teachers is positive, so much so that they are even willing to sacrifice future income opportunities just to become teachers. For example, Gilderoy Lockhart, the celebrity author of seven books (Rowling, 1999a, pp. 28– 29, 38), becomes a professor at Hogwarts (Rowling, 1999a, p. 64). The value of education is further underscored by the high status of the school headmaster. The current headmaster, for example, is considered by many to be the greatest wizard of his time (Rowling, 1998, p. 66) (Note: See the number of Hogwarts’ former headmasters that are included in the list of the most famous wizards: www. hp-lexicon.org/wizards/card_wizards.html, accessed June 6, 2022.). POTTERIAN EDUCATION SYSTEM: INVESTMENT IN HUMAN CAPITAL Given the central role of education in the Potterian economy, we could consider the role of the Hog- warts School as a mechanism that contributes to the Potterians’ economic growth via endogenous accumula- tion of human capital (Uzawa, 1965; Lucas Jr., 1988). In Lucas’ model, human capital accumulation is modeled in the same way as physical capital accumulation is modeled in the Solow model. However, there is no knowledge accumulation in the Potterian economy. Thus, the stock of human capital is essentially fixed. Moreover, the contribution of education to the wizards’ productivity after they graduate and start working, most of them for the Potterian government, is unclear. Since there is no evidence of significant physical capital investment in the Potterian economy, the contribution of the wizards’ schooling to the productivity of physical capital is also doubtful. Along with the evidence that the Potterian economy is not growing (Snir and Levy, 2010), this suggests that the endogenous human capital accumulation mechanism does not play a role in the Potterian economy. As Long (2005) notes, ‘If only “He- Who-Must-Not-Be-Named” understood neo-classical endogenous growth theory, it might have been a different story.’ The Potterian education system is not problem-free, however. Hogwarts is under the supervision of the Min- istry of Magic and, thus, subject to the influence of politi- cians. The government can interfere with the school cur- riculum at will (Rowling, 2003, p. 229). Another weakness of Hogwarts is that its curriculum has not been revised for hundreds of years. Consequently, the graduates do not know more than their predecessors. The subjects and classes they take are those that were taken by the students of previous generations (Rowling, 1999a, p. 161). Similarly, the textbooks they use were also used by their parents and even grandparents. For example, the used copy of Advanced Potion-Making that Harry Potter bor- rowed from Professor Slughorn (Rowling, 2005, pp. 123, 125) was also used by Professor Snape’s mom, Eileen Prince (Rowling, 2005, p. 417), and by the Half-Blood Prince—Professor Snape (Rowling, 2005, p. 126), who was a classmate of Harry’s parents (Rowling, 2005, p. 360). The old-fashioned curriculum does not encourage innovative and creative thinking (Snir and Levy, 2010). The Potterian students focus primarily on practical skills but learn very little theory, literature, arts or philosophy. Most lack cre- ative skills and cannot think originally or independently (Rowling, 2000, p. 437). oi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 It follows that a naïve reader of Harry Potter would get a distorted view of economics. Consider some of the lessons that the principles of Potterian economics teach: governments are corrupt, wasteful and not trustwor- thy; markets are not fair because transactions are zero- sum; political process is not transparent; government is big and controls much of the production; governments cannot provide security, the most basic public good; in the marketplace, the wealthy have advantage over the poor; markets encourage crony capitalism; capitalists want to enslave the proletariat; bankers are goblins; busi- nessmen are deceptive and devious; wealthy people are mean and unethical; there is monopoly on information; power is concentrated; wealth is used to influence the government; domestic producers should be protected from foreign competition even if they are inefficient; international trade is not good; ignorance about foreign- ers, foreign cultures and foreign languages is the norm; lack of institutions offering financial intermediary ser- vices is the norm; no interest payments are expected on deposits; commodity money, coin denominations and attributes, and their exchange rates, all lead to higher transaction costs; paper checks are non-existent; arbi- trage opportunities are not exploited; monopolies are common; economic growth is not important; economic stagnation is not something that should bother us; tech- nological progress is not essential; innovative and cre- ative thinking is rare; prices are expected to remain unchanged; capital investment is non-existent; human capital does not accumulate; public employees should expect life-time job-security regardless of how inefficient In addition, the Potterian model lacks discussion of important aspects of real-world economies and societies. One important theme that is absent from the world of Harry Potter is religion. There are only two occasions where religion-related acts take place. One is the Christ- mas dinner party, which is held annually at the Hog- warts (e.g. Rowling, 1998, pp. 131–132). The second is the inscription Harry finds on his parents’ gravestone: ‘The last enemy that shall be destroyed is death’ (Rowling, 2007, pp. 216–217), which comes from the New Testament, 1 Corinthians 15:26, King James Version. Taxes of any kind are absent as well. The word ‘tax’ is mentioned in the seven-book series only once, in Rowling (2000, p. CONCLUSION: SUMMARY, POSSIBLE OBJECTIONS AND CAVEATS This paper is motivated, first by the evidence that much of the general knowledge about economic issues is trans- mitted and learned through various print and electronic media, and second, by the recent findings in psychol- ogy and neuroscience about the powerful influence that literary works, particularly fiction, have on the human mind. Building on these findings, we analyze the Potte- rian economy to assess the economic ideas and insights that these books convey to the readers, i.e. the principles of Potterian economics. Potterians do not have elementary schools or insti- tutes of higher education like universities. Upon gradu- ating from Hogwarts, wizards choose a profession and usually stick to it (Rowling, 1999a, pp. 161–162). Since there is no incentive to study further, Potterians’ stock of knowledge does not increase beyond what is acquired in the school. Their education system, therefore, resembles the Marxian system. Education is compulsory and all students receive the same education. Yet even in this We find that the Potterian model is not in line with a single economic model. Rather, it is a mix of ideas from various models. These include Marxian notions of class struggle and equality, public choice aspects of inefficient and corrupt government, sticky prices in the Keynesian 25 Levy and Snir spirit, full employment of the type found in Classical models, etc. the main employer in the Potterian economy. Therefore, it would have no difficulty collecting such taxes. Because the Potterian economics mixes ideas from different models and worldviews, many elements of the Potterian model are mutually inconsistent and contradictory, and thus perhaps even confusing. For example, the Potterian economic model is critical of market-based systems, yet it belittles government. The government is corrupt, yet it has public support. Many welfare-improving and mutually beneficial transactions do not take place, and there are no credit markets because of biases and prejudices, yet the books are often viewed and described as rejecting stereotypes. Potterian money is made of precious metals, yet its purchasing power is unrelated to its commodity value. The Potterian wizards value education, yet their school curriculum excludes theoretical subjects, their knowledge does not exceed much of their parents’ knowledge and they do not have institutes of high education, such as universities or colleges. CONCLUSION: SUMMARY, POSSIBLE OBJECTIONS AND CAVEATS According to Ross (2015), ‘“In a July 2015 story, a jour- nalist estimated it would cost more than $43,000 a year to go to Hogwarts, including all the Diagon Alley necessities like wands, robes, books and so on.” “My friends and I are having a super-intense debate about the cost of tuition at Hogwarts. Thanks.” “Rowling quickly shut down the idea on Twitter, clarifying that magical education is, in fact, free”: “There’s no tuition fee! The Ministry of Magic covers the cost of all magical education!”’ (Source: A. Ross, ‘The 50 Most Important Things We’ve Learned from J.K. Rowling,’ Time, July 30, 2015.) Thus,according to J.K.Rowling,there are no tuition fees at Hogwarts. But then the absence of taxes is particu- larly puzzling because, in any economy, taxes would be essential for funding public expenditures, such as public education. How is ‘free education’ funded? Who pays for it? The lack of taxes cannot be explained by the medieval structure of the Potterian society either, since taxes are mentioned numerous times in the Bible, in both the Old Testament (e.g. Exodus 30:11–16) and the New Testament (e.g. Romans 13:1–7). It is well known also that taxes were collected in Ancient Greece (e.g. Sosin, 2014), as well as in the Roman Empire (e.g. Hopkins, 1980). The absence of taxes in the Potterian economy is therefore mystifying, difficult to understand and a significant distortion. Moreover, the Potterian model misses many deep and fundamental aspects of economic analysis. For exam- ple, the Potterian bank does not serve as an intermedi- ary between savers and investors, the Potterian money lacks some key attributes (e.g. divisibility, portability, and homogeneity) that are essential for it to serve as an efficient medium of exchange or store of value, arbitrage opportunities are not exploited and efficiency-improving transactions go unnoticed. oi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 In light of the evidence about the potential influence of popular fiction on readers’ views and opin- ions, these can influence and shape the public’s under- standing of economic issues and matters. g Perhaps more importantly, however, these distorted views of the economy can potentially contribute to the public’s biases, misconceptions and more generally to their economic illiteracy, as documented by Caplan (2007), Rubin (2003) and others. Because most of the public never take college- or university-level courses in economics, they are susceptible to these kinds of subtle influences (Salonikov et al., 2022). It is likely, therefore, that the public that is exposed to such views and sentiments about economics, economic institutions, economic decisions and their outcomes, can be persuaded more easily by populist arguments against certain policies, against businessmen, against bankers and other beneficial service providers, against authorities (e.g. the central bank), etc. We shall note two possible objections to our inter- pretation. First, one could argue that because many of the Potterian economic principles coincide with popular folk economics, we should not expect them to be con- sistent with professional economists’ models, views or insights. For example, Harry Potter books are liked by many adults that read them, but they were primarily written for children. That might explain, one could argue, why the Potterian economy is so simple, with no capital markets, no taxes, etc. Potterian principles of economics pay attention to the Potterians’ unequal income distribu- tion, emphasizing the affluence of the upper-class wiz- ards and the poverty of the lower-class wizards, perhaps because children can relate to poor vs rich, but not to deeper notions of incentives and efficiency. These sim- plifying assumptions are necessary to make the books children-friendly. Moreover, the confusing insights the Harry Potter books convey about economics and economic matters by mixing various conflicting and mutually inconsistent messages, observations and arguments can lead to a process where a naïve citizen might lose faith in both market institutions as well as in government authority. The results might be apathy and indifference, which should be troubling. Following this line of argument, other aspects of the Potterian world can be explained by the fact that the books belong to a fantasy genre, and the events they describe are set in medieval times. Indeed, wizards, gob- lins, dragons and other similar creatures are typically associated with medieval times. oi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 3), in the context of the ownership of Riddle House: ‘The wealthy man who owned the Riddle House these days neither lived there nor put it to any use; they said in the village that he kept it for “tax reasons,” though nobody was very clear what these might be.’ Thus, if the wizards are subject to some taxes like ordinary muggles are, the author does not give us any information about it. It could be that taxes in the Potterian economy come in the form of constraints and limitations, for example, limitations on what the Potterian wizards can and can- not do. Such limitations could be interpreted as a tax. That, however, still leaves unanswered the question: why do Potterians choose this kind of ‘limitation-tax’ over income tax, which is more standard? The puzzle is par- ticularly interesting given that the Potterian government, that is the Ministry of Magic, is quite powerful, and it is Oxford Open Economics, 2022, Vol. 1, No. 1 26 and propagation of these biases and norms may be taking place from the period of early youth, because this is the age group in which fiction’s influence is likely to be par- ticularly strong and long-lasting. Further, an entire gen- eration of children and youngsters grew up along with Harry Potter, closely following his adventures for over 20 years since the first book in the series was published in 1998. Therefore, given the books extraordinarily large worldwide audience, and given that a significant propor- tion of the readers are kids, these biased and distorted views about many fundamental economic issues could potentially persist, and even spread further. A folk eco- nomic interpretation of the Potterian model, therefore, suggests that popular intermediaries might be playing an important role in spreading biases and ignorance on important economic issues. and unproductive they are; the default employer is the public sector, not the private sector; downward social mobility is the norm; there are no taxes yet education is free; high education is not critical; curriculum can remain unchanged for centuries; only practical skills are valued, theoretical knowledge is of no value; there is a constant class struggle; and this is only a partial list. Many of these lessons seem shallow and uninformed characterizations of markets, market institutions and arrangements and market participants and their motives and goals. oi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 However, on some aspects of the Potterian world, there is less consensus and thus they are subject to different interpretations. These observations and arguments are important. However, the books’ broad, near-universal success suggests that in addition to telling a story, which seems to appeal to a large and diverse audience, the author may have also been able to capture the popular beliefs and sentiments of hundreds of millions of people across the globe. In other words, people from a broad and varied range of backgrounds, values, cultures, societies, religions and places, irrespective of age and gender, all seem to be able to relate to the Potterian story and the sentiments it conveys, consciously and perhaps subconsciously. For example, in an essay published in June 2004 in the French daily Le Monde, Ilias Yocaris, a professor of literary theory and French literature, argued that ‘the fantastic universe of Harry Potter is a capitalist universe.’ In July 2004, however, the same newspaper published an article by Isabelle Smadja, a professor of philosophy, in which she argues exactly the opposite: ‘Far from being a capitalist lackey, Harry Potter is, in fact, the first fictional hero of the anti-globalist, anti-free market, pro-Third World, “Seattle” generation . . . [Smadja] suggests that Yocaris has been confused by the fact that the Potter books are, themselves, such a global, commercial and marketing success. Examination of the text suggests that they are, in fact, a “ferocious critique of consumer society and the world of free enterprise.”’ (Source: http://www.nzherald.co.nz/lifestyle/news/article.cfm? c_id=6&objectid=3576571, accessed June 6, 2022. New York Times translated Ilias Yocaris’ article to English and published it on its editorial page in July 18, 2004, available at http://www.nytimes.com/2004/07/18/ opinion/harry-potter-market-wiz.html, accessed June 6, 2022.) Under this reasoning, we argue, Potterian economics can still inform us about the formation of folk economics, even if from the author’s point of view, it merely reflects her personal opinion based on her private life experi- ences, or even if the entire project was intended just for children’s bedtime reading. For similar reasons, the value of studying Potterian economics is not diminished even if the author is ‘expressive’ (Hillman, 2010b), i.e. she is expressing not necessarily her own beliefs about eco- nomic matters, but rather her beliefs about her readers’ beliefs. oi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 That can explain also the use of commodity money, why goblins are unfriendly, etc. These findings imply that popular literature along with other information intermediaries may be contributing to the public’s lack of understanding of many economic issues, as some studies of economic and financial literacy have documented recently. But we should note that some of the biases we identify, many of them stereotypical, are not new, as they have been around for centuries (Harap, 2003). For example, the negative portrayal of bankers has been a recurring theme in popular literature for a long time. Similarly, populist views and criticisms of governments, class struggles and other social ills and institutions are not new. Similarly, the stagnation of the Potterian economy is necessary for making the story credible, as it takes place during the medieval period, a period in which the world was stagnating because there was no increase in capital stock or population, and there was little technological progress. The corrupt and inefficient government could be a literary device similar to those used in comic books such as ‘Superman’ or ‘Batman’: corrupt characters are required to have a need for a hero in the form of Harry Potter and his sidekicks. These observations suggest that in addition to directly influencing the public views and opinions, Harry Pot- ter books could be reinforcing the existing norms and beliefs. This reinforcement mechanism might be play- ing an important role in transmitting the biases and other social treats through generations, via the process of cultural transmission of values (Bisin and Verdier, 2000, 2008; Necker and Voskort, 2014). Moreover, the formation The second type of argument could be that Potte- rian economics merely captures the author’s subjec- tive opinions and views, which reflect her personal experiences and biases about economic issues, and the financial consequences of her life experience as a divorced, unemployed and welfare-dependent sin- gle parent, followed by phenomenal success (Source: 27 Levy and Snir \| Levy and Snir https://www.businessinsider.com/the-rags-to-riches- story-of-jk-rowling-2015-5, accessed June 6, 2022.). As a caveat, we should note that the interpretation of Potterian economics, which we lay out in this paper, is based on our understanding of the economics of Harry Potter. Many of the ideas and the interpretations we offer seem non-controversial and consistent with the interpretations of other scholars and writers cited above, most of them outside the field of economics. oi/10.1093/ooec/odac004/6646895 by Emory University Libraries user on 04 August 2022 Thus, regardless of the motivation of the author for writing these books, and regardless of whether the books reflect her private opinions, or her beliefs about her readers’ opinions, interpreting the Potterian economic model as a reflection of folk economics suggests that popular intermediaries could potentially play an impor- tant role in spreading biases, distorted opinions and igno- rance on important economic issues. Thus, rather than dismissing the ‘mishmash’ of mutually inconsistent and contradictory economic ideas found in the Harry Potter books, we suggest taking them seriously to try and under- stand their sources and persistence. ) We agree that some of the aspects of the Potterian economy may be subject to a different interpretation than ours. We believe, however, that one main reason for these discrepancies is precisely what we argued above— principles of Potterian economics are a mix of ideas from different models, not always consistent with each other, quite often mutually orthogonal and contradictory. A story that contains a mix of ideas and worldviews that are inconsistent with each other will likely lead to different interpretations, depending on readers’ points of view. As Gary Becker is quoted in the August 21, 2011, New York Times article (p. SR5), writers do not always understand the intricacies of how markets work and function. It is not easy to write an adventure story that contains all social and market institutions and get it fully right. It is similar to writing a formal economic model: it requires a solid and deep understanding of economic ideas. But this is not limited to economics, however, as it applies to other fields of knowledge as well. Therefore, it is not surprising that some of the most successful fiction authors, it turns out, have spent an enormous amount of time on research and study (Francis, 2012; Popoola, 2015). For example, according to Freedman (2005, p. xiii), Isaac Asimov was the best physicist among non-physicists, and the best zoologist among non-zoologists, hinting at the seriousness in which Asimov did his research before writing his books and essays. Similarly, it took J.R.R. Tolkien 17 years to complete The Lord of the Rings (Source: middle-earth.xenite.org/2011/09/16/how-long- did-it-take-j-r-r-tolkien-to-write-the-lord-of-the-rings/, accessed June 6, 2022.). Before we end, consider the following thought exper- iment. Acknowledgments We thank an anonymous reviewer for helpful comments and the editor Amrita Dhillon for guidance. Many colleagues and students have commented on earlier versions of this paper. In particular, we would like to thank Zurab Abramishvili, Markus Brückner, Roger Congleton and Pierre-Guillaume Méon, the discussants at the Silvaplana Workshop on Political Economy. In addition, we thank Anat Alexandron, Sima Amram, Paul Anglin, Katrina Babb, Anindya Banerjee, Bob Barsky, Yoram Bauman, Mark Bergen, Sabrina Artinger, Helen Casey, Allan Chen, Ong EeCheng, Raphael Franck, Max Gillman, Allen Goodman, Danielle Gurevitch, Yuval Heller, Arye Hillman, Daniel Houser, Miriam Krausz, Christoph Kuzmics, Frank Lechner, Steven Levitt, Sarit Levy, Lorence Maimony, Asher Meir, Hugo Mialon, Igal Milchtaich, Lavinia Moldovan, Shmuel Nitzan, Chryssa Papathanassiou, Ron Peretz, Paul Rubin, Adi Schnytzer, Matthew Shapiro, Ainit Snir, the late Heinrich Ursprung, Timothy Wong, and the seminar participants at Bar- Ilan University, at the Silvaplana Workshop on Political Economy, at the International School of Economics Tbilisi (ISET), at the National University of Singapore, at the American Economic Association Conference on Teaching and Research in Economic Education, and at the American Economic Association’s annual conference, for comments, suggestions and conversations about the economics of magic. Avihai Levy and Eliav Livneh, two part-time wizards, provided research assistance. Earlier versions of this paper were circulated under the titles ‘Abracadabra! Social Norms and Public Perceptions through Harry Potter’s Looking Glasses’ and ‘Popular Perceptions and Political Economy in the Contrived World of Harry Potter.’ All errors are ours. Future studies should analyze the economic content of other literary texts to better understand how popu- lar opinions about the economy are influenced by fic- tion and media, and how they form and change over time (Cowen, 2008; Miller and Watts, 2011). For example, the Potterian model points to a change in the image of the government as reflected in fiction. For example, Tolkien’s Lord of the Rings, published in the 1950s (Tolkien, 1993), depicts the government as efficient and benevo- lent. In contrast to this idealistic and perhaps naïve view, the Harry Potter books portray the government officials as corrupt, dishonest, incompetent and unkind, imply- ing that today’s public is perhaps more realistic. Inter- estingly, this change occurred during the period when economists moved away from studying normative mod- els of the government to studying models that emphasize the role of private and group-specific incentives (Hillman, 2009). Acknowledgments ) As another example, Adhia (2013) provides evidence of ideological change in India. Since the 1980s, he notes, the popular sentiments in India have evolved from con- demning profits as anti-social to accepting—and even applauding—business success. Applying the method of content analysis to Hindi films, he finds that rich mer- chant characters have changed from being depicted as villains to being depicted as heroes (Note: The methodol- ogy we have employed in this paper is similar in spirit to content analysis, a widely used empirical methodology employed in other social sciences and humanities. See Levy et al. (2002) for an example and a brief survey.). These developments point to changes that modern soci- eties experience and suggest fruitful avenues for future research. Data Availability We believe this point highlights a key advantage of this paper. It shows why the Potterian economy resonates with people. Despite its shortcomings and inaccuracies, it is consistent with folk economics, which while perhaps problematic for human flourishing in a Smithian sense, it captures and reflects people’s views on many economic and social issues. It is stable equilibrium, just not one that will get us to today’s level of prosperity in the developed world, even with Wizarding powers. Data availability does not apply in this case. The online supplementary appendix contains a detailed reference to all economic themes, topics and issues we have identified in the Harry Potter books, along with the quotations of the relevant texts from the books, and their exact locations. Conflict of Interest None declared. SUPPLEMENTARY DATA Aaronovitch, D. (2010) Voodoo Histories: The Role of the Conspiracy Theory in Shaping Modern History, New York, NY: Riverhead. Adhia, N. (2013) ‘The Role of Ideological Change in India’s Economic Liberalization’, The Journal of Socio-Economics, 44: 103–11. Albrecht, M. (1954) ‘The Relationship of Literature and Society’, American Journal of Sociology, 59: 425–36. Aaronovitch, D. (2010) Voodoo Histories: The Role of the Conspiracy Theory in Shaping Modern History, New York, NY: Riverhead. 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(2010) Books Are Windows, Books Are Mirrors: Multicul- tural Collections for Children and Young Adults Opening New Worlds. ManuscriptMidrand Grad Institute. Potterian Economics Last Revision: June 9, 2022 Last Revision: June 9, 2022 The tables below list the economic ideas, principles, concepts, observations, and events that we identify in the Potterian economic model and the corresponding quotes from the Harry Potter books. Many of the economic ideas appear in the Harry Potter books more than once. For example, various make and model broomsticks which are listed in Appendix E, and which we discuss in the paper in section 12 in the context of technological innovations, are mentioned in the books numerous times. To construct these tables, we tried to identify the episodes and the locations in the story where these ideas appear for the first time. We shall note that the list is not exhaustive and there are many more economic ideas in the world of Harry Potter. 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(2004) ‘Managerial and Customer Costs of Price Adjustment: Direct Evidence From Industrial Markets’, Review of Economics and Statistics, 86: 514–33. Tullock, G. (1959) ‘Problems of Majority Voting’, Journal of Political Economy, 67: 571–9. Contents Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 1 This appendix contains a list of the ideas found in Potterian economics which are discussed in the body of the paper. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 2 Appendix A. Economic Ideas1 Economic Idea Quote Reference Commodity money denominations “The gold ones are Galleons,” he explained. “Seventeen silver Sickles to a Galleon and twenty-nine Knuts to a Sickle, it’s easy enough.” Rowling, 1998, p. 49 Foreign currency “You’re not the first one who’s had trouble with money,” said Mr. Roberts, scrutinizing Mr. Weasley closely.” Rowling, 2000, p. 50 Coin values are independent of their values as a commodity “I had two try and pay me with great gold coins the size of hubcaps ten minutes ago.” Rowling, 2000, p. 50 Commodity money - heavy and cumbersome “Ron purchased a dancing shamrock hat and a large green rosette, he also bought a small figure of Viktor Krum, the Bulgarian Seeker…“Wow, look at these!” said Harry…“Omnioculars,” said the saleswizard eagerly…”Bargain - ten Galleons each.”…“Three pairs,” said Harry firmly to the wizard…Their money bags considerably lighter, they went back to the tents. Rowling, 2000, pp. 60–61 Counterfeit money “Well, let’s check how yeh’ve done!” said Hagrid. “Count yer coins! An’ there’s no point tryin’ ter steal any, Goyle,” he added, his beetle-black eyes narrowed. “It’s leprechaun gold. Vanishes after a few hours.” “I know that, Harry, but if she wakes up and the locket’s gone – I need to duplicate it – Geminio! There…That should fool her…” “They have added Germino…Curse!” said Griphook. “Everything you touch will…multiply, but the copies are worthless – and if you continue to handle the treasure, you will eventually be crushed to death by the weight of expanding gold!” Rowling, 2000, p. 350 Rowling, 2007, p. 173 Rowling, 2007, p. 356 Constraints on Converting metals into gold “Sorcerer’s Stone…will transform any metal into pure gold…but the only Stone currently in existence belongs to Mr. Nicolas Flamel…who celebrated his six hundred and sixty-fifth birthday last year.” Rowling, 1998, p. 143 Opportunity cost of using precious metals for making coins “And now Wormtail was whimpering. He pulled a long, thin, shining silver dagger from inside his cloak.” “Harry walked up the worn stone steps, staring at the newly materialized door. Its black paint was shabby and scratched. The silver doorknocker was in the form of a Rowling, 2000, p. 413 Rowling, 2003, p. 45 1 This appendix contains a list of the ideas found in Potterian economics which are discussed in the body of the paper. Appendix A. Economic Ideas1 3 twisted serpent. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 There was no keyhole or letterbox.” “Dumbledore gave a great sniff as he took a golden watch from his pocket and examined it.” Rowling, 1998, p. 8 Transaction cost of withdrawing money from the bank “An’ I’ve also got a letter here from Professor Dumbledore,”… “Very well,” he said…“I will have someone take you down…” Griphook whistled and a small cart came…hurtled through a maze of twisting passages…left, right, right, left, middle fork, right, left,…The rattling cart seemed to know its own way… they plunged even deeper “…I think I’m gonna be sick.”…Hagrid…had to lean against the wall to stop his knees from trembling…Inside were mounds of gold coins…silver…bronze Knuts…He turned to Griphook. “…can we go more slowly?” “One speed only,” said Griphook. They were going even deeper now and gathering speed. The air became colder and colder…One wild cart ride later they stood…outside Gringotts.” Rowling, 1998, pp. 47–49 The amount of money that Harry withdraws from the bank completely fills his bag Once Harry had refilled his money bag with gold Galleons, silver Sickles, and bronze Knuts from his vault at Gringotts, he had to exercise a lot of self-control not to spend the whole lot at once. Rowling, 1999b, p. 31 Cash (Clower) constraint “I haven’t got any money — and you heard Uncle Vernon last night…he won’t pay for me to go and learn magic.” Rowling, 1998, p. 41 Wizards’ difficulties in handling foreign currency “They had reached the station. There was a train to London in five minutes’ time. Hagrid, who didn’t understand “Muggle money,” as he called it, gave the bills to Harry so he could buy their tickets.” “Help me, Harry,” he muttered, pulling a roll of Muggle money... “This one’s a - a - a ten? Ah yes, I see the little number on it now…So this is a five?” “A twenty,” Harry corrected him... “Ah yes, so it is…I don’t know, these little bits of paper…” “You foreign?” said Mr. Roberts as Mr. Weasley returned with the correct notes. “Foreign?” repeated Mr. Weasley, puzzled. “You’re not the first one who’s had trouble with money” “Stored in an underground vault at Gringotts in London was a small fortune that his parents had left him. Of course, it was only in the Wizarding world that he had money; you couldn’t use Galleons, Sickles, and Knuts in Muggle shops.” Rowling, 1998, p. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 43 Rowling, 2000, p. 50 Rowling, 1999a, p. 30 4 Just-below (or psychological) prices “A plump woman outside an Apothecary was shaking her head as they passed, saying, “Dragon liver, sixteen Sickles an ounce, they’re mad…” Rowling, 1998, p. 46 Sticky prices I – Daily Prophet costs 1 Knut for 7 years “Hermione, however, had to move her orange juice aside quickly to make way for a large damp barn owl bearing a sodden Daily Prophet in its beak. “What are you still getting that for?” said Harry irritably, thinking of Seamus as Hermione placed a Knut in the leather pouch on the owl’s leg and it took off again.” Rowling, 2003, p. 167 Sticky prices II – Floo Powder’s price is the same for over 100 years “No shortage of Floo powder has ever been reported, nor does anybody know anyone who makes it. Its price has remained constant for one hundred years: two Sickles a scoop. Every wizard household carries a stock of Floo powder, usually conveniently located in a box or vase on the mantelpiece.” pottermore head.tumb lr.com/pos t/1021758 02190/floo -powder, accessed on May, 31, 2015 Gringotts bank – a monopoly run by goblins “They didn’ keep their gold in the house, boy! Nah, first stop fer us is Gringotts. Wizards’ bank.” “Wizards have banks?” “Just the one. Gringotts. Run by goblins.” Rowling, 1998, p. 41 Goblins – greedy bankers “They passed a group of goblins who were cackling over a sack of gold that they had undoubtedly won betting on the match, and who seemed quite unperturbed by the trouble at the campsite.” Rowling, 2000, p. 81 Counterfeit money – even school boys can do that “Hermione soon devised a very clever method of communicating the time and date of the next meeting… She gave each of the members of the D.A. a fake Galleon …“You see the numerals around the edge of the coins? ... On real Galleons that’s just a serial number referring to the goblin who cast the coin. On these fake coins, though, the numbers will change to reflect the time and date of the next meeting. The coins will grow hot when the date changes.” Rowling, 2003, p. 297 Introduction of counterfeit gold Galleons – makes people extra cautious “Yeah…I prefer your way,” said Harry, grinning, as he slipped his Galleon into his pocket. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 “I suppose the only danger with these is that we might accidentally spend them.” “Fat chance,” said Ron, who was examining his own fake Galleon with a slightly mournful air, “I haven’t got any real Galleons to confuse it with.” Rowling, 2003, p. 298 Withdrawing money from the bank – the Gringotts “Morning,” said Hagrid to a free goblin. “We’ve come ter take some money outta Mr. Harry Potter’s safe.” “You have his key, sir?”…“Got it,” said Hagrid at last, holding up a tiny golden key. The goblin looked at it closely. “That seems to be in order.” Rowling, 1998, p. 47 Services “When the cart stopped Griphook unlocked the door Rowling 5 Gringotts - safe keeping Heaps of little bronze Knuts. “All yours,” smiled Hagrid …“The gold ones are Galleons,” he explained. “Seventeen silver Sickles to a Galleon and twentynine Knuts to a Sickle, it’s easy enough. Right, that should be enough fer a couple o’ terms, we’ll keep the rest safe for yeh.” “Harry. Gringotts is the safest place in the world fer anything yeh want ter keep safe.” 48–49 Rowling, 1998, p. 41 Services offered at Gringotts – exchange of wizard money for precious stones “A pair of goblins bowed them through the silver doors and they were in a vast marble hall. About a hundred more goblins were sitting on high stools behind a long counter… examining precious stones through eyeglasses.” Rowling 1998, p. 47 Services offered at Gringotts – exchange of wizard money for Muggle- money “You be careful, Arthur,” said Mrs. Weasley sharply as they were bowed into the bank by a goblin at the door. “…Hermione’s parents…were standing nervously at the counter that ran all along the great marble hall…“But you’re Muggles!” said Mr. Weasley delightedly…“What’s that you’ve got there? Oh, you’re changing Muggle money. Molly, look!” He pointed excitedly at the ten- pound notes in Mr. Granger’s hand.” Rowling, 1999a, p. 37 Wizards face difficulties in using Muggle money “There was a train to London in five minutes’ time. Hagrid, who didn’t understand “Muggle money,” as he called it, gave the bills to Harry so he could buy their tickets.” “Help me, Harry,” he muttered, pulling a roll of Muggle money... “This one’s a - a - a ten? Ah yes, I see the little number on it now…So this is a five?” “A twenty,” Harry corrected him... Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 “Ah yes, so it is…I don’t know, these little bits of paper…” Rowling, 1998, p. 43 Rowling, 2000, p. 50 Wizards’ and Muggles’ lack of interaction “But what does a Ministry of Magic do?” “Well, their main job is to keep it from the Muggles that there’s still witches an’ wizards up an’ down the country.” “Hey, Harry,”…“have you heard?...Sirius Black’s been sighted.” “Where?” said Harry and Ron quickly. “Not too far from here,” said Seamus…“It was a Muggle who saw him…‘Course, she didn’t really understand. The Muggles think he’s just an ordinary criminal, don’t they?” “You foreign?” said Mr. Roberts as Mr. Weasley returned with the correct notes. “Foreign?” repeated Mr. Weasley, puzzled. “You’re not the first one who’s had trouble with Rowling, 1998, p. 42 Rowling, 1999b, p. 80 Rowling, 2000, p. 50 6 money” Wizards need to borrow from illegal usurers or friends “Turns out he’s [Ludo Bagman] in big trouble with the goblins. Borrowed loads of gold off them. A gang of them cornered him in the woods after the World Cup and took all the gold he had, and it still wasn’t enough to cover all his debts. They followed him all the way to Hogwarts to keep an eye on him.” “Harry, you help yourself to anything you want…No charge.” “I can’t do that!” said Harry, who had already pulled out his money bag to pay…“You don’t pay here,” said Fred firmly, waving away Harry’s gold…“You gave us our start-up loan, we haven’t forgotten,” said George sternly.” Rowling, 2000, p. 471 Rowling, 2005, p. 78 Fred and George Weasley borrow from Harry Potter “Harry…had forced the Weasley twins to take the thousand Galleons prize money he had won in the Triwizard Tournament to help them realize their ambition to open a joke shop.” Rowling, 2003, p. 79 Wizards borrow from Goblins “Turns out he’s [Ludo Bagman] in big trouble with the goblins. Borrowed loads of gold off them. A gang of them cornered him in the woods after the World Cup and took all the gold he had, and it still wasn’t enough to cover all his debts. They followed him all the way to Hogwarts to keep an eye on him.” Rowling, 2000, p. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 471 Weasley twins consider gambling to obtain funds needed for opening their Joke-Shop “We’ll bet thirty-seven Galleons, fifteen Sickles, three Knuts,” said Fred as he and George quickly pooled all their money, “that Ireland wins - but Viktor Krum gets the Snitch. Oh and we’ll throw in a fake wand.”…“Boys,” said Mr. Weasley under his breath, “I don’t want you betting…That’s all your savings” Rowling, 2000, p. 57 Government depends on donations from wealthy individuals “Mr. Weasley and Mr. Malfoy looked at each other… Malfoy’s cold gray eyes swept over Mr. Weasley, and then up and down the row. “Good lord, Arthur,” he said softly. “What did you have to sell to get seats in the Top Box? Surely your house wouldn’t have fetched this much?” Fudge, who wasn’t listening, said, “Lucius has just given a very generous contribution to St. Mungo’s Hospital for Magical Maladies and Injuries, Arthur. He’s here as my guest.” Rowling, 2000, p. 66 Gringotts’ employees offer private usury services “Turns out he’s [Ludo Bagman] in big trouble with the goblins. Borrowed loads of gold off them. A gang of them cornered him in the woods after the World Cup and took all the gold he had, and it still wasn’t enough to cover all his debts. They followed him all the way to Hogwarts to keep an eye on him.” Rowling, 2000, p. 471 7 7 Wizards that make windfall gains spend them immediately “The clipping had clearly come out of the wizarding newspaper, the Daily Prophet…Harry picked up the clipping…and read:…Arthur Weasley…has won the annual Daily Prophet Grand Prize Galleon Draw. A delighted Mr. Weasley told the Daily Prophet, “We will be spending the gold on a summer holiday in Egypt.” Rowling, 1999b, p. 5 Wizards view financial service providers as immoral “Wizards have banks?” “Just the one. Gringotts. Run by goblins.” Harry dropped the bit of sausage he was holding. “Goblins?” “Yeah — so yeh’d be mad ter try an’ rob it, I’ll tell yeh that. Never mess with goblins, Harry. “They passed a group of goblins who were cackling over a sack of gold that they had undoubtedly won betting on the match, and who seemed quite unperturbed by the trouble at the campsite.” “Absolute nightmare,” said Bagman to Harry in an undertone, noticing Harry watching the goblins too. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 “Their English isn’t too good…it’s like being back with all the Bulgarians at the Quidditch World Cup…but at least they used sign language another human could recognize.” “The goblins play as dirty as him. They say you drew with Diggory, and Bagman was betting you’d win outright. So Bagman had to run for it. He did run for it right after the third task.” Rowling, 1998, p. 41 Rowling, 2000, p. 81 Rowling, 2000, p. 287 Rowling, 2000, p. 471 Goblins’ inferior image inhibits most forms of interaction between wizards and Goblins “If there was a wizard of whom I would believe that they did not seek personal gain,” said Griphook finally, “it would be you, Harry Potter. Goblins … are not used to… the respect that you have shown this night. Not from wand-carriers.” “Then I have to say this,” Bill went on. “If you have struck any kind of bargain with Griphook, and most particularly if that bargain involves treasure, you must be exceptionally careful. Goblin notions of ownership, payment, and repayment are not the same as human ones.” …However, there is a belief among some goblins, and those at Gringotts are perhaps most prone to it, that wizards cannot be trusted in matters of gold and treasure, that they have no respect for goblin ownership.” Rowling, 2007, p. 323 Rowling, 2007, p. 342 Rent-seeking - Wealthy individuals often fund public goods “I’ll leave a note for Dumbledore when I drop you off, he ought to know Malfoys been talking to Fudge again.” “What private business have they got together, anyway?” “Gold, I expect,” said Mr. Weasley angrily. “Malfoy’s been giving generously to all sorts of things for years… Rowling, 2003, p. 116 8 and thus exert influence on public officials and public policy gets him in with the right people…then he can ask favors …delay laws he doesn’t want passed…oh, he’s very well- connected, Lucius Malfoy.” Newspaper reports portray the officials positively, who reciprocate by making decisions favoring the reporters and wealthy wizards “Rita…said…“All right, Fudge is leaning on the Prophet, but it comes to the same thing. They won’t print a story that shows Harry in a good light. Nobody wants to read it. It’s against the public mood.” Rowling 2003, p. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 423 The Minister of Magic is appointed, not elected “Newly appointed Minister of Magic, Rufus Scrimgeour, spoke today of the tough new measures taken by his Ministry to ensure the safety of students returning to Hogwarts School of Witchcraft and Wizardry this autumn.” Rowling 2005, p. 27 Government – Ministry of Magic “Hagrid read his newspaper, the Daily Prophet. “Ministry o’ Magic messin’ things up as usual,” Hagrid muttered, turning the page. “There’s a Ministry of Magic?” Harry asked...“’Course,” said Hagrid. “They wanted Dumbledore fer Minister, o’ course, but he’d never leave Hogwarts, so old Cornelius Fudge got the job. Bungler if ever there was one. So he pelts Dumbledore with owls every morning, askin’ fer advice.” Rowling, 1998, p. 42 Inept public employees “You wouldn’t believe how many people, even people who work at the Ministry, can’t do a decent Shield Charm” Rowling, 2005, p. 78 Bribery “It was Umbridge’s lie that brought the blood surging into Harry’s brain and obliterated his sense of caution – that the locket she had taken as a bribe from a petty criminal was being used to bolster her own pure-blood credentials.” “When you stripped this house of all the valuables you could find,” Harry began again, “you took a bunch of stuff from the kitchen cupboard. There was a locket there.” Harry’s mouth was suddenly dry: He could sense Ron and Hermione’s tension and excitement too. “What did you do with it?” “Why?” asked Mundungus. “Is it valuable?” “You’ve still got it!” cried Hermione. “No, he hasn’t,” said Ron shrewdly. “He’s wondering whether he should have asked more money for it ” “More?” said Mundungus Rowling, 2007, p. 172 Rowling, 2007, p. 144 9 “That wouldn’t have been effing difficult…bleedin’ gave it away, di’n’ I? No choice.” “What do you mean?” “I was selling in Diagon Alley and she come up to me and asks if I’ve got a license for trading in magical artifacts. Bleedin’ snoop. She was gonna fine me, but she took a fancy to the locket an’ told me she’d take it and let me off that time, and to fink meself lucky.” “Who was this woman?” asked Harry. “I dunno, some Ministry hag.” Nepotism is common “What does your dad do at the Ministry of Magic, anyway?” “He works in the most boring department,” said Ron. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 “The Misuse of Muggle Artifacts Office.” “The what?” “It’s all to do with bewitching things that are Muggle-made, you know, in case they end up back in a Muggle shop or house. Like, last year, some old witch died and her tea set was sold to an antiques shop. This Muggle woman bought it, took it home, and tried to serve her friends tea in it. It was a nightmare — Dad was working overtime for weeks.” “You are sweet,” beamed Mrs. Weasley…“Yes, Rufus Scrimgeour has set up several new offices in response to the present situation, and Arthur’s heading the Office for the Detection and Confiscation of Counterfeit Defensive Spells and Protective Objects.” “I’ve been promoted,” Percy said before Harry could even ask, and from his tone, he might have been announcing his election as supreme ruler of the universe. “I’m now Mr. Crouch’s personal assistant, and I’m here representing him.” “In a surprise move last night the Ministry of Magic passed new legislation… “‘The Minister has been growing uneasy about goings-on at Hogwarts for some time,’ said junior Assistant to the Minister, Percy Weasley.” “D’you know what you want to do after Hogwarts?” Harry asked the other two... “Not really,” said Ron slowly. “Except…well…” He looked slightly sheepish. “What?” Harry urged him. ‘“Well, it’d be cool to be an Auror [at the Ministry of Magic],’ “ said Ron in an off-hand voice. “Yeah, it would,” said Harry fervently. “But they’re, like, the elite,” said Ron.” “Good training for when we’re all Aurors,” said Ron excitedly, attempting the Impediment Curse on a wasp that had buzzed into the room and making it stop dead in Rowling, 1999a, p. 20 Rowling, 2005, p. 56 Rowling, 2000, p. 268 Rowling, 2003, p. 229 Rowling, 2003, p. 170 Rowling, 2000, p. 392 “That wouldn’t have been effing difficul it away, di’n’ I? No choice.” “What do y selling in Diagon Alley and she come up I’ve got a license for trading in magical a snoop. She was gonna fine me, but she to locket an’ told me she’d take it and let m and to fink meself lucky.” “Who was thi Harry. “I dunno, some Ministry hag.” Nepotism is common “What does your dad do at the Ministry anyway?” “He works in the most boring Ron. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 “The Misuse of Muggle Artifacts O what?” “It’s all to do with bewitching th Muggle-made, you know, in case they en Muggle shop or house. Like, last year, so died and her tea set was sold to an antiqu Muggle woman bought it, took it home, her friends tea in it. It was a nightmare — working overtime for weeks.” “You are sweet,” beamed Mrs. Weasley… Scrimgeour has set up several new office the present situation, and Arthur’s headin the Detection and Confiscation of Count Spells and Protective Objects.” “I’ve been promoted,” Percy said before ask, and from his tone, he might have be election as supreme ruler of the universe Crouch’s personal assistant, and I’m her him.” “In a surprise move last night the Minist passed new legislation… “‘The Minister growing uneasy about goings-on at Hogw time,’ said junior Assistant to the Minist Weasley.” “D’you know what you want to do after Harry asked the other two... “Not really, “Except…well…” He looked slightly sh Harry urged him. ‘“Well, it’d be cool to the Ministry of Magic],’ “ said Ron in an “Yeah, it would,” said Harry fervently. “ the elite,” said Ron.” “Good training for when we’re all Auror excitedly, attempting the Impediment Cu that had buzzed into the room and makin “That wouldn’t have been effing difficult…bleedin’ gave it away, di’n’ I? No choice.” “What do you mean?” “I was selling in Diagon Alley and she come up to me and asks if I’ve got a license for trading in magical artifacts. Bleedin’ snoop. She was gonna fine me, but she took a fancy to the locket an’ told me she’d take it and let me off that time, and to fink meself lucky.” “Who was this woman?” asked Harry. “I dunno, some Ministry hag.” Nepotism is common “What does your dad do at the Ministry of Magic, anyway?” “He works in the most boring department,” said Ron. “The Misuse of Muggle Artifacts Office.” “The what?” “It’s all to do with bewitching things that are Muggle-made, you know, in case they end up back in a Muggle shop or house. Like, last year, some old witch died and her tea set was sold to an antiques shop. This Muggle woman bought it, took it home, and tried to serve her friends tea in it. It was a nightmare — Dad was working overtime for weeks.” Rowling, 1999a, p. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 20 “You are sweet,” beamed Mrs. Weasley…“Yes, Rufus Scrimgeour has set up several new offices in response to the present situation, and Arthur’s heading the Office for the Detection and Confiscation of Counterfeit Defensive Spells and Protective Objects.” 10 midair.” Rent-seeking: Wealthy wizards that fund public officials’ office expenditures have access to the officials and influence their decisions “Malfoy’s been giving generously to all sorts of things for years… gets him in with the right people…then he can ask favors …delay laws he doesn’t want passed…oh, he’s very well-connected, Lucius Malfoy.” Rowling, 2003, p. 116 Mr. Malfoy knows in advance about the Ministry’s planned raid “Mr. Malfoy, what a pleasure to see you again,” said Mr. Borgin in a voice as oily as his hair. “Delighted — and young Master Malfoy, too — charmed. How may I be of assistance? I must show you, just in today, and very reasonably priced —” “I’m not buying today, Mr. Borgin, but selling,” said Mr. Malfoy. “Selling?” The smile faded slightly from Mr. Borgin’s face. “You have heard, of course, that the Ministry is conducting more raids,” said Mr. Malfoy, taking a roll of parchment from his inside pocket and unraveling it for Mr. Borgin to read. “I have a few — ah — items at home that might embarrass me, if the Ministry were to call…” Mr. Borgin fixed a pair of pince-nez to his nose and looked down the list. “The Ministry wouldn’t presume to trouble you, sir, surely?” Mr. Malfoy’s lip curled. “I have not been visited yet. The name Malfoy still commands a certain respect, yet the Ministry grows ever more meddlesome. There are rumors about a new Muggle Protection Act — no doubt that flea- bitten, Muggle-loving fool Arthur Weasley is behind it — ” Harry felt a hot surge of anger. “— and as you see, certain of these poisons might make it appear —” “I understand, sir, of course,” said Mr. Borgin. “Let me see…“I am in something of a hurry, Borgin, I have important business elsewhere today —” …They started to haggle. “Done,” said Mr. Malfoy at the counter. “Come, Draco —”… “Good day to you, Mr. Borgin. I’ll expect you at the manor tomorrow to pick up the goods.” Rowling, 1999a, pp. 33–34 Junior public officials make “Percy hurried forward with his hand outstretched. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 Apparently his disapproval of the way Ludo Bagman ran Rowling, 2000, p. and influence their decisions Mr. Malfoy knows in advance about the Ministry’s planned raid “Mr. Malfoy, what a pleasure to see you again,” said Mr. Borgin in a voice as oily as his hair. “Delighted — and young Master Malfoy, too — charmed. How may I be of assistance? I must show you, just in today, and very reasonably priced —” “I’m not buying today, Mr. Borgin, but selling,” said Mr. Malfoy. “Selling?” The smile faded slightly from Mr. Borgin’s face. “You have heard, of course, that the Ministry is conducting more raids,” said Mr. Malfoy, taking a roll of parchment from his inside pocket and unraveling it for Mr. Borgin to read. “I have a few — ah — items at home that might embarrass me, if the Ministry were to call…” Mr. Borgin fixed a pair of pince-nez to his nose and looked down the list. “The Ministry wouldn’t presume to trouble you, sir, surely?” Mr. Malfoy’s lip curled. “I have not been visited yet. The name Malfoy still commands a certain respect, yet the Ministry grows ever more meddlesome. There are rumors about a new Muggle Protection Act — no doubt that flea- bitten, Muggle-loving fool Arthur Weasley is behind it — ” Harry felt a hot surge of anger. “— and as you see, certain of these poisons might make it appear —” “I understand, sir, of course,” said Mr. Borgin. “Let me see…“I am in something of a hurry, Borgin, I have important business elsewhere today —” …They started to haggle. “Done,” said Mr. Malfoy at the counter. “Come, Draco —”… “Good day to you, Mr. Borgin. I’ll expect you at the manor tomorrow to pick up the goods.” Rowling, 1999a, pp. 33–34 Junior public officials make efforts to please their superiors “Percy hurried forward with his hand outstretched. Apparently his disapproval of the way Ludo Bagman ran his department did not prevent him from wanting to make a good impression.” “Mr. Crouch!” said Percy breathlessly, sunk into a kind of halfbow that made him look like a hunchback. “Would you like a cup of tea?” “Oh,” said Mr. Crouch, looking Rowling, 2000, p. 56 Rowling, 2000, pp. 58–59 11 over at Percy in mild surprise. “Yes - thank you, Weatherby.” Fred and George choked into their own cups. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 Percy, very pink around the ears, busied himself with the kettle.” “I just can’t justify taking more time off at the moment,” he told them. “Mr. Crouch is really starting to rely on me.” “Yeah, you know what, Percy?” said George seriously. “I reckon he’ll know your name soon.” “Are you sure you wouldn’t like to stay at Hogwarts tonight, Barty?” “No, Dumbledore, I must get back to the Ministry,” said Mr. Crouch. “It is a very busy, very difficult time at the moment…I’ve left young Weatherby in charge…Very enthusiastic…a little overenthusiastic, if truth be told…” “What do they think they’re doing, annoying senior Ministry members?” Percy hissed, watching Fred and George suspiciously. “No respect…” Ludo Bagman shook off Fred and George fairly quickly, however, and, spotting Harry, waved and came over to their table. “I hope my brothers weren’t bothering you, Mr. Bagman?” said Percy at once.” Rowling, 2000, p. 104 Rowling, 2000, p. 182 Rowling, 2000, p. 273. Many offices are overstaffed with low productivity workers “You realize Bertha Jorkins has been missing for over a month now? Went on holiday to Albania and never came back?” Rowling, 2000, p. 40 Hogwarts’ Healer, Madam Pomfrey cures them all “How exactly did it happen, Harry?” Harry retold the story “…and then I got the bezoar down his throat and his breathing eased up a bit, Slughorn ran for help, McGonagall and Madam Pomfrey turned up, and they brought Ron up here. They reckon he’ll be all right. Madam Pomfrey says he’ll have to stay here a week or so…keep taking essence of rue…” Rowling, 2005, p. 263 Private Property and inheritance “Ministry, is it?” said the older man, looking down at Ogden. “Correct!” said Ogden angrily, dabbing his face. “And you, I take it, are Mr. Gaunt?” “S’right,” said Gaunt. “Got you in the face, did he?” “Yes, he did!” snapped Ogden. “Should’ve made your presence known, shouldn’t you?” said Gaunt aggressively. “This is private property. Can’t just walk in here and not expect my son to defend himself.” “When the cart stopped at last beside a small door in the passage wall, Hagrid got out and had to lean against the Rowling, 2005, p. 132 Rowling, 1998, p. 12 wall to stop his knees from trembling. Griphook unlocked the door. A lot of green smoke came billowing out, and as it cleared, Harry gasped. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 Griphook unlocked the door. A lot of green smoke came billowing out, and as it cleared, Harry gasped. Inside were mounds of gold coins. Columns of silver. Heaps of little bronze Knuts. “All yours,” smiled Hagrid. All Harry’s — it was incredible. The Dursleys couldn’t have known about this or they’d have had it from him faster than blinking. How often had they complained how much Harry cost them to keep? And all the time there had been a small fortune belonging to him, buried deep under London.” “Stored in an underground vault at Gringotts in London was a small fortune that his parents had left him. Of course, it was only in the Wizarding world that he had money; you couldn’t use Galleons, Sickles, and Knuts in Muggle shops.” “You see,” Dumbledore said, turning back to Harry and again speaking as though Uncle Vernon had not uttered, “if you have indeed inherited the house, you have also inherited —”…“As you can see, Harry,” said Dumbledore loudly, over Kreacher’s continued croaks of “wont, won’t, won’t,” “Kreacher is showing a certain reluctance to pass into your ownership.”… “Give him an order,” said Dumbledore. “If he has passed into your ownership, he will have to obey.”…“Well, that simplifies matters,” said Dumbledore cheerfully. “It means that Sirius knew what he was doing. You are the rightful owner of number twelve, Grimmauld Place and of Kreacher.” “Can’t the Order control Mundungus?” Harry demanded of the other two in a furious whisper. “Can’t they at least stop him stealing everything that’s not fixed down when he’s at headquarters?” “Shh!” said Hermione desperately, looking around to make sure nobody was listening; there were a couple of warlocks sitting close by who were staring at Harry with great interest, and Zabini was lolling against a pillar not far away. “Harry, I’d be annoyed too, I know it’s your things he’s stealing—” “Harry gagged on his butterbeer; he had momentarily forgotten that he owned number twelve, Grimmauld Place. “Yeah, it’s my stuff!” he said. “No wonder he wasn’t pleased to see me! W ll I’ i t t ll D bl d h t’ i h ’ 48 Rowling, 1999a, p. 30 Rowling, 2005, pp. 33–34 Rowling, 2005, p. 161 wall to stop his knees from trembling. Griphook unlocked the door. A lot of green smoke came billowing out, and as it cleared, Harry gasped. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 Inside were mounds of gold coins. Columns of silver. Heaps of little bronze Knuts. “All yours,” smiled Hagrid. All Harry’s — it was incredible. The Dursleys couldn’t have known about this or they’d have had it from him faster than blinking. How often had they complained how much Harry cost them to keep? And all the time there had been a small fortune belonging to him, buried deep under London.” “Stored in an underground vault at Gringotts in London was a small fortune that his parents had left him. Of course, it was only in the Wizarding world that he had money; you couldn’t use Galleons, Sickles, and Knuts in Muggle shops.” “You see,” Dumbledore said, turning back to Harry and again speaking as though Uncle Vernon had not uttered, “if you have indeed inherited the house, you have also inherited —”…“As you can see, Harry,” said Dumbledore loudly, over Kreacher’s continued croaks of “wont, won’t, won’t,” “Kreacher is showing a certain reluctance to pass into your ownership.”… “Give him an order,” said Dumbledore. “If he has passed into your ownership, he will have to obey.”…“Well, that simplifies matters,” said Dumbledore cheerfully. “It means that Sirius knew what he was doing. You are the rightful owner of number twelve, Grimmauld Place and of Kreacher.” “Can’t the Order control Mundungus?” Harry demanded of the other two in a furious whisper. “Can’t they at least stop him stealing everything that’s not fixed down when he’s at headquarters?” “Shh!” said Hermione desperately, looking around to make sure nobody was listening; there were a couple of warlocks sitting close by who were staring at Harry with great interest, and Zabini was lolling against a pillar not far away. “Harry, I’d be annoyed too, I know it’s your things he’s stealing—” “Harry gagged on his butterbeer; he had momentarily forgotten that he owned number twelve, Grimmauld Place. “Yeah, it’s my stuff!” he said. “No wonder he wasn’t pleased to see me! Well, I’m going to tell Dumbledore what’s going on, he’s the only one who scares Mundungus.” 48 Rowling, 1999a, p. 30 Rowling, 2005, pp. 33–34 Rowling, 2005, p. 161 he “I have some questions for the three of you, and I think it will be best if we do it individually…“We’re not going anywhere ” said Harry while Hermione nodded Rowling, 2007, pp. 80-85 wall to stop his knees from trembling. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 “‘To Miss Hermione Jean Granger, I leave my copy of The Tales of Beedle the Bard, in the hope that she will find it entertaining and instructive.’” Scrimgeour now pulled out of the bag a small book … Harry saw that the title was in runes; he had never learned to read them As he looked a tear splashed onto the all.”…“I am here, as I’m sure you know, because of Albus Dumbledore’s will.” Harry, Ron, and Hermione looked at one another. “A surprise, apparently! You were not aware then that Dumbledore had left you anything?” “A-all of us?” said Ron, “Me and Hermione too?” “Yes, all of –” But Harry interrupted. “Dumbledore died over a month ago. Why has it taken this long to give us what he left us?” “Isn’t it obvious?” said Hermione, before Scrimgeour could answer. “They wanted to examine whatever he’s left us. You had no right to do that!” she said, and her voice trembled slightly. “I had every right,” said Scrimgeour dismissively. “The Decree for Justifiable Confiscation gives the Ministry the power the confiscate the contents of a will –” “That law was created to stop wizards passing on Dark artifacts,” said Hermione, “and the Ministry is supposed to have powerful evidence that the deceased’s possessions are illegal before seizing them! …Harry spoke: “So why have you decided to let us have our things now? Can’t think of a pretext to keep them?” “No, it’ll be because thirty-one days are up,” said Hermione at once. “They can’t keep the objects longer than that unless they can prove they’re dangerous. Right?” “… how do you account for the fact that he remembered you in his will? He made exceptionally few personal bequests. … Why do you think you were singled out?” “I…dunno,” said Ron…Scrimgeour… removed a scroll of parchment which he unrolled and read aloud. “‘The Last Will and Testament of Albus Percival Wulfric Brian Dumbledore’…Yes, here we are…‘To Ronald Bilius Weasley, I leave my Deluminator, in the hope that he will remember me when he uses it.’” Scrimgeour took from the bag an object … leaned forward and passed the Deluminator to Ron, who took it and turned it over in the fingers looking stunned. “That is a valuable object,” said Scrimgeour, watching Ron. … To what use did he think you would put to the Deluminator, Mr. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 Inside were mounds of gold coins. Columns of silver. Heaps of little bronze Knuts. “All yours,” smiled Hagrid. All Harry’s — it was incredible. The Dursleys couldn’t have known about this or they’d have had it from him faster than blinking. How often had they complained how much Harry cost them to keep? And all the time there had been a small fortune belonging to him, buried deep under London.” 48 “Stored in an underground vault at Gringotts in London was a small fortune that his parents had left him. Of course, it was only in the Wizarding world that he had money; you couldn’t use Galleons, Sickles, and Knuts in Muggle shops.” “You see,” Dumbledore said, turning back to Harry and again speaking as though Uncle Vernon had not uttered, “if you have indeed inherited the house, you have also inherited —”…“As you can see, Harry,” said Dumbledore loudly, over Kreacher’s continued croaks of “wont, won’t, won’t,” “Kreacher is showing a certain reluctance to pass into your ownership.”… “Give him an order,” said Dumbledore. “If he has passed into your ownership, he will have to obey.”…“Well, that simplifies matters,” said Dumbledore cheerfully. “It means that Sirius knew what he was doing. You are the rightful owner of number twelve, Grimmauld Place and of Kreacher.” “Can’t the Order control Mundungus?” Harry demanded of the other two in a furious whisper. “Can’t they at least stop him stealing everything that’s not fixed down when he’s at headquarters?” “Shh!” said Hermione desperately, looking around to make sure nobody was listening; there were a couple of warlocks sitting close by who were staring at Harry with great interest, and Zabini was lolling against a pillar not far away. “Harry, I’d be annoyed too, I know it’s your things he’s stealing—” “Harry gagged on his butterbeer; he had momentarily forgotten that he owned number twelve, Grimmauld Place. “Yeah, it’s my stuff!” he said. “No wonder he wasn’t pleased to see me! Well, I’m going to tell Dumbledore what’s going on, he’s the only one who scares Mundungus.” Rowling 2005, p 161 “I have some questions for the three of you, and I think it will be best if we do it individually…“We’re not going anywhere,” said Harry, while Hermione nodded vigorously. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 “You can speak to us together, or not at Rowling 2007, p 80-85 13 all.”…“I am here, as I’m sure you know, because of Albus Dumbledore’s will.” Harry, Ron, and Hermione looked at one another. “A surprise, apparently! You were not aware then that Dumbledore had left you anything?” “A-all of us?” said Ron, “Me and Hermione too?” “Yes, all of –” But Harry interrupted. “Dumbledore died over a month ago. Why has it taken this long to give us what he left us?” “Isn’t it obvious?” said Hermione, before Scrimgeour could answer. “They wanted to examine whatever he’s left us. You had no right to do that!” she said, and her voice trembled slightly. “I had every right,” said Scrimgeour dismissively. “The Decree for Justifiable Confiscation gives the Ministry the power the confiscate the contents of a will –” “That law was created to stop wizards passing on Dark artifacts,” said Hermione, “and the Ministry is supposed to have powerful evidence that the deceased’s possessions are illegal before seizing them! …Harry spoke: “So why have you decided to let us have our things now? Can’t think of a pretext to keep them?” “No, it’ll be because thirty-one days are up,” said Hermione at once. “They can’t keep the objects longer than that unless they can prove they’re dangerous. Right?” “… how do you account for the fact that he remembered you in his will? He made exceptionally few personal bequests. … Why do you think you were singled out?” “I…dunno,” said Ron…Scrimgeour… removed a scroll of parchment which he unrolled and read aloud. “‘The Last Will and Testament of Albus Percival Wulfric Brian Dumbledore’…Yes, here we are…‘To Ronald Bilius Weasley, I leave my Deluminator, in the hope that he will remember me when he uses it.’” Scrimgeour took from the bag an object … leaned forward and passed the Deluminator to Ron, who took it and turned it over in the fingers looking stunned. “That is a valuable object,” said Scrimgeour, watching Ron. … To what use did he think you would put to the Deluminator, Mr. Weasley?” “Put out lights, I s’pose,” mumbled Ron. “What else could I do with it?” Evidently Scrimgeour had no suggestions. After squinting at Ron for a moment or tow, he turned back to Dumbledore’s will. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 Weasley?” “Put out lights, I s’pose,” mumbled Ron. “What else could I do with it?” Evidently Scrimgeour had no suggestions. After squinting at Ron for a moment or tow, he turned back to Dumbledore’s will. “‘To Miss Hermione Jean Granger, I leave my copy of The Tales of Beedle the Bard, in the hope that she will find it entertaining and instructive.’” Scrimgeour now pulled out of the bag a small book … Harry saw that the title was in runes; he had never learned to read them. As he looked, a tear splashed onto the embossed symbols. “Why do you think Dumbledore left you that book, Miss Granger?” asked Scrimgeour. “He…he knew I liked books,” said Hermione in a thick 14 voice, mopping her eyes with her sleeve. “But why that particular book?” “I don’t know. He must have thought I’d enjoy it.” “Did you ever discuss codes, or any means of passing secret messages, with Dumbledore?” “No, I didn’t,” said Hermione,…Scrimgeour turned back to the will. “‘To Harry James Potter,’” he read, and Harry’s insides contracted with a sudden excitement, “‘I leave the Snitch he caught in his first Quidditch match at Hogwarts, as a reminder of the rewards of perseverance and skill.’” As Scrimgeour pulled out the tiny, walnut-sized golden ball, …“Why did Dumbledore leave you this Snitch?” asked Scrimgeour. “No idea,” said Harry…“What …could it be?” “I’m asking the questions,” said Scrimgeour,.“I notice that your birthday cake is in the shape of a Snitch,” Scrimgeour said to Harry…“I don’t think there’s anything hidden in the icing,” said Scrimgeour, “but a Snitch would be a very good hiding place for a small object…“Because Snitches have flesh memories,” she said…“Correct,” said Scrimgeour…“It occurs to me that Dumbledore, who had prodigious magical skill, whatever his other faults, might have enchanted this Snitch so that it will open only for you.”…“Take it,” said Scrimgeour quietly…“That’s all, then, is it?” asked Hermione, making to raise herself off the sofa. “Not quite,” said Scrimgeour, who looked bad tempered now. “Dumbledore left you a second bequest, Potter.” “What is it?” asked Harry…“The sword of Godric Gryffindor,” he said…“So where is it?” Harry asked suspiciously. “Unfortunately,” said Scrimgeour, “that sword was not Dumbledore’s to give away. The sword of Godric Gryffindor is an important historical artifact, and as such, belongs –” “It belongs to Harry!” said Hermione hotly. 15 15 Contract “Finally, I wish to impress upon any compete that this tournament is not to lightly. Once a champion has been se of Fire, he or she is obliged to see the to the end. The placing of your name constitutes a binding, magical contrac change of heart once you have becom be very sure, therefore, that you are w prepared to play before you drop you goblet. Now, I think it is time for bed all.” “Empty threat, Karkaroff,” growled a door. “You can’t leave your champio compete. They’ve all got to compete. contract, like Dumbledore said. Conv “I – I think everybody should write th so we know who was here. But I also deep breath, “that we all ought to agr what we’re doing. So if you sign, you tell Umbridge or anybody else what w the last person – Zacharias – had sign the parchment back and slipped it car There was an odd feeling in the group though they had just signed some kin “How do I look?” said Hepzibah, turn admire the various angles of her face “Lovely, madam,” squeaked Hokey. H assume that it was down in Hokey’s c lie through her teeth when asked this Hepzibah Smith looked a long way fr opinion. Unbreakable Vow “So Snape was offering to help him?” offering to help him!” said Harry. “H Malfoy’s mother to protect him, that Unbreakable Oath or something —” “ Vow?” said Ron, looking stunned. “N have…Are you sure?” “Yes, I’m sure what does it mean?” “Well, you can’t Unbreakable Vow…” “I’d worked th myself, funnily enough. What happen then?” “You die,” said Ron simply. “ tried to get me to make one when I w nearly did too, I was holding hands w everything when Dad found us. He w Ron. ct “Finally, I wish to impress upon any of you wishing to compete that this tournament is not to be entered into lightly. Once a champion has been selected by the Goblet of Fire, he or she is obliged to see the tournament through to the end. The placing of your name in the goblet constitutes a binding, magical contract. There can be no change of heart once you have become a champion. Please be very sure, therefore, that you are wholeheartedly prepared to play before you drop your name into the goblet. 15 Now, I think it is time for bed. Good night to you all.” “Empty threat, Karkaroff,” growled a voice from near the door. “You can’t leave your champion now. He’s got to compete. They’ve all got to compete. Binding magical contract, like Dumbledore said. Convenient, eh?” “I – I think everybody should write their name down, just so we know who was here. But I also think,” she took a deep breath, “that we all ought to agree not to shout about what we’re doing. So if you sign, you’re agreeing not to tell Umbridge or anybody else what we’re up to.”…When the last person – Zacharias – had signed, Hermione took the parchment back and slipped it carefully into her bag. There was an odd feeling in the group now. It was as though they had just signed some kind of contract.” “How do I look?” said Hepzibah, turning her head to admire the various angles of her face in the mirror. “Lovely, madam,” squeaked Hokey. Harry could only assume that it was down in Hokey’s contract that she must lie through her teeth when asked this question, because Hepzibah Smith looked a long way from lovely in his opinion. Rowling, 2000, p. 166 Rowling, 2000, p. 179 Rowling, 2003, p. 259 Rowling, 2005, pp. 285–286 akable “So Snape was offering to help him?”…“Yes, Snape was offering to help him!” said Harry. “He said he’d promised Malfoy’s mother to protect him, that he’d made an Unbreakable Oath or something —” “An Unbreakable Vow?” said Ron, looking stunned. “Nah, he can’t have…Are you sure?” “Yes, I’m sure,” said Harry. “Why, what does it mean?” “Well, you can’t break an Unbreakable Vow…” “I’d worked that much out for myself, funnily enough. What happens if you break it, then?” “You die,” said Ron simply. “Fred and George tried to get me to make one when I was about five. I nearly did too I was holding hands with Fred and Rowling, 2005, p. 214 constitutes a binding, magical contract. There can be no change of heart once you have become a champion. Please be very sure, therefore, that you are wholeheartedly prepared to play before you drop your name into the goblet. Now, I think it is time for bed. Good night to you all.” “Empty threat, Karkaroff,” growled a voice from near the door. “You can’t leave your champion now. Contents Appendix A. Economic Ideas p. 2 Appendix B. Additional Economic Ideas p. 30 Appendix C. Round Prices p. 33 Appendix D. Convenient Prices p. 35 Appendix E. Broomstick Industry – Technological Progress p. 37 “It chose him, he was the one who found it, it came to him out of the Sorting Hat –” “According to reliable historical sources, the sword may present itself to any worthy Gryffindor,” said Scrimgeour. “That does not make it the exclusive property of Mr. Potter, whatever Dumbledore may have decided.” …“You go too far!” shouted Scrimgeour, standing up: Harry jumped to his feet too. …“No! D’you want to give him an excuse to arrest us?” “Remembered you’re not at school, have you?” said Scrimgeour breathing hard into Harry’s face. “Remembered that I am not Dumbledore, who forgave your insolence and insubordination? You may wear that scar like a crown, Potter, but it is not up to a seventeen- year-old boy to tell me how to do my job! It’s time you learned some respect!”…“I don’t like your methods, Mi i ” id H “Remembered that I am not Dumbledore, who forgave your insolence and insubordination? You may wear that scar like a crown, Potter, but it is not up to a seventeen- year-old boy to tell me how to do my job! It’s time you learned some respect!”…“I don’t like your methods, Minister,” said Harry. 15 W short weeks since I was asked to leave the [the court of the Ministry of Magic], it has become the practice to hold a full crimina with a simple matter of underage magic!” Hogwarts’ Head Mistress and High Inquisitor amasses absolute power and authority I contacted the Minister at once, and he qu me that the High Inquisitor has to have the pupils of privileges, or she – that is to say less authority than common teachers!…I w our amendment…hem, hem…‘High Inqui henceforth have supreme authority over al sanctions and removal of privileges pertai students of Hogwarts, and the power to al punishments, sanctions and removals of p have been ordered by other staff members Cornelius Fudge, Minister of Magic, Orde Class, etc., etc.’” She rolled up the parchm back into her handbag, still smiling. “So… will have to ban these two from playing Q again,” she said, looking from Harry to Ge again. Harry felt the Snitch fluttering mad “Ban us?” he said, and his voice sounded distant. “From playing …ever again?” “Y think a lifelong ban ought to do the trick,” her smile widening still further as she wat struggle to comprehend what she had said Weasley here. And I think, to be safe, this twin ought to be stopped, too – if his team restrained him, I feel sure he would have a Mr. Malfoy as well. I will want their broo The Ministry decides to change the time of Harry’s court hearing but informs him on this change at the last minute A cold male voice rang across the courtroom. “You’re late.” “Sorry,” said Harry nervously “I — I didn’t know the time had been changed.” “That is not the Wizengamot’s [the court of the Ministry of Magic] fault,” said the voice. “An owl was sent to you this morning. Take your seat.” Rowling, 2003, p. 103 According to the Minister of Magic, laws can be changed “The Ministry does not have the power to expel Hogwarts students, Cornelius, as I reminded you on the night of the second of August,” said Dumbledore. “Nor does it have the right to confiscate wands until charges have been successfully proven…In your admirable haste to ensure that the law is upheld, you appear, inadvertently I am sure, to have overlooked a few laws yourself.” “Laws can be changed,” said Fudge savagely. 15 112 I contacted the Minister at once, and he quite agreed with me that the High Inquisitor has to have the power to strip pupils of privileges, or she – that is to say, I – would have less authority than common teachers!…I was reading out our amendment…hem, hem…‘High Inquisitor will henceforth have supreme authority over all punishments, sanctions and removal of privileges pertaining to the students of Hogwarts, and the power to alter such punishments, sanctions and removals of privileges as may have been ordered by other staff members. Signed, Cornelius Fudge, Minister of Magic, Order of Merlin First Class, etc., etc.’” She rolled up the parchment and put it back into her handbag, still smiling. “So…I really think I will have to ban these two from playing Quidditch ever again,” she said, looking from Harry to George and back again. Harry felt the Snitch fluttering madly in his hand. “Ban us?” he said, and his voice sounded strangely distant. “From playing …ever again?” “Yes, Mr. Potter, I think a lifelong ban ought to do the trick,” said Umbridge, her smile widening still further as she watched him struggle to comprehend what she had said. “You and Mr. Weasley here. And I think, to be safe, this young man’s twin ought to be stopped, too – if his teammates had not restrained him, I feel sure he would have attacked young Mr. Malfoy as well. I will want their broomsticks Rowling, 2003, pp. 310–311. The Ministry decides to change the time of Harry’s court hearing but informs him on this change at the last minute A cold male voice rang across the courtro late.” “Sorry,” said Harry nervously “I — the time had been changed.” “That is not t Wizengamot’s [the court of the Ministry o said the voice. “An owl was sent to you th Take your seat.” According to the Minister of Magic, laws can be changed “The Ministry does not have the power to students, Cornelius, as I reminded you on second of August,” said Dumbledore. “No the right to confiscate wands until charges successfully proven…In your admirable h that the law is upheld, you appear, inadver to have overlooked a few laws yourself.” changed,” said Fudge savagely. “Of cours Dumbledore, inclining his head. “And you to be making many changes, Cornelius. 15 “Of course they can,” said Dumbledore, inclining his head. “And you certainly seem to be making many changes, Cornelius. Why, in the few short weeks since I was asked to leave the Wizengamot [the court of the Ministry of Magic], it has already become the practice to hold a full criminal trial to deal with a simple matter of underage magic!” Rowling, 2003, p. 112 Hogwarts’ Head Mistress and High Inquisitor amasses absolute power and authority I contacted the Minister at once, and he quite agreed with me that the High Inquisitor has to have the power to strip pupils of privileges, or she – that is to say, I – would have less authority than common teachers!…I was reading out our amendment…hem, hem…‘High Inquisitor will henceforth have supreme authority over all punishments, sanctions and removal of privileges pertaining to the students of Hogwarts, and the power to alter such punishments, sanctions and removals of privileges as may have been ordered by other staff members. Signed, Cornelius Fudge, Minister of Magic, Order of Merlin First Class, etc., etc.’” She rolled up the parchment and put it back into her handbag, still smiling. “So…I really think I will have to ban these two from playing Quidditch ever again,” she said, looking from Harry to George and back again. Harry felt the Snitch fluttering madly in his hand. “Ban us?” he said, and his voice sounded strangely distant. “From playing …ever again?” “Yes, Mr. Potter, I think a lifelong ban ought to do the trick,” said Umbridge, her smile widening still further as she watched him Rowling, 2003, pp. 310–311. “The Ministry does not have the power to expel Hogwarts students, Cornelius, as I reminded you on the night of the second of August,” said Dumbledore. “Nor does it have the right to confiscate wands until charges have been successfully proven…In your admirable haste to ensure that the law is upheld, you appear, inadvertently I am sure, to have overlooked a few laws yourself.” “Laws can be changed,” said Fudge savagely. “Of course they can,” said Dumbledore, inclining his head. “And you certainly seem to be making many changes, Cornelius. 15 He’s got to compete. They’ve all got to compete. Binding magical contract, like Dumbledore said. Convenient, eh?” “I – I think everybody should write their name down, just so we know who was here. But I also think,” she took a deep breath, “that we all ought to agree not to shout about what we’re doing. So if you sign, you’re agreeing not to tell Umbridge or anybody else what we’re up to.”…When the last person – Zacharias – had signed, Hermione took the parchment back and slipped it carefully into her bag. There was an odd feeling in the group now. It was as though they had just signed some kind of contract.” Rowling, 2000, p. 179 Rowling, 2003, p. 259 16 A cold male voice rang across the courtroom. “You’re late.” “Sorry,” said Harry nervously “I — I didn’t know the time had been changed.” “That is not the Wizengamot’s [the court of the Ministry of Magic] fault,” said the voice. “An owl was sent to you this morning. Take your seat.” Rowling, 2003, p. 103 “The Ministry does not have the power to expel Hogwarts students, Cornelius, as I reminded you on the night of the second of August,” said Dumbledore. “Nor does it have the right to confiscate wands until charges have been successfully proven…In your admirable haste to ensure that the law is upheld, you appear, inadvertently I am sure, to have overlooked a few laws yourself.” “Laws can be changed,” said Fudge savagely. “Of course they can,” said Dumbledore, inclining his head. “And you certainly seem to be making many changes, Cornelius. Why, in the few short weeks since I was asked to leave the Wizengamot [the court of the Ministry of Magic], it has already become the practice to hold a full criminal trial to deal with a simple matter of underage magic!” Rowling, 2003, p. 15 Why, in the few short weeks since I was asked to leave the Wizengamot [the court of the Ministry of Magic], it has already become the practice to hold a full criminal trial to deal with a simple matter of underage magic!” Hogwarts’ Head Mistress and High Inquisitor amasses absolute power and authority I contacted the Minister at once, and he quite agreed with me that the High Inquisitor has to have the power to strip pupils of privileges, or she – that is to say, I – would have less authority than common teachers!…I was reading out our amendment…hem, hem…‘High Inquisitor will henceforth have supreme authority over all punishments, sanctions and removal of privileges pertaining to the students of Hogwarts, and the power to alter such punishments, sanctions and removals of privileges as may have been ordered by other staff members. Signed, Cornelius Fudge, Minister of Magic, Order of Merlin First Class, etc., etc.’” She rolled up the parchment and put it back into her handbag, still smiling. “So…I really think I will have to ban these two from playing Quidditch ever again,” she said, looking from Harry to George and back again. Harry felt the Snitch fluttering madly in his hand. “Ban us?” he said, and his voice sounded strangely distant. “From playing …ever again?” “Yes, Mr. Potter, I think a lifelong ban ought to do the trick,” said Umbridge, her smile widening still further as she watched him struggle to comprehend what she had said. “You and Mr. Weasley here. And I think, to be safe, this young man’s twin ought to be stopped, too – if his teammates had not restrained him, I feel sure he would have attacked young Mr. Malfoy as well. I will want their broomsticks Hogwarts’ Head Mistress and High Inquisitor amasses absolute power and authority I 17 confiscated, of course; I shall keep them safely in my office, to make sure there is no infringement of my ban. Selective enforcement of the laws by the Ministry of Magic “So all that remains,” said Fudge, now buttering himself a second crumpet, “is to decide where you’re going to spend the last two weeks of your vacation. I suggest you take a room here at the Leaky Cauldron and…” “Hang on,” blurted Harry. “What about my punishment?” Fudge blinked. “Punishment?” “I broke the law!” Harry said. 15 The nearest one, which had been erected outside Flourish and Blotts, under a striped, stained awning, had a cardboard sign pinned to its front: AMULETS - Effective Against Werewolves, Dementors, and Inferi! A seedy-looking little wizard was rattling armfuls of silver symbols on chains at passersby. “One for your little girl, madam?” he called at Mrs. Weasley as they passed, leering at Ginny. “Protect her pretty neck?” “If I were on duty…” said Mr. Weasley, glaring angrily at the amulet seller. “Yes, but don’t go arresting anyone now, dear, we’re in a hurry,” said Mrs. Weasley, nervously consulting a list. “Well, you see, in all the panic about You-Know-Who, odd things have been cropping up for sale everywhere, things that are supposed to guard against You-Know-Who and the Death Eaters. You can imagine the kind of thing…so-called protective potions that are really gravy with a bit of bubotuber pus added, or instructions for defensive jinxes that actually make your ears fall off…Well, in the main the perpetrators are just people like Mundungus Fletcher, who’ve never done an honest day’s work in their lives and are taking advantage of how frightened everybody is.” Rowling, 2003, p. 377 Rowling, 2005, p. 73 Rowling, 2005, p. 56 New businesses open rarely “It’s this joke shop idea they’ve got,” said Ron. “I thought they were only saying it to annoy Mum, but they really mean it, they want to start one. They’ve only got a year left at Hogwarts, they keep going on about how it’s time to think about their future, and Dad can’t help them, and they need gold to get started.” Rowling, 2000, pp. 366–367 Attempts by an entrepreneur to import flying carpets are “He wants a word with you about your embargo on flying carpets.” Mr. Weasley heaved a deep sigh…“Carpets are defined as a Muggle Artifact by the Registry of Proscribed Charmable Objects”…“Well, they’ll never replace brooms Rowling, 2000, p. 59 won’t like that…” On the other hand, a number of shabby-looking stalls had sprung up along the street. The nearest one, which had been erected outside Flourish and Blotts, under a striped, stained awning, had a cardboard sign pinned to its front: AMULETS - Effective Against Werewolves, Dementors, and Inferi! A seedy-looking little wizard was rattling armfuls of silver symbols on chains at passersby. “One for your little girl, madam?” he called at Mrs. 15 80 Selective enforcement of the laws by the Ministry of Magic 18 won’t like that…” Once they had eaten their Christmas lunch, th Harry and Hermione were planning to pay M another visit, escorted by Mad-Eye and Lupi Mundungus turned up in time for Christmas trifle, having managed to ‘borrow’ a car for t as the Underground did not run on Christmas car, which Harry doubted very much had bee the knowledge or consent of it's owner, had h Enlarging Spell put upon it as the Weasley's Anglia; On the other hand, a number of shabby-looki sprung up along the street. The nearest one, w been erected outside Flourish and Blotts, und stained awning, had a cardboard sign pinned AMULETS - Effective Against Werewolves, and Inferi! A seedy-looking little wizard wa armfuls of silver symbols on chains at passer your little girl, madam?” he called at Mrs. W they passed, leering at Ginny. “Protect her pr “If I were on duty…” said Mr. Weasley, glar the amulet seller. “Yes, but don’t go arresting now, dear, we’re in a hurry,” said Mrs. Weas nervously consulting a list. “Well, you see, in all the panic about You-K odd things have been cropping up for sale ev things that are supposed to guard against You and the Death Eaters. You can imagine the k thing…so-called protective potions that are r with a bit of bubotuber pus added, or instruct defensive jinxes that actually make your ears off…Well, in the main the perpetrators are ju Mundungus Fletcher, who’ve never done an work in their lives and are taking advantage o frightened everybody is.” New businesses open rarely “It’s this joke shop idea they’ve got,” said Ro they were only saying it to annoy Mum, but t mean it, they want to start one. They’ve only left at Hogwarts, they keep going on about h to think about their future, and Dad can’t hel they need gold to get started.” Attempts by an entrepreneur to import flying carpets are “He wants a word with you about your emba carpets.” Mr. Weasley heaved a deep sigh…“ defined as a Muggle Artifact by the Registry Charmable Objects”…“Well, they’ll never re won’t like that…” Once they had eaten their Christmas lunch, the Weasleys, Harry and Hermione were planning to pay Mr. Weasley another visit, escorted by Mad-Eye and Lupin. 15 “The Decree for the Restriction of Underage Wizardry!” “Oh, my dear boy, we’re not going to punish you for a little thing like that!” cried Fudge, waving his crumpet impatiently. “It was an accident! We don’t send people to Azkaban just for blowing up their aunts!” Rowling, 1999b, p. 28 Half-blood and mud-blood wizards must be questioned to make sure that they did not “steal” magic powers “Muggle-born Register!” she read aloud. “‘The Ministry of Magic is undertaking a survey of so-called “Muggle- borns” to better understand how they came to possess magical secrets. “‘Recent research undertaken by the Department of Mysteries reveals that magic can only be passed from person to person when Wizards reproduce. Where no proven Wizarding ancestry exists, therefore, the so-called Muggle-born is likely to have obtained magical power by theft or force. “‘The Ministry is determined to root out such usurpers of magical power, and to this end has issued an invitation to every so-called Muggle-born to present themselves for interview by the newly appointed Muggle-born Registration Commission.’” “People won’t let this happen,” said Ron. “It is happening, Ron,” said Lupin. “Muggle-borns are being rounded up as we speak.” “But how are they supposed to have ‘stolen’ magic?” said Ron. “It’s mental, if you could steal magic there wouldn’t be any Squibs, would there?” “I know,” said Lupin. “Nevertheless, unless you can prove that you have at least one close Wizarding relative, you are now deemed to have obtained your magical power illegally and must suffer the punishment.” Rowling, 2007, p. 136 Criminal activities “And you off buying stolen cauldrons! Didn’t I tell you not to go? Didn’t I!” “I – well, I –” Mundungus looked deeply uncomfortable. “It — it was a very good business opportunity, see –” “Mundungus!” said Hermione. “What’s he brought all those cauldrons for?” “Probably looking for a safe place to keep them,” said Harry. “Isn’t that what he was doing the night he was supposed to be tailing me? Picking up dodgy cauldrons?” “Yeah, you’re right!” said Fred, as the front door opened; Mundungus heaved his cauldrons through it and disappeared from view. “Blimey, Mum Rowling, 2003, p. 18 Rowling, 2003, p. 15 The Harry doubted very much had been taken with ge or consent of it's owner, had had a similar pell put upon it as the Weasley's old Ford r hand, a number of shabby-looking stalls had ong the street. The nearest one, which had d outside Flourish and Blotts, under a striped, ing, had a cardboard sign pinned to its front: - Effective Against Werewolves, Dementors, A seedy-looking little wizard was rattling ilver symbols on chains at passersby. “One for rl, madam?” he called at Mrs. Weasley as leering at Ginny. “Protect her pretty neck?” n duty…” said Mr. Weasley, glaring angrily at eller. “Yes, but don’t go arresting anyone we’re in a hurry,” said Mrs. Weasley, onsulting a list. see, in all the panic about You-Know-Who, have been cropping up for sale everywhere, re supposed to guard against You-Know-Who th Eaters. You can imagine the kind of alled protective potions that are really gravy bubotuber pus added, or instructions for nxes that actually make your ears fall n the main the perpetrators are just people like Fletcher, who’ve never done an honest day’s r lives and are taking advantage of how verybody is.” Rowling, 2003, p. 377 Rowling, 2005, p. 73 Rowling, 2005, p. 56 ke shop idea they’ve got,” said Ron. “I thought nly saying it to annoy Mum, but they really y want to start one. They’ve only got a year warts, they keep going on about how it’s time ut their future, and Dad can’t help them, and old to get started.” Rowling, 2000, pp. 366–367 word with you about your embargo on flying r. Weasley heaved a deep sigh…“Carpets are Muggle Artifact by the Registry of Proscribed Objects”…“Well, they’ll never replace brooms Rowling, 2000, p. 59 won’t like that…” Once they had eaten their Christmas lunch, the Weasleys, Harry and Hermione were planning to pay Mr. Weasley another visit, escorted by Mad-Eye and Lupin. Mundungus turned up in time for Christmas pudding and trifle, having managed to ‘borrow’ a car for the occasion, as the Underground did not run on Christmas Day. The car, which Harry doubted very much had been taken with the knowledge or consent of it's owner, had had a similar Enlarging Spell put upon it as the Weasley's old Ford Anglia; On the other hand, a number of shabby-looking stalls had sprung up along the street. 15 Mundungus turned up in time for Christmas pudding and trifle, having managed to ‘borrow’ a car for the occasion, as the Underground did not run on Christmas Day. The car, which Harry doubted very much had been taken with the knowledge or consent of it's owner, had had a similar Enlarging Spell put upon it as the Weasley's old Ford Anglia; On the other hand, a number of shabby-looking stalls had sprung up along the street. The nearest one, which had been erected outside Flourish and Blotts, under a striped, stained awning, had a cardboard sign pinned to its front: AMULETS - Effective Against Werewolves, Dementors, and Inferi! A seedy-looking little wizard was rattling armfuls of silver symbols on chains at passersby. “One for your little girl, madam?” he called at Mrs. Weasley as they passed, leering at Ginny. “Protect her pretty neck?” “If I were on duty…” said Mr. Weasley, glaring angrily at the amulet seller. “Yes, but don’t go arresting anyone now, dear, we’re in a hurry,” said Mrs. Weasley, nervously consulting a list. “Well, you see, in all the panic about You-Know-Who, odd things have been cropping up for sale everywhere, things that are supposed to guard against You-Know-Who and the Death Eaters. You can imagine the kind of thing…so-called protective potions that are really gravy with a bit of bubotuber pus added, or instructions for defensive jinxes that actually make your ears fall off…Well, in the main the perpetrators are just people like Mundungus Fletcher, who’ve never done an honest day’s work in their lives and are taking advantage of how frightened everybody is.” Rowling, 2003, p. 377 Rowling, 2005, p. 73 Rowling, 2005, p. 56 sses arely “It’s this joke shop idea they’ve got,” said Ron. “I thought they were only saying it to annoy Mum, but they really mean it, they want to start one. They’ve only got a year left at Hogwarts, they keep going on about how it’s time to think about their future, and Dad can’t help them, and they need gold to get started.” Rowling, 2000, pp. 366–367 hat…” ad eaten their Christmas lunch, the Weasleys, Hermione were planning to pay Mr. Weasley t, escorted by Mad-Eye and Lupin. turned up in time for Christmas pudding and g managed to ‘borrow’ a car for the occasion, rground did not run on Christmas Day. 15 Weasley as they passed, leering at Ginny. “Protect her pretty neck?” “If I were on duty…” said Mr. Weasley, glaring angrily at the amulet seller. “Yes, but don’t go arresting anyone now, dear, we’re in a hurry,” said Mrs. Weasley, nervously consulting a list. 19 blocked in Britain, will they?” said Bagman. “Ali thinks there’s a niche in the market for a family vehicle,” said Mr. Crouch. “I remember my grandfather had an Axminster that could seat twelve - but that was before carpets were banned, of course.” The Squibbler – unreliable tabloids “Of course not,” said Hermione scathingly, before Harry could answer. “The Quibbler’s rubbish, everyone knows that.” Rowling, 2003, p. 144 Daily Prophet publishes biased information “Harry…was talking more than he’d talked in days - about how no one believed he hadn’t entered the tournament of his own free will, how Rita Skeeter had lied about him in the Daily Prophet” “They’re trying to discredit him,” said Lupin. “Didn’t you see the Daily Prophet last week? They reported that he’d been voted out of the Chairmanship of the International Confederation of Wizards because he’s getting old and losing his grip, but it’s not true” Rowling, 2000, p. 213 Rowling 2003, p. 72 Harry Potter knows nothing about the Weasleys’ Wizard Wheezes – the Weasley twins’ joke shop “What are Weasleys’ Wizard Wheezes?” Harry asked… Ron and Ginny both laughed, although Hermione didn’t. “Mum found this stack of order forms when she was cleaning Fred and George’s room,” said Ron quietly. “Great long price lists for stuff they’ve invented. Joke stuff, you know. Fake wands and trick sweets,…I never knew they’d been inventing all that”…“and, you know, they were planning to sell it at Hogwarts to make some money” Rowling 2000, p. 36 The wizards have only one wand-maker “Don’ mention it,” said Hagrid gruffly. “Don’ expect you’ve had a lotta presents from them Dursleys. Just Ollivanders left now — only place fer wands, Ollivanders, and yeh gotta have the best wand.” Rowling, 1998, p. 53 Opening the market to caldron imports is expected to drive the prices down “What are you working on?” said Harry. “A report for the Department of International Magical Cooperation,” said Percy smugly. “We’re trying to standardize cauldron thickness. 15 Some of these foreign imports are just a shade too thin - leakages have been increasing at a rate of almost three percent a year”… “unless some sort of international law is imposed we might well find the market flooded with flimsy, shallow bottomed products that seriously endanger -” Rowling, 2000, pp. 36–37 Kids eat the same candies and collect the same cards as their parents “Madam Rosmertas finest oak-matured mead,” said Dumbledore, raising his glass to Harry, who caught hold of his own and sipped. He had never tasted anything like it before, but enjoyed it immensely...Harry could not suppress a suspicion that Dumbledore was rather enjoying himself. Rowling, 2005, p. 31 Zonko’s Joke “The walk into Hogsmeade was not enjoyable…More Rowling, 20 Shop goes out of business than once Harry wondered whether they might not have had a better time in the warm common room, and when they finally reached Hogsmeade and saw that Zonko’s Joke Shop had been boarded up, Harry took it as confirmation that this trip was not destined to be fun.” 2005, pp. 158–159 Lack of competition limits the Potterians’ choice although there’s demand for new products “Mum found this stack of order forms when she was cleaning Fred and George’s room,” said Ron quietly. “Great long price lists for stuff they’ve invented. Joke stuff, you know. Fake wands and trick sweets, loads of stuff. It was brilliant, I never knew they’d been inventing all that…” “But the common room was packed and full of shrieks of laughter and excitement; Fred and George were demonstrating their latest bit of joke shop merchandise.” “…they headed farther along the street in search of Weasleys’ Wizard Wheezes, the joke shop run by Fred and George.”…“And he and Harry led the way into the shop. It was packed with customers; Harry could not get near the shelves.” “We’ve just developed this more serious line,” said Fred…“Well, we thought Shield Hats were a bit of a laugh, you know, challenge your mate to jinx you while wearing it and watch his face when the jinx just bounces off. But the Ministry bought five hundred for all its support staff! 15 And we’re still getting massive orders!” “So we’ve expanded into a range of Shield Cloaks, Shield Gloves…” “And then we thought we’d get into the whole area of Defense Against the Dark Arts, because it’s such a money spinner,” continued George enthusiastically. “This is cool. Look, Instant Darkness Powder… Handy if you want to make a quick escape.” “And our Decoy Detonators…” Rowling, 2000, p. 36 Rowling, 2003, p. 403 Rowling, 2005, p. 76 Rowling, 2005, p. 78 Wizards view muggle-made goods as inferior “Why would anyone bother making door keys shrink?” said George. “Just Muggle-baiting,” sighed Mr. Weasley. “Sell them a key that keeps shrinking to nothing so they can never find it when they need it. Of course, it’s very hard to convict anyone because no Muggle would admit their key keeps shrinking — they’ll insist they just keep losing it.” “Harry left Hermione dabbing her black eye with paste Rowling 1999a, p. 25 Rowling, 21 earner…” Wealthy wizards enjoy a luxurious life style, and own almost all the assets and capital “Judging by the fact that Draco Malfoy usually had the best of everything, his family was rolling in wizard gold; he could just see Malfoy strutting around a large manor house.” Rowling, 1999a, p. 19 Voldemort comes from a well- established family that was stripped of its assets “That old man was —?” “Voldemort’s grandfather, yes,” said Dumbledore. “Marvolo, his son, Morfin, and his daughter, Merope, were the last of the Gaunts, a very ancient Wizarding family noted for a vein of instability and violence that flourished through the generations due to their habit of marrying their own cousins. Lack of sense coupled with a great liking for grandeur meant that the family gold was squandered several generations before Marvolo was born. He, as you saw, was left in squalor and Poverty.” Rowling, 2005, pp. 138 Businessmen’s negative image “Mum wants them to go into the Ministry of Magic like Dad, and they told her all they want to do is open a joke shop.” “Mrs. Weasley…did not think running a joke shop was a suitable career for two of her sons.” Rowling, 2000, p. 36 Rowling, 2003, p. 79 Wizards with muggle predecessors, are considered by wealthy wizards a threat because of their different culture “Harry knew exactly what was making Mr. Malfoy’s lip curl like that. 15 The Malfoys prided themselves on being purebloods; in other words, they considered anyone of Muggle descent, like Hermione, second-class. However, under the gaze of the Minister of Magic, Mr. Malfoy didn’t dare say anything.” Rowling, 2000, p. 66 Wealthy pure- blood wizards consider themselves superior to mud-blood wizards “I would have thought you’d be ashamed that a girl of no wizard family beat you in every exam,” snapped Mr. Malfoy.” “Malfoy called her ‘Mudblood,’ Hagrid”…Hagrid looked outraged…“But I don’t know what it means. I could tell it was really rude, of course.” “It’s about the most insulting thing he could think of,” gasped Ron…“Mudblood’s a really foul name for someone who is Muggle-born — you know, non-magic parents. There are some wizards — like Malfoy’s family — who think they’re better than everyone else because they’re what people call pure- Rowling, 1999a, p. 34 Rowling, 1999a, pp. 72–74 22 22 blood.” “If you’re wondering what the smell is, Mother, a Mudblood just walked in,” said Draco Malfoy.” “You’re lying, filthy Mudblood, and I know it! You have been inside my vault at Gringotts! Tell the truth, tell the truth!” Rowling, 2005, p. 74 Rowling, 2007, p. 308 Wealthy pure- blood wizards consider themselves superior to half-blood wizards “Shut your mouth!” Bellatrix shrieked. “You dare speak his name with your unworthy lips, you dare besmirch it with your half-blood’s tongue, you dare” “…they thought Voldemort had the right idea, they were all for the purification of the wizarding race, getting rid of Muggle-borns and having pure-bloods in charge. They weren’t alone, either, there were quite a few people, before Voldemort showed his true colors, who thought he had the right idea about things” “He’d play up the pure-blood side so he could get in with Lucius Malfoy and the rest of them…he’s just like Voldemort. Pure-blood mother, Muggle father…ashamed of his parentage” Rowling, 2003, p. 584 Rowling, 2003, p. 84 Rowling, 2005, p. 417 Wealthy wizards associated with middle class wizards are often disinherited by their families “The tapestry looked immensely old…the golden thread with which it was embroidered still glinted brightly enough to show them a sprawling family tree dating back (as far as Harry could tell) to the Middle Ages. 15 Large words at the very top of the tapestry read: “The Noble and Most Ancient House of Black Toujours pur.” “You’re not on here!” said Harry, after scanning the bottom of the tree closely. “I used to be there,” said Sirius, pointing at a small, round, charred hole in the tapestry, rather like a cigarette burn. “My sweet old mother blasted me off after …I’d had enough.” “Where did you go?” asked Harry, staring at him. “Your dad’s place,” said Sirius…“when I was seventeen I got a place of my own. My Uncle Alphard had left me a decent bit of gold – he’s been wiped off here, too, that’s probably why” Rowling, 2003, pp. 83–84 Intermarriages further block upward mobility “The pure-blood families are all interrelated,” said Sirius. “If you’re only going to let your sons and daughters marry pure-bloods your choice is very limited; there are hardly any of us left. Molly and I are cousins by marriage and Arthur’s something like my second cousin once removed.” Rowling, 2003, p. 85 Cultural “There was a greater variety of dishes in front of them Rowling, 23 barriers – wizards lack basic knowledge about other people’s customs and traditions than Harry had ever seen, including several that were definitely foreign. “What’s that?” said Ron, pointing at a large dish...“Bouillabaisse,” said Hermione…“It’s French,” said Hermione, “I had it on holiday summer before last. It’s very nice.” “I’ll take your word for it,” said Ron, helping himself to black pudding.” “Excuse me, are you wanting ze bouillabaisse?” It was the girl from Beauxbatons…Ron…stared up at her, opened his mouth to reply, but nothing came out except a faint gurgling noise. “Yeah, have it,” said Harry, pushing the dish toward the girl. “You ‘ave finished wiz it?” “Yeah,” Ron said breathlessly. “Yeah, it was excellent.”...Ron was still goggling at the girl as though he had never seen one before.” “When the second course arrived they noticed a number of unfamiliar desserts too. Ron examined an odd sort of pale blancmange closely, then moved it carefully a few inches to his right, so that it would be clearly visible from the Ravenclaw table.” “I’ll be havin’ a few words with her, an’ all,” said Hagrid grimly, stomping up the stairs. “The less you lot ‘ave ter do with these foreigners, the happier yeh’ll be. Yeh can trust any of ‘em.” 2000, pp. 15 162–163 Rowling, 2000, p. 163 Rowling, 2000, p. 164 Rowling, 2000, p. 363 When a leading English wand- maker disappears, wizards can’t find another wand-maker “Talking of Diagon Alley,” said Mr. Weasley, “looks like Ollivander’s gone too.” “The wandmaker?” said Ginny, looking startled. “That’s the one. Shop’s empty. No sign of a struggle. No one knows whether he left voluntarily or was kidnapped.” “But what’ll people do for wands?” Rowling, 2005, p. 70 Trade in Muggle goods takes place only under very special circumstances and only for very specific goods “But what does a Ministry of Magic do?” “Well, their main job is to keep it from the Muggles that there’s still witches an’ wizards up an’ down the country.” “What does your dad do at the Ministry of Magic, anyway?” “He works in the most boring department,” said Ron. “The Misuse of Muggle Artifacts Office.” “The what?” “It’s all to do with bewitching things that are Muggle-made, you know, in case they end up back in a Muggle shop or house. Like, last year, some old witch died and her tea set was sold to an antiques shop. This Muggle woman bought it, took it home, and tried to serve her friends tea in it. It was a nightmare — Dad was working overtime for weeks.” Rowling, 1998, p. 42 Rowling, 1999a, p. 20 24 24 “Harry…followed Fred toward the back of the shop, where he saw a stand of card and rope tricks. “Muggle magic tricks!” said Fred happily, pointing them out. “For freaks like Dad, you know, who love Muggle stuff. It’s not a big earner…” Rowling, 2005, p. 77 A junior official uses quality as a pretext to block an importation of considerably cheaper foreign goods “What are you working on?” said Harry. “A report for the Department of International Magical Cooperation,” said Percy smugly. “We’re trying to standardize cauldron thickness. Some of these foreign imports are just a shade too thin - leakages have been increasing at a rate of almost three percent a year”… “unless some sort of international law is imposed we might well find the market flooded with flimsy, shallow bottomed products that seriously endanger -” “He wants a word with you about your embargo on flying carpets.” Mr. 15 Weasley heaved a deep sigh…“Carpets are defined as a Muggle Artifact by the Registry of Proscribed Charmable Objects”…“Well, they’ll never replace brooms in Britain, will they?” said Bagman. “Ali thinks there’s a niche in the market for a family vehicle,” said Mr. Crouch. “I remember my grandfather had an Axminster that could seat twelve - but that was before carpets were banned, of course.” Rowling, 2000, pp. 36–37 Rowling, 2000, p. 59 Senior wizard officials are driven by ego- rents for power “Fudge… [the current minister of magic] is frightened of Dumbledore?” said Harry incredulousy. “Frightened of what he is up to… Fudge thinks… Dumbledore wants to be minister of magic… Deep down Fudge knows Dumbledore is much cleverer than he is… but it seems that he's become fond of power.” “A well… said Thicknesse [the presiding minister of magic]. "If you ask me, the blood traitors are as bad as the mudbloods.” Rowling, 2003, p. 89 Rowling, 2007, p. 247 Despite the wizards’ prejudices against any type of humanoids, they are willing to employ elves in large numbers, who work hard under terrible “He is wanting paying for his work, sir.” “Paying?” said Harry blankly. “Well - why shouldn’t he be paid?” Winky looked quite horrified…“House-elves is not paid, sir!”… “No, no, no. I says to Dobby, I says, go find yourself a nice family and settle down, Dobby. He is getting up to all sorts of high jinks, sir, what is unbecoming to a house- elf… “Well, it’s about time he had a bit of fun,” said Harry. “House-elves is not supposed to have fun, Harry Potter,” said Winky firmly, from behind her hands. “House-elves does what they is told. I is not liking heights at all, Harry Potter”…“but my master sends me to the Top Box and I comes, sir.”…“Why’s he sent you up here, if he Rowling, 2000, p. 64 25 s and ithout knows you don’t like heights?” said Harry, frowning. “Master - master wants me to save him a seat, Harry Potter. He is very busy,”…“Winky does what she is told. Winky is a good house-elf.” “You know, house-elves get a very raw deal!” said Hermione indignantly. “It’s slavery, that’s what it is! That Mr. 15 Crouch made her go up to the top of the stadium, and she was terrified, and he’s got her bewitched so she can’t even run when they start trampling tents! Why doesn’t anyone do something about it?” “Well, the elves are happy, aren’t they?” Ron said. “You heard old Winky back at the match… ‘House-elves is not supposed to have fun’…that’s what she likes, being bossed around…” “You may rest assured that she will be punished,” Mr. Crouch added coldly. “M-m-master…” Winky stammered, looking up at Mr. Crouch, her eyes brimming with tears. “M-m-master, p-p-please…” Mr. Crouch stared back, his face somehow sharpened, each line upon it more deeply etched. There was no pity in his gaze. “Winky has behaved tonight in a manner I would not have believed possible,” he said slowly. “I told her to remain in the tent. I told her to stay there while I went to sort out the trouble. And I find that she disobeyed me. This means clothes.” “No!” shrieked Winky, prostrating herself at Mr. Crouch’s feet. “No, master! Not clothes, not clothes!” Rowling, 2000, p. 80 Rowling, 2000, p. 89 signs wo ier ent hem “But he cannot now give testimony, Cornelius,” said Dumbledore.…“He cannot give evidence about why he killed those people.” “Why he killed them? Well, that’s no mystery, is it?” blustered Fudge. “He was a raving lunatic! From what Minerva and Severus have told me, he seems to have thought he was doing it all on You Know- Who’s instructions!” “Lord Voldemort was giving him instructions, Cornelius,” Dumbledore said. “Those peoples deaths were mere by-products of a plan to restore Voldemort to full strength again. The plan succeeded. Voldemort has been restored to his body.” Fudge…began to sputter, still goggling at Dumbledore. “You-Know- Who …returned? Preposterous. Come now, Dumbledore …” “As Minerva and Severus have doubtless told you,” said Dumbledore, “we heard Barty Crouch confess. Under the influence of Veritaserum, he told us how he was smuggled out of Azkaban, and how Voldemort…went to free him from his father and used him to capture Harry. The plan worked, I tell you. Crouch has helped Voldemort Rowling, 2000, pp. 453–454 knows you don’t like heights?” said Harry, frowning. “Master - master wants me to save him a seat, Harry Potter. He is very busy,”…“Winky does what she is told. conditions and almost without pay 15 453–454 26 now, come now…certainly, Cro himself to be acting upon You- to take the word of a lunatic lik Professor Slughorn complains that due to the war prices are sky- high “There was a final plunk from t and prices are sky-high at the m Cost born by the public for the government inefficiency - the price paid for goods sold by swindlers that offer a false sense of security “…a number of shabby-looking the street. The nearest one…had to its front: AMULETS, Effecti Dementors, and Inferi! A seed rattling armfuls of silver symbo “One for your little girl, madam Weasley as they passed, leering pretty neck?” “If I were on duty glaring angrily at the amulet sel arresting anyone now, dear, we Weasley…” “You wouldn’t believe how ma who work at the Ministry, can’t Charm,” said George. “‘Course teaching them, Harry.” “…the M hundred for all its support staff massive orders!” “So we’ve exp Shield Cloaks, Shield Gloves… thought we’d get into the whole the Dark Arts, because it’s such continued George… “This is co Powder, we’re importing it from to make a quick escape.” The usually crowded “Leaky Cauldron” bar is empty because even the most loyal consumers seem to have lost their appetite “The Leaky Cauldron was, for t memory, completely empty. On wizened and toothless, remaine “The bar of the Leaky Cauldron Tom, the stooped and toothless glasses behind the bar counter; having a muttered conversation at Hermione and drew back into Lestrange,” murmured Tom, an inclined his head subserviently. The bar owner notices Hagrid. From the “The Leaky Cauldron was, for t memory, completely empty. On wizened and toothless, remaine now, come now…certainly, Crouch may have believed himself to be acting upon You-Know-Who’s orders - but to take the word of a lunatic like that, Dumbledore…” Professor Slughorn complains that due to the war prices are sky- high “There was a final plunk from the piano,…My last bottle, and prices are sky-high at the moment.” Rowling, 2005, p. 43 Cost born by the public for the government inefficiency - the price paid for goods sold by swindlers that offer a false sense of security “…a number of shabby-looking stalls had sprung up along the street. The nearest one…had a cardboard sign pinned to its front: AMULETS, Effective Against Werewolves, Dementors, and Inferi! 15 Winky is a good house-elf.” “You know, house-elves get a very raw deal!” said Hermione indignantly. “It’s slavery, that’s what it is! That Mr. Crouch made her go up to the top of the stadium, and she was terrified, and he’s got her bewitched so she can’t even run when they start trampling tents! Why doesn’t anyone do something about it?” “Well, the elves are happy, aren’t they?” Ron said. “You heard old Winky back at the match… ‘House-elves is not supposed to have fun’…that’s what she likes, being bossed around…” “You may rest assured that she will be punished,” Mr. Crouch added coldly. “M-m-master…” Winky stammered, looking up at Mr. Crouch, her eyes brimming with tears. “M-m-master, p-p-please…” Mr. Crouch stared back, his face somehow sharpened, each line upon it more deeply etched. There was no pity in his gaze. “Winky has behaved tonight in a manner I would not have believed possible,” he said slowly. “I told her to remain in the tent. I told her to stay there while I went to sort out the trouble. And I find that she disobeyed me. This means clothes.” “No!” shrieked Winky, prostrating herself at Mr. Crouch’s feet. “No, master! Not clothes, not clothes!” “But he cannot now give testimony, Cornelius,” said Dumbledore.…“He cannot give evidence about why he killed those people.” “Why he killed them? Well, that’s no mystery, is it?” blustered Fudge. “He was a raving lunatic! From what Minerva and Severus have told me, he seems to have thought he was doing it all on You Know- Who’s instructions!” “Lord Voldemort was giving him instructions, Cornelius,” Dumbledore said. “Those peoples deaths were mere by-products of a plan to restore Voldemort to full strength again. The plan succeeded. Voldemort has been restored to his body.” Fudge…began to sputter, still goggling at Dumbledore. “You-Know- Who …returned? Preposterous. Come now, Dumbledore …” “As Minerva and Severus have doubtless told you,” said Dumbledore, “we heard Barty Crouch confess. Under the influence of Veritaserum, he told us how he was smuggled out of Azkaban, and how Voldemort…went to free him from his father and used him to capture Harry. The plan worked, I tell you. Crouch has helped Voldemort to return.” “See here, Dumbledore…you - you can’t seriously believe that You-Know-Who’s back? Come Rowling, 2000, pp. 15 72 Cost born by the public for the government inefficiency - the price paid for goods sold by swindlers that offer a false sense of security 27 barman’s reaction it is clear that Hagrid is one of his last loyal customers looked up hopefully as they entered, but before he could speak, Hagrid said importantly, “Jus’ passin’ through today, Tom, sure yeh understand, Hogwarts business, yeh know.” Makes and Models of the broomsticks used by the Potterian wizards See Table A5 in this appendix. Wealthy wizards are even willing to give up on some of their income just to become teachers “There was also a list of the new books he’d need for the coming year. SECOND-YEAR STUDENTS WILL REQUIRE: Standard Book of Spells Grade 2 by Miranda Goshawk Break with a Banshee by Gilderoy Lockhart Gadding with Ghouls by Gilderoy Lockhart Holidays with Hags by Gilderoy Lockhart 43 Travels with Trolls by Gilderoy Lockhart Voyages with Vampires by Gilderoy Lockhart Wanderings with Werewolves by Gilderoy Lockhart Year with the Yeti by Gilderoy Lockhart Fred, who had finished his own list, peered over at Harry’s. “You’ve been told to get all Lockhart’s books, too!” he said.” An hour later, they headed for Flourish and Blotts… bookshop…a large crowd jostling outside the doors, trying to get in. The reason for this was proclaimed by a large banner stretched across the upper windows: GILDEROY LOCKHART will be signing copies of his autobiography MAGICAL ME today 12:30P.M.to 4:30P.M. “We can actually meet him!” Hermione squealed. “I mean, he’s written almost the whole booklist!”…A harassed looking wizard stood at the door, saying, “Calmly, please, ladies…Don’t push, there…mind the books, now…” “They had reached Lockhart’s classroom…When the whole class was seated, Lockhart cleared his throat loudly and silence fell. He reached forward, picked up Neville Longbottom’s copy of Travels with Trolls, and held it up to show his own, winking portrait on the front. “Me,” he said, pointing at it and winking as well. “Gilderoy Lockhart... “I see you’ve all bought a complete set of my books — well done.” Rowling, 1999a, pp. 28–29 Rowling, 1999a, p. 38 Rowling, 1999a, p. 15 A seedy-looking little wizard was rattling armfuls of silver symbols on chains at passersby. “One for your little girl, madam?” he called at Mrs. Weasley as they passed, leering at Ginny. “Protect her pretty neck?” “If I were on duty…” said Mr. Weasley, glaring angrily at the amulet seller. “Yes, but don’t go arresting anyone now, dear, we’re in a hurry,” said Mrs. Weasley…” “You wouldn’t believe how many people, even people who work at the Ministry, can’t do a decent Shield Charm,” said George. “‘Course, they didn’t have you teaching them, Harry.” “…the Ministry bought five hundred for all its support staff! And we’re still getting massive orders!” “So we’ve expanded into a range of Shield Cloaks, Shield Gloves…” “…“And then we thought we’d get into the whole area of Defense Against the Dark Arts, because it’s such a money spinner,” continued George… “This is cool. Look, Instant Darkness Powder, we’re importing it from Peru. Handy if you want to make a quick escape.” Rowling, 2005, p. 73 Rowling, 2005, p. 78 The usually crowded “Leaky Cauldron” bar is empty because even the most loyal consumers seem to have lost their appetite “The Leaky Cauldron was, for the first time in Harry’s memory, completely empty. Only Tom the landlord, wizened and toothless, remained of the old crowd.” “The bar of the Leaky Cauldron was nearly deserted. Tom, the stooped and toothless landlord, was polishing glasses behind the bar counter; a couple of warlocks having a muttered conversation in the far corner glanced at Hermione and drew back into the shadows. “Madam Lestrange,” murmured Tom, and as Hermione paused he inclined his head subserviently.” Rowling, 2005, p. 72 Rowling, 2007, p. 347 The bar owner notices Hagrid. From the “The Leaky Cauldron was, for the first time in Harry’s memory, completely empty. Only Tom the landlord, wizened and toothless remained of the old crowd He Rowling, 2005, p. 15 64 barman’s reaction it is clear that Hagrid is one of his last loyal customers looked up hopefully as they entered, but before he could speak, Hagrid said importantly, “Jus’ passin’ through today, Tom, sure yeh understand, Hogwarts business, yeh know.” Makes and Models of the broomsticks used by the Potterian wizards See Table A5 in this appendix. Wealthy wizards are even willing to give up on some of their income just to become teachers “There was also a list of the new books he’d need for the coming year. SECOND-YEAR STUDENTS WILL REQUIRE: Standard Book of Spells Grade 2 by Miranda Goshawk Break with a Banshee by Gilderoy Lockhart Gadding with Ghouls by Gilderoy Lockhart Holidays with Hags by Gilderoy Lockhart 43 Travels with Trolls by Gilderoy Lockhart Voyages with Vampires by Gilderoy Lockhart Wanderings with Werewolves by Gilderoy Lockhart Year with the Yeti by Gilderoy Lockhart Fred, who had finished his own list, peered over at Harry’s. “You’ve been told to get all Lockhart’s books, too!” he said.” Rowling, 1999a, pp. 28–29 An hour later, they headed for Flourish and Blotts… bookshop…a large crowd jostling outside the doors, trying to get in. The reason for this was proclaimed by a large banner stretched across the upper windows: GILDEROY LOCKHART will be signing copies of his autobiography MAGICAL ME today 12:30P.M.to 4:30P.M. “We can actually meet him!” Hermione squealed. “I mean, he’s written almost the whole booklist!”…A harassed looking wizard stood at the door, saying, “Calmly, please, ladies…Don’t push, there…mind the books, now…” “They had reached Lockhart’s classroom…When the whole class was seated, Lockhart cleared his throat loudly and silence fell. He reached forward, picked up Neville Longbottom’s copy of Travels with Trolls, and held it up to show his own, winking portrait on the front. “Me,” he said, pointing at it and winking as well. “Gilderoy Lockhart... 15 “I see you’ve all bought a complete set of my books — well done.” 28 The current headmaster is considered by many to be the greatest wizard of his time Harry unwrapped his Chocolate Frog and picked up the card…Harry turned over his card and read: ALBUS DUMBLEDORE CURRENTLY HEADMASTER OF HOGWARTS Considered by many the greatest wizard of modern times, Dumbledore is particularly famous for his defeat of the dark wizard Grindelwald in 1945, for the discovery of the twelve uses of dragon’s blood, and his work on alchemy with his partner, Nicolas Flamel. Professor Dumbledore enjoys chamber music and tenpin bowling. Rowling, 1998, p. 66 The government can interfere with the school curriculum and governance Daily Prophet: “In a surprise move last night the Ministry of Magic passed new legislation giving itself an unprecedented level of control at Hogwarts School “ “…the passing of Educational Decree Number Twenty- three…creates the new position of Hogwarts High Inquisitor…‘The Inquisitor will have powers to inspect her fellow educators and make sure that they are coming up to scratch.” Rowling, 2003, p. 229 The Hogwarts’ graduates don’t know more than their predecessors “The time had come to choose their subjects for the third year…“I just want to give up Potions,” said Harry. “We can’t,” said Ron gloomily. “We keep all our old subjects, or I’d’ve ditched Defense Against the Dark Arts.” “Harry bent swiftly over the tattered book Slughorn had lent him. To his annoyance he saw that the previous owner had scribbled all over the pages, so that the margins were as black as the printed portions. Bending low to decipher the ingredients (even here, the previous owner had made annotations and crossed things out)…” “I just tried a few of the tips written in the margins, honestly, Ginny, there’s nothing funny-” “Harry bent low to retrieve the book, and as he did so, he saw…scribbled along the bottom of the back cover in the same small, cramped handwriting…This book is the property of the Half Blood Prince.” “It’s just that I was right about Eileen Prince once owning the book. 15 You see…she was Snape’s mother!”… “I was going through the rest of the old Prophets and there was a tiny announcement about Eileen Prince marrying a man called Tobias Snape, and then later an announcement saying that she’d given birth…“Snape must have been proud of being ‘half a Prince’, you see? Tobias Snape was a Muggle from what it said in the Prophet” “Yeah, that Rowling, 1999a, p. 161 Rowling, 2005, p. 123 Rowling, 2005, p. 125 Rowling, 2005, p. 126 Rowling, 2005, p. 417 29 29 could get in with Lucius Malfoy and the rest of hem… Pure-blood mother, Muggle father…ashamed of his parentage, trying to make himself feared using the Dark Arts, gave himself an impressive new name…the Half- Blood Prince - how could Dumbledore have missed —?” “…that the parents he would destroy in his murderous quest were people that Professor Snape knew, that they were your mother and father —” Rowling, 2005, p. 360 Potterian students lack creative skills and cannot think originally, which diminishes entrepreneurial spirit “It hasn’t been easy, Harry, guiding you through these tasks without arousing suspicion. I have had to use every ounce of cunning I possess, so that my hand would not be detectable in your success. Dumbledore would have been very suspicious if you had managed everything too easily. As long as you got into that maze, preferably with a decent head start - then, I knew, I would have a chance of getting rid of the other champions and leaving your way clear. But I also had to contend with your stupidity. The second task…that was when I was most afraid we would fail. I was keeping watch on you, Potter. I knew you hadn’t worked out the egg’s clue, so I had to give you another hint” Rowling, 2000, p. 434 After Graduation from Hogwarts, wizards choose a profession The time had come to choose their subjects for the third year…Percy Weasley was eager to share his experience. “Depends where you want to go, Harry,” he said. “It’s never too early to think about the future, so I’d recommend Divination. People say Muggle Studies is a soft option, but I personally think wizards should have a thorough understanding of the non-magical community, particularly if they’re thinking of working in close contact with them — look at my father, he has to deal with Muggle business all the time. 15 My brother Charlie was always more of an outdoor type, so he went for Care of Magical Creatures. Play to your strengths, Harry.” Rowling, 1999a, pp. 161–162 Goblins can easily distinguish between the fake and real Galleons “The long counter was manned by goblins sitting on high stools serving the first customers of the day. Hermione, Ron, and Travers headed toward an old goblin who was examining a thick gold coin through an eyeglass…The goblin tossed the coin he was holding aside, said to nobody in particular, “Leprechaun,” and then greeted Travers.” Rowling, 2007, p. 351 Inflation of home prices “This house!” shrieked Uncle Vernon, the vein his forehead starting to pulse. “Our house! House prices are skyrocketing around here! Rowling, 2007, p. 20 Inflation “Yes, dragon,” repeated the wizard conversationally. “My last bottle, and prices are sky-high at the moment. Still, it might be reusable.” Rowling, 2005, p. 43 could get in with Lucius Malfoy and the rest of hem… Pure-blood mother, Muggle father…ashamed of his parentage, trying to make himself feared using the Dark Arts, gave himself an impressive new name…the Half- Blood Prince - how could Dumbledore have missed —?” “…that the parents he would destroy in his murderous quest were people that Professor Snape knew, that they were your mother and father —” Rowling, 2005, p. 360 Potterian students lack creative skills and cannot think originally, which diminishes entrepreneurial spirit “It hasn’t been easy, Harry, guiding you through these tasks without arousing suspicion. I have had to use every ounce of cunning I possess, so that my hand would not be detectable in your success. Dumbledore would have been very suspicious if you had managed everything too easily. As long as you got into that maze, preferably with a decent head start - then, I knew, I would have a chance of getting rid of the other champions and leaving your way clear. But I also had to contend with your stupidity. The second task…that was when I was most afraid we would fail. I was keeping watch on you, Potter. I knew you hadn’t worked out the egg’s clue, so I had to give you another hint” Rowling, 2000, p. 434 After Graduation from Hogwarts, wizards choose a profession The time had come to choose their subjects for the third year…Percy Weasley was eager to share his experience. 15 “Depends where you want to go, Harry,” he said. “It’s never too early to think about the future, so I’d recommend Divination. People say Muggle Studies is a soft option, but I personally think wizards should have a thorough understanding of the non-magical community, particularly if they’re thinking of working in close contact with them — look at my father, he has to deal with Muggle business all the time. My brother Charlie was always more of an outdoor type, so he went for Care of Magical Creatures. Play to your strengths, Harry.” Rowling, 1999a, pp. 161–162 Goblins can easily distinguish between the fake and real Galleons “The long counter was manned by goblins sitting on high stools serving the first customers of the day. Hermione, Ron, and Travers headed toward an old goblin who was examining a thick gold coin through an eyeglass…The goblin tossed the coin he was holding aside, said to nobody in particular, “Leprechaun,” and then greeted Travers.” Rowling, 2007, p. 351 Inflation of home prices “This house!” shrieked Uncle Vernon, the vein his forehead starting to pulse. “Our house! House prices are skyrocketing around here! Rowling, 2007, p. 20 Inflation “Yes, dragon,” repeated the wizard conversationally. “My last bottle, and prices are sky-high at the moment. Still, it might be reusable.” Rowling, 2005, p. 43 30 Appendix B. Additional Economic Ideas2 Appendix B. Additional Economic Ideas2 Appendix B. Additional Economic Ideas Additional Economic Ideas Quote Reference Rational ignorance- inattention “Haven’t…you been getting the Daily Prophet!” Hermione asked nervously. “Yeah, I have!” said Harry. “Have you – er – been reading it thoroughly?” Hermione asked…“Not cover to cover,” said Harry defensively. Rowling, 2003, p. 55 Barter exchange “Ron had taken out a lumpy package and unwrapped it. There were four sandwiches inside. He pulled one of them apart and said, “She always forgets I don’t like corned beef.” “Swap you for one of these,” said Harry, holding up a pasty.” “Though the goblins of Gringotts will consider it base treachery, I have decided to help you – for payment.” …“I want the sword. 2 The list in this appendix cites additional ideas found in Potterian economics but are not discussed explicitly in the paper because of space limitations. 15 The sword of Godric Gryffindor.”… The sword is the price of my hire, take it or leave it!” “A large sign had been affixed to the Gryffindor noticeboard; so large it covered everything else on it – the lists of secondhand spellbooks for sale…the offers to barter certain Chocolate Frog Cards for others” Rowling, 1998, p. 66 Rowling, 2007, pp. 334–335 Rowling, 2003, p. 262 Barter exchange – a lack of double coincidence of wants “I have decided to help you – for payment.” …“How much do you want? I’ve got gold.” “Not gold,” said Griphook. “I have gold.” Rowling, 2007, pp. 334 Implicit contract – a handshake “I have your word, Harry Potter, that you will give me the sword of Gryffindor if I help you?” “Yes,” said Harry. “Then shake,” said the goblin, holding out his hand.” Rowling, 2007, p. 337 Secondhand, used goods’ market “Mrs. Weasley and Ginny were going to a secondhand robe shop.” “I thought they’d bring out the color of your eyes, dear,” said Mrs. Weasley fondly. “Well, they’re okay!” said Ron angrily, looking at Harry’s robes. “Why couldn’t I have some like that?” “Because…well, I had to get yours secondhand, and there wasn’t a lot of choice!” said Mrs. Weasley” Rowling, 1999a, p. 37 Rowling, 2000, p. 101 Diminishing marginal utility “The leprechaun gold I gave you…Why didn’t you tell me it disappeared?”…“I dunno…I never noticed it had gone...“Must be nice,” Ron said…“To have so much Rowling, 2000, p. 351 2 The list in this appendix cites additional ideas found in Potterian economics but are not discussed explicitly in the paper be 31 money you don’t notice if a pocketful of Galleons goes missing.” Budget constraint “I haven’t got any money — and you heard Uncle Vernon last night…he won’t pay for me to go and learn magic.” Rowling, 1998, p. 41 Non-tradable goods “We need them for the Skiving Snackboxes but they’re a Class C Non-Tradable Substance so we’ve been having a bit of trouble getting hold of them.” Rowling, 2003, p. 128 Price adjustment - Haggling and negotiating over price “In that case, perhaps we can return to my list,” said Mr. Malfoy shortly. “I am in something of a hurry, Borgin, I have important business elsewhere today —” They started to haggle.” “Look what Dung’s got us,” said George...“Venomous Tentacula seeds,” said George. 15 “Ten Galleons the lot, then Dung?” said Fred. “Wiv all the trouble I went to get ‘em?” said Mundungus, his saggy, bloodshot eyes stretching even wider. “I’m sorry, lads, but I’m not taking a Knut under twenty.”…Mundungus looked nervously over his shoulder…he grunted. “All right, lads, ten it is, if you’ll take ‘em quick.” Rowling, 1999a, p. 34 Rowling, 2003, pp. 128–129 Fund raising, donations “All proceeds from the Fountain of Magical Brethren will be given to St. Mungo’s Hospital for magical maladies and injuries.” Rowling, 2003, p. 95 Consumption smoothing “Once Harry had refilled his money bag with gold Galleons, silver Sickles, and bronze Knuts from his vault at Gringotts, he had to exercise a lot of self-control not to spend the whole lot at once.” Rowling, 1999b, p. 31 Black market “Meanwhile, a flourishing black-market trade in aids to concentration, mental agility and wakefulness had sprung up among the fifth- and seventh-years. Harry and Ron were much tempted by the bottle of Baruffio’s Brain Elixir offered to them by Ravenclaw sixth-year Eddie Carmichael, who swore it was solely responsible for the nine ‘Outstanding’ OWLs he had gained the previous summer and was offering a whole pint for a mere twelve Galleons.” Rowling, 2003, p. 527 Marketing and market research “We’re going to use it to do a bit of market research, find out exactly what the average Hogwarts student requires from a joke shop, carefully evaluate the results of our research, then produce products to fit the demand.” Rowling, 2003, p. 169 Minimum wage “Our short-term aims," said Hermione…"are to secure house-elves fair wages and working conditions.” Rowling, 2000, p. 145 Representative government “Our long-term aims include…trying to get an elf into the Department for the Regulation and Control of Magical Creatures, because they're shockingly underrepresented.” Rowling, 2000, p. 145 32 Opening the market to caldron imports is expected to drive the prices down “What are you working on?” said Harry. “A report for the Department of International Magical Cooperation,” said Percy smugly. “We’re trying to standardize cauldron thickness. Some of these foreign imports are just a shade too thin - leakages have been increasing at a rate of almost three percent a year”… “unless some sort of international law is imposed we might well find the market flooded with flimsy, shallow bottomed products that seriously endanger -” Rowling, 2000, pp. 15 36–37 Fine for traffic violation “Daily Prophet…said: Inquiry at the Ministry of Magic - Arthur Weasley, Head of the Misuse of Muggle Artifacts Office, was today fined fifty Galleons for bewitching a Muggle car.” Rowling, 1999a, p. 142 Import restrictions- regulations “He wants a word with you about your embargo on flying carpets.” Mr. Weasley heaved a deep sigh…“Carpets are defined as a Muggle Artifact by the Registry of Proscribed Charmable Objects”…“Well, they’ll never replace brooms in Britain, will they?” said Bagman. “Ali thinks there’s a niche in the market for a family vehicle,” said Mr. Crouch. “I remember my grandfather had an Axminster that could seat twelve - but that was before carpets were banned, of course.” Rowling, 2000, p. 59 Product Standardization “We’re trying to standardize cauldron thickness. Some of these foreign imports are just a shade too thin - leakages have been increasing at a rate of almost three percent a year” Rowling, 2000, p. 36 Gambling market “Now, now, Penny, no sabotage!” said Percy heartily as she examined the Firebolt closely. “Penelope and I have got a bet on,” he told the team. “Ten Galleons on the outcome of the match!” Rowling, 1999b, p. 164 Scarcity “As Harry might have told you, the final of the Quidditch World Cup takes place this Monday night, and my husband, Arthur, has just managed to get prime tickets through his connections at the Department of Magical Games and Sports.” Rowling, 2000, p. 20 Saving “Boys,” said Mr. Weasley under his breath, “I don’t want you betting…That’s all your savings” Rowling, 2000, p. 57 Corruption “As Harry might have told you, the final of the Quidditch World Cup takes place this Monday night, and my husband, Arthur, has just managed to get prime tickets through his connections at the Department of Magical Games and Sports.” Rowling, 2000, p. 20 33 Appendix C. Round Prices3 Appendix C. Round Prices3 Round Prices Quote Reference 1,500 Galleons – cursed opal necklace at Borgin and Burkes “Is this necklace for sale?” she asked, pausing beside a glass-fronted case. “If you’ve got one and a half thousand Galleons,” said Mr. Borgin coldly.” Rowling, 2005, p. 3 This appendix offers a list of the goods and services with round prices, as discussed in the paper, in section 6. 15 83 1,000 Galleons – prize for winning the Triwizard tournament “An impartial judge will decide which students are most worthy to compete for the Triwizard Cup, the glory of their school, and a thousand Galleons personal prize money.” Rowling, 2000, pp. 121–122 1,000 Galleons – A bounty on the head of escaped Death Eaters “A large poster had been stuck up in the window… and Harry found himself staring once more at the pictures of the ten escaped Death Eaters. The poster, By Order of the Ministry of Magic, offered a thousand-Galleon reward to any witch or wizard with information leading to the recapture of any of the convicts pictured” Rowling, 2003, p. 416 700 Galleons – Prize from Daily Prophet drawing “I couldn’t believe it when Dad won the Daily Prophet Draw. Seven hundred galleons! Most of it’s gone on this trip, but they’re going to buy me a new wand for next year.” Rowling, 1999b, p. 6 500 Galleons – Goblin-made armor at Borgin and Burkes “Mr. Burke would like to make an improved offer for the goblin-made armor,” said Voldemort. “Five hundred Galleons, he feels it is a more than fair.” Rowling, 2005, p. 286 100 Galleons – Acromantula Venom, per pint “I mean, it’s almost impossible to get venom from an acromantula while it’s alive…” “Slughorn seemed to be talking more to himself than Harry now. “…seems an awful waste not to collect it…might get a hundred Galleons a pint” Rowling, 2005, p. 316 20 Galleons – Deflagration Deluxe “Hermione, it’s…twenty [Galleons] for the Deflagration Deluxe…” Rowling, 2003, p. 472 10 Galleons – Hermione’s birthday present “I’ve still got ten Galleons,” she said, checking her purse. “It’s my birthday in September, and Mum and Dad gave me some money to get myself an early birthday present.” Rowling, 1999b, p. 36 10 Galleons – Omnioculars “Omnioculars,” said the saleswizard eagerly. “You can replay action… slow everything down…and they flash up a play-by- play breakdown if you need it. Bargain - ten Galleons each.” Rowling, 2000, p. 60 10 Galleons – “Professor Dumbledore offered Dobby ten Galleons a Rowling, 3 This appendix offers a list of the goods and services with round prices, as discussed in the paper, in section 6. 34 Weekly pay to house-elf week, and weekends off,” said Dobby” 2000, p. 15 244 10 Galleons – Metamorph- Medals for changing your appearance, 100,000 disguises “Some idiot’s started selling Metamorph-Medals. Just sling them around your neck and you’ll be able to change your appearance at will. A hundred thousand disguises, all for ten Galleons!” Rowling, 2005, p. 58 10 Galleons – Bet on the outcome of a Quidditch match “Now, now, Penny, no sabotage!” said Percy heartily as she examined the Firebolt closely. “Penelope and I have got a bet on,” he told the team. “Ten Galleons on the outcome of the match!” Rowling, 1999b, p. 164 10 Galleons – Slytherin’s Locket “She didn’t seem to have any idea how much it was worth. Happy to get ten Galleons for it. Best bargain we ever made!” Rowling, 2005, p. 171 10 Galleons – Unicorn hair “Not long after this, Hagrid became tearful again and pressed the whole unicorn tail upon Slughorn, who pocketed it with cries of, “To friendship! To generosity! To ten Galleons a hair!” Rowling, 2005, p. 320 5 Galleons – Basic Blaze box “Hermione, it’s five Galleons for your Basic Blaze box” Rowling, 2003, p. 472 5 Galleons – Rubber chicken wand “Bagman didn’t seem to think the wand was rubbish at all…his boyish face shone with excitement as…the wand gave a loud squawk and turned into a rubber chicken… “Excellent! I haven’t seen one that convincing in years! I’d pay five Galleons for that!” Rowling, 2000, p. 57 5 Knuts per scoop – Glittery-Black Beetle Eyes “Harry himself examined…glittery-black beetle eyes (five Knuts a scoop).” Rowling, 1998, p. 52 35 Appendix D. Convenient Prices4 Convenient Prices Quote Reference 21 Galleons – Silver Unicorn Horn “Harry himself examined silver unicorn horns at twenty- one Galleons each” Rowling, 1998, p. 52 16 Galleons – Human skull at Borgin and Burkes “And…what about this lovely…um…skull?” “Sixteen Galleons.” Rowling, 2005, p. 83 12 Galleons – 12-week course of Apparition lessons “Apparition Lessons: If you are seventeen years of age…you are eligible for a twelve-week course of Apparition Lessons from a Ministry of Magic Apparition instructor…Cost: 12 Galleons.” Rowling, 2005, p. 233 9 Galleons – New copy of Advanced Potion-Making “He pulled the old copy of Advanced Potion-Making… There sat the Prince’s copy, disguised as a new book, and there sat the fresh copy from Flourish and Blotts, looking thoroughly secondhand. 15 “I’ll give Slughorn back the new one, he can’t complain, it cost nine Galleons.” Rowling, 2005, p. 144 7 Galleons – New wand from Ollivanders “Harry shivered. He wasn’t sure he liked Mr. Ollivander too much. He paid seven gold Galleons for his wand, and Mr. Ollivander bowed them from his shop.” Rowling, 1998, p. 55 2 Galleons – Headless hat Fred and George were demonstrating their latest bit of joke shop merchandise. “Headless Hats!” shouted George, as Fred waved a pointed hat decorated with a fluffy pink feather at the watching students. “Two Galleons each” Rowling, 2003, p. 403 16 Sickles – Dragon Liver, per ounce “A plump woman outside an Apothecary was shaking her head as they passed, saying, “Dragon liver, sixteen Sickles an ounce, they’re mad…” Rowling, 1998, p. 46 11 Sickles – Night bus to London “Listen, how much would it be to get to London?” “Eleven Sickles,” said Stan.” Rowling, 1999b, p. 22 7 Sickles – Canary Cream “Canary Creams!” Fred shouted to the excitable crowd. “George and I invented them – seven Sickles each, a bargain!” Rowling, 2000, p. 236 4 Sickles – Hot water and toothbrush in the color of your choice “Listen, how much would it be to get to London?” “Eleven Sickles,” said Stan, “but…for fifteen you get an ’ot-water bottle an’ a toofbrush in the color of your choice.” Rowling, 1999b, p. 22 2 Sickles – Hot chocolate “Listen, how much would it be to get to London?” “Eleven Sickles,” said Stan, “…but for firteen you get ’ot Rowling, 1999b, p. 4 This appendix offers a list of the goods and services with convenient prices as discussed in the paper in section 6 36 on the night bus chocolate” 22 2 Sickles – Membership in S.P.E.W “It’s S-P-E-W. Stands for the Society for the Promotion of Elfish Welfare.”…“We start by recruiting members,” said Hermione happily. “I thought two Sickles to join” Rowling, 2000, pp. 144–145 36 on the night bus chocolate” 22 2 Sickles – Membership in S.P.E.W “It’s S-P-E-W. Stands for the Society for the Promotion of Elfish Welfare.”…“We start by recruiting members,” said Hermione happily. “I thought two Sickles to join” Rowling, 2000, pp. 144–145 36 on the night bus chocolate” 22 2 Sickles – Membership in S.P.E.W “It’s S-P-E-W. Stands for the Society for the Promotion of Elfish Welfare.”…“We start by recruiting members,” said Hermione happily. 15 “I thought two Sickles to join” Rowling, 2000, pp. 144–145 37 E. Broomstick Makes and Models – Technological Innovations in the Potteria Economy5 Appendix E. Broomstick Makes and Models – Technological Innovations in the Potter Economy5 Broomstick makes and models Quote Reference Cleansweep-5 “As for the old Cleansweeps” — he smiled nastily at Fred and George, who were both clutching Cleansweep Fives —“sweeps the board with them.” Rowling, 1999a, p. 71 Cleansweep-6 “Pausing every few pages [of The Quibbler magazine:], he read…“an interview with a wizard who claimed to have flown to the moon on a Cleansweep Six and brought back a bag of moon frogs to prove it.” Rowling, 2003, p. 143 Cleansweep-7 “He’s just the build for a Seeker, too,” said Wood… “Light —speedy—we’ll have to get him a decent broom, Professor — a Nimbus Two Thousand or a Cleansweep Seven, I’d say.” Rowling, 1998, p. 98 Cleansweep-11 “He…almost walked into Ron, who was…clutching his broomstick. He gave a great leap of surprise when he saw Harry and attempted to hide his new Cleansweep Eleven behind his back.” Rowling, 2003, p. 202 Nimbus-2000 “Even Harry, who knew nothing about the different brooms, thought it looked wonderful. Sleek and shiny, with a mahogany handle, it had a long tail of neat, straight twigs and Nimbus Two Thousand written in gold near the top.” “They took turns riding Harry’s Numbus-2000, which was easily the best broom” Rowling, 1998, p. 108 Rowling, 1999a, p. 30 Nimbus-2001 “Let me show you the generous gift he’s made to the Slytherin team.” All seven of them held out their broomsticks. Seven highly polished, brand-new handles and seven sets of fine gold lettering spelling the words Nimbus Two Thousand and One… “Very latest model. Only came out last month,” said Flint…“I believe it outstrips the old Two Thousand series by a considerable amount.” Rowling, 1999a, p. 71 Comet-260 “What did you say you’ve got at home, Malfoy, a Comet Two Sixty?” Ron grinned at Harry. “Comets look flashy, but they’re not in the same league as the Nimbus.” Rowling, 1998, p. 107 Comet-290 “Ron was rhapsodizing about his new broom…“…nought to seventy in ten seconds, not bad, is it? When you think the Comet Two Ninety’s only nought to sixty and that’s with a decent tailwind according to Which Broomstick?” Rowling, 2003, p. 5 Broomstick models and makes are discussed in the paper in the context of technological progress, in section 14. 15 128 Shooting Star “They took turns riding Harry’s Nimbus Two Thousand, Rowling, 5 Broomstick models and makes are discussed in the paper in the context of technological progress, in section 14. 38 which was easily the best broom; Ron’s old Shooting Star was often outstripped by passing butterflies.” “Harry borrowed a copy of Which Broomstick from Wood, and decided to spend the day reading up on the different makes. He had been riding one of the school brooms at team practice, an ancient Shooting Star, which was very slow and jerky.” 1999a, p. 30 Rowling, 1999b, p. 121 Bluebottle “At…the field…was a gigantic blackboard…watching it, Harry saw that it was flashing advertisements across the field. The Bluebottle: A Broom for All the Family - safe, reliable, and with Built-in Anti-Burglar Buzzer.” Rowling, 2000, pp. 62–63 Silver Arrow “Look at the balance on it! If the Nimbus series has a fault, it’s a slight list to the tail end — you often find they develop a drag after a few years. They’ve updated the handle too, a bit slimmer than the Cleansweeps, reminds me of the old Silver Arrows — a pity they’ve stopped making them. I learned to fly on one, and a very fine old broom it was too…” Rowling, 1999b, p. 162 Firebolt “Harry…was able to read the sign next to the broom: “THE FIREBOLT: This state-of-the-art racing broom sports a stream-lined, superfine handle of ash, treated with a diamond-hard polish and hand-numbered with its own registration number. Each individually selected birch twig in the broomtail has been honed to aerodynamic perfection, giving the Firebolt unsurpassable balance and pinpoint precision. The Firebolt has an acceleration of 150 miles an hour in ten seconds and incorporates an unbreakable braking charm. Price on request.” Harry didn’t like to think how much gold the Firebolt would cost.” “He had never wanted anything as much in his whole life — but he had never lost a Quidditch match on his Nimbus Two Thousand, and what was the point in emptying his Gringotts vault for the Firebolt, when he had a very good broom already? Harry didn’t ask for the price, but he returned, almost every day after that, just to look at the Firebolt.” “On the other hand, she rides a Comet-260, which is going to look like a joke next to the Firebolt.” Rowling, 1999b, p. 32 Rowling, 1999b, p. 39 39 15 32 Rowling, 1999b, p. 162 References Rowling, J.K., 1998. Harry Potter and the Philosopher’s Stone. Bloomsbury, London, UK. Rowling, J.K., 1999a. Harry Potter and the Chamber of Secrets. Bloomsbury, London, UK. Rowling, J.K., 1999b. Harry Potter and the Prisoner of Azkaban. Bloomsbury, London, UK. 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https://openalex.org/W2808704696	https://hal.uca.fr/hal-01858850/file/pdf	English	null	Use of Psychotropic Medications and Illegal Drugs, and Related Consequences Among French Pharmacy Students – SCEP Study: A Nationwide Cross-Sectional Study	Frontiers in pharmacology	2,018	cc-by	11,395	To cite this version: David Balayssac, Bruno Pereira, Maxime Darfeuille, Pierre Cuq, Laurent Vernhet, et al.. Use of Psychotropic Medications and Illegal Drugs, and Related Consequences Among French Pharmacy Students – SCEP Study: A Nationwide Cross-Sectional Study. Frontiers in Pharmacology, 2018, 9, pp.725. 10.3389/fphar.2018.00725. hal-01858850 Edited by: Edited by: Francisco Lopez-Munoz, Universidad Camilo José Cela, Spain Edited by: Francisco Lopez-Munoz, Universidad Camilo José Cela, Spain Background: The use of psychotropic medications and illegal drugs is a worldwide public health issue, leading to addiction, psychiatric and somatic disorders, and death. Pharmacy students are more exposed to psychotropic medications than other students (non-medical), which could lead to an overuse. The main objective of this study was to assess the prevalence of psychotropic drug use (medications and illegal drugs) by French pharmacy students, by carrying out a nationwide cross-sectional study. The relation of these medications and illegal drug use with several comorbidities and academic achievement was also assessed. Francisco Lopez-Munoz, Universidad Camilo José Cela, Spain , p Reviewed by: F. Javier Alvarez, Universidad de Valladolid, Spain John Martin Corkery, University of Hertfordshire, United Kingdom Correspondence: David Balayssac dbalayssac@chu-clermontferrand.fr Reviewed by: F. Javier Alvarez, Universidad de Valladolid, Spain John Martin Corkery, University of Hertfordshire, United Kingdom Correspondence: David Balayssac dbalayssac@chu-clermontferrand.fr Correspondence: David Balayssac dbalayssac@chu-clermontferrand.fr Methods: This online survey was performed by emails sent to all French pharmacy faculties over a period of 66 days (March 16, 2016 to May 20, 2016). The survey assessed the prevalence of uses of psychotropic medications and illegal drugs during the last 3 months. These uses were compared to student characteristics (personal and university) and comorbidities (anxiety, depression, stress, and fatigue). Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Received: 23 April 2018 Accepted: 15 June 2018 Published: 17 July 2018 Citation: Balayssac D, Pereira B, Darfeuille M, Cuq P, Vernhet L, Collin A, Vennat B and Authier N (2018) Use Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Received: 23 April 2018 Accepted: 15 June 2018 Published: 17 July 2018 Received: 23 April 2018 Accepted: 15 June 2018 Published: 17 July 2018 Results: Of the 2,609 questionnaires received, 2,575 were completed and useable for the analysis. Among French pharmacy students and during the 3 last months, 9.4% have used psychotropic medications, 21.5% illegal drugs and 3.3% both psychotropic medications and illegal drugs. Psychotropic medications were used in the cases of a medical prescription (49.0%), a self-medication (42.4%) or a non-medical intent (26.3%). Use of Psychotropic Medications and Illegal Drugs, and Related Consequences Among French Pharmacy Students – SCEP Study: A Nationwide Cross-Sectional Study David Balayssac1, Bruno Pereira2, Maxime Darfeuille3, Pierre Cuq4, Laurent Vernhet5, 1 Inserm U1107, NEURO-DOL, Faculté de Pharmacie, Laboratoire de Toxicologie, Université Clermont Auvergne, CHU Clermont-Ferrand, Délégation à la Recherche Clinique et à l’Innovation, Clermont-Ferrand, France, 2 CHU Clermont-Ferrand, Délégation à la Recherche Clinique et à l’Innovation, Clermont-Ferrand, France, 3 Faculté de Pharmacie, Laboratoire de Toxicologie, Université Clermont Auvergne, Clermont-Ferrand, France, 4 Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, ENSCM, Faculté de Pharmacie, Université de Montpellier, Montpellier, France, 5 UMR Inserm 1085, Institut de Recherche sur la Santé, l’Environnement et le Travail (IRSET), Université de Rennes 1, Rennes, France, 6 Inserm U1107, NEURO-DOL, Faculté de Pharmacie, Laboratoire de Toxicologie, Université Clermont Auvergne, Clermont-Ferrand, France, 7 ACCePPT, Faculté de Pharmacie, Université Clermont Auvergne, Clermont-Ferrand, France, 8 Inserm U1107, NEURO-DOL, Université Clermont Auvergne, Faculté de Médecine, CHU Clermont-Ferrand, Pharmacologie Médicale, Clermont-Ferrand, France HAL Id: hal-01858850 https://uca.hal.science/hal-01858850v1 Submitted on 5 Oct 2018 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. ORIGINAL RESEARCH published: 17 July 2018 doi: 10.3389/fphar.2018.00725 Edited by: Stress scores of the last 7 days were higher for psychotropic medication users compared to non-users and illegal drug users. Proportions of anxiety and depression at the time of answer were higher for psychotropic medication users than for non-users and illegal drug users. Fatigue scores of the last 7 days were lower for illegal drug users compared to non-users and self-medicated students. Annual average marks of the last INTRODUCTION cannabis daily (Sachs et al., 2015). Cannabis use can induce acute and long-term disorders, such as cardiovascular disorders (tachycardia, myocardial infarction and cardiomyopathies, etc.), respiratory disorders (airway inﬂammation, chronic bronchitis, emphysema, etc.), lung cancer, immunosuppression, sleep disorders (somnolence and decrease of the sleep quality, etc.), cognitive disorders (memory impairment, deﬁcits in visual attention, verbal ﬂuency, etc.) and substance use disorders (Sachs et al., 2015; Blanco et al., 2016). Cannabis use may be associated with psychiatric disorders, but data from the scientiﬁc literature remains more ambiguous (Sachs et al., 2015; Blanco et al., 2016; Hill, 2017). The use of psychotropic medications and illegal drugs is a worldwide public health issue leading to addiction, psychiatric and somatic disorders, and death (United Nations Oﬃce on Drugs and Crime [UNODC], 2016). Among psychotropic medications, benzodiazepines, Z-drugs (zolpidem and zopiclone) and opioids are the main drugs used and misused (Casati et al., 2012). The prevalence of use of medically prescribed benzodiazepines was about 9.7% of young adults in 2013 in France (Bénard-Laribière et al., 2016). Benzodiazepines and Z-drugs are used by more than 5.2% of American people between the ages of 18 and 80 years old (Soyka, 2017). Besides the risk of addiction for chronic users, benzodiazepines are responsible for several adverse eﬀects such as drowsiness, lethargy, fatigue, excessive sedation, stupor, “hangover eﬀects” the next day, disturbances of concentration and attention, symptom rebound after discontinuation (i.e., recurrence of the original disorder, most commonly a sleep disorder), hypotonia, ataxia, impaired driving ability, falls (fractures) and death from overdose (3.1 deaths per 100,000 American adults in 2013) (Bachhuber et al., 2016; Soyka, 2017). Although initially presented as superior to benzodiazepines, Z-drugs are not likely diﬀerent to benzodiazepines regarding either eﬀectiveness or safety (Holbrook, 2004). Misuse of opioid medications is currently a growing problem worldwide (Morley et al., 2017). In the United States in 2005, 4–5% of the adult population (9.6– 11.5 million people) were prescribed long-term opioid therapy (Boudreau et al., 2009). Thanks to their pain relieving and sedative properties opioid medications may also produce feelings of pleasure, contentedness, and satisfaction, which may lead to misuse, abuse and addiction (Pergolizzi et al., 2017). Young individuals are more at risk of inappropriate opioid use (Peck et al., 2016). Misuse and addiction may lead to death by overdose. In the United States, the epidemic of opioid-overdose led to 42,000 deaths in 2016 (Oquendo and Volkow, 2018). Citation: Citation: Balayssac D, Pereira B, Darfeuille M, Cuq P, Vernhet L, Collin A, Vennat B and Authier N (2018) Use of Psychotropic Medications and Illegal Drugs, and Related Consequences Among French Pharmacy Students – SCEP Study: A Nationwide Cross-Sectional Study. Front. Pharmacol. 9:725. doi: 10.3389/fphar.2018.00725 July 2018 \| Volume 9 \| Article 725 1 Frontiers in Pharmacology \| www.frontiersin.org Balayssac et al. Psychotropic Drugs and Pharmacy Students year, attendance and perception of study difﬁculty were lower for illegal drug users than for non-users. Conclusion: French pharmacy students were less exposed to psychotropic medications and illegal drugs than the general French population. However, in comparison to other students in other countries, the use of psychotropic medications seemed to be lower, but with a proportionally higher use of anxiety/sedative medications and a lower use of opioid medications. Keywords: psychotropic drugs, street drugs, pharmacy student, self-medication, comorbidity Frontiers in Pharmacology \| www.frontiersin.org INTRODUCTION Pharmacy studies in France last 6 years and begin with a diﬃcult competitive exam at the end of the 1st year with a limited number of places for each university (numerus clausus). French pharmacy studies are aimed at preparing future pharmacists for several roles, such as prevention, screening, diagnosis, treatment and patient follow-up; the dispensing and administration of medications, products and medical devices, and giving pharmaceutical advice; guidance regarding the healthcare system and the medico-social sector; and health education (Journal République Française, 2009). French pharmacy students attend university courses on pharmacology, toxicology and clinical pharmacy, including psychotropic medications and illegal drugs. These courses integrate the beneﬁt/risk ratio of psychotropic medication use, so that these future pharmacists will be able to protect their patients from drug misuse and abuse. The risk of illegal drug use and therapeutic strategies to prevent drug addiction are also taught to pharmacy students. We hypothesize that this knowledge could have two consequences on student behavior. On the one hand, it is expected that these students would be better informed on risks and thus be protected against the use and misuse of these psychotropic medications and drugs. On the other hand, this knowledge may comfort students in the use of these drugs, as they consider that they know what they are doing and are aware of the risks. It is known that medical students tend to have greater knowledge of appropriate self-medication, are more conﬁdent and have greater awareness of the eﬀects of self-medication, and tend to practice it more often and appropriately (Durrieu et al., 2007; James et al., 2008). Medical and pharmacy students have a high prevalence of self-medication practices (78–88%) (Lukovic et al., 2014; Patil et al., 2014; Alkhatatbeh et al., 2016). In India, the prevalence of self-medication amongst medical In 2014, it was estimated that 5% of the world population between 15 and 64 years old have used at least one illegal drug (United Nations Oﬃce on Drugs and Crime [UNODC], 2016). Cannabis remains by far the most widely cultivated, traﬃcked and abused “illegal” (depending on the country) drug worldwide (WHO, 2018). But despite its wide availability, cannabis use is not safe. Cannabis addiction will aﬀect 10% of individuals who start using cannabis and 25–50% of individuals who use July 2018 \| Volume 9 \| Article 725 Frontiers in Pharmacology \| www.frontiersin.org 2 Psychotropic Drugs and Pharmacy Students Balayssac et al. Survey Protocol The primary outcome measured was psychotropic drug use (medications and illegal drugs) during the 3 last months (list of medications and illegal drugs, Supplementary Table 1), indication of use, type of prescription for medication (medical or self- medication) and frequency of use (nota bene: French pharmacists are not allowed to prescribe drugs). Secondary outcomes were the assessment of several comorbidities, such as anxiety and depression symptoms using the hospital anxiety and depression scale (HADS) at the time of the answer, considering the following thresholds, normal (total score ≤7), borderline or suggestive of possible anxiety/depression (total score of 8–10) and indicative of anxiety/depression (total score ≥11) (Zigmond and Snaith, 1983), stress of the last 7 days using a visual analogic scale (0–100 VAS: 0 no stress and 100 maximum stress imaginable) (Pottier et al., 2011) and fatigue of the last 7 days using a visual analogic scale (0–100 VAS: 0 no fatigue and 100 maximum fatigue imaginable) (Punja et al., 2014). Age and sex were recorded for the socio-demographic factors. Information on university studies was recorded with the present year of study, the annual average mark of last year (usually in France, marks are rated on a 20-point scale and assessed as follows: not validated <10/20, pass grade 10–12/20, fairly good grade 12– 14/20, good grade 14–16/20 and very good grade >16/20), grade repetition, study diﬃculty perceived by the student (0–100 VAS score, 0: easy study and 100 very hard study) and attendance of the student (0–100 VAS score, 0: no attendance and 100: attendance to all the courses). Daily consumptions of cigarettes and electronic cigarettes, occasional alcohol consumption and the consumption of alcohol higher than the limits of 21 units per week for males and 14 units per weeks for females (deﬁned as hazardous alcohol consumption) were also recorded. Approximately 20 min were required to complete the online survey. Thus, the main objective of this study was to assess the prevalence of psychotropic drug use (medications and illegal drugs) during the last 3 months and its related medical indication among French pharmacy students, by performing a nationwide cross-sectional study. The secondary objectives focused on the association between these medications (medical prescription and self-medication) and illegal drug use with several comorbidities (anxiety and depression at the time of the answer, stress and fatigue during the last 7 days) and academic achievement. 1https://redcap.chu-clermontferrand.fr/surveys/?s=4YR8HMH8XM INTRODUCTION students increases from the ﬁrst (50%) to the ﬁnal year of study (90.5%) (Kasulkar and Gupta, 2015). In comparison, self- medication is about 35% in the adult Brazilian population (within the last 15 days) (Domingues et al., 2015), 20% in the adult Spanish populations (within the last 15 days) (Carrasco-Garrido et al., 2010), 50% in adolescents (meta-analysis) (Gualano et al., 2015) and 38% in the elderly (systematic review) (Jerez-Roig et al., 2014). Moreover, French pharmacy students have easy access to medications during their internships in community pharmacies and hospitals (pharmacy and clinical departments), and student jobs in community pharmacies. However, little is known of the prevalence of psychotropic medications and illegal drugs used by pharmacy students. libertés, No.0160) in accordance with French law and approved by the local ethics committee (No. 2016/CE23, March 22, 2016, Comité de Protection des Personnes sud-est 6, IRB: 00008526). The participants’ consent was obtained with the answer to the survey. Frontiers in Pharmacology \| www.frontiersin.org Study Design For example, it is well known that alcohol 1https://redcap.chu-clermontferrand.fr/surveys/?s=4YR8HMH8XM July 2018 \| Volume 9 \| Article 725 Frontiers in Pharmacology \| www.frontiersin.org 3 Psychotropic Drugs and Pharmacy Students Balayssac et al. psychotropic medications], 21.5% (554/2,575) illegal drugs [18.2% (468/2,575) only illegal drugs] and 3.3% (86/2,575) both psychotropic medications and illegal drugs. It is important to note that 72.4% (1,864/2,575) of pharmacy students declared that they did not use any psychotropic medications or illegal drugs (deﬁned in the study as non-users). Psychotropic medications were used in the cases of a medical prescription (49.0%, 119/243), a self-medication (42.4%, 103/243) or a non-medical intent (26.3%, 64/243). consumption is frequently under-estimated by respondents (Stockwell et al., 2016). The quality of the questionnaire has been discussed and designed by a multidisciplinary group of pharmacist, addictologist, biostatistician, toxicologist, and epidemiologist. Sample Description Q1:23, Q3: 93 Year repetition (N = 2,551) 40.9% 1st year repetition (N = 1,025) 66.3% 2nd year repetition (N = 1,025) 18.5% 3rd year repetition (N = 1,025) 9.0% 4th year repetition (N = 1,025) 3.1% 5th year repetition (N = 1,025) 2.7% 6th year repetition (N = 1,025) 0.3% Tobacco consumption (daily) (N = 2,563) 18.7% e-cigarette consumption (daily) (N = 2,549) 1.8% Alcohol consumption (occasionally) (N = 2,156) 84.0% Hazardous alcohol consumption, male (N = 768) 8.9% Hazardous alcohol consumption, female (N = 1,388) 3.8% The survey lasting 66 days resulted in the collection of 2,609 answers of which 2,575 were assessable according to the inclusion/exclusion criteria. The description of the pharmacy students who answered the survey is presented in Table 1. According to the estimation of the number of pharmacy students in France (N = 18,000), the response rate was close to 14%. Study Design We conducted a cross-sectional nationwide online survey in French pharmacy faculties to assess psychotropic drug use by pharmacy students from March 16, 2016 to May 20, 2016 (66 days). The internet address of the survey1 (Supplementary Data Sheet 1) was sent by email to all the French Pharmacy students (metropolitan France) through the student associations of the French pharmacy faculties and the national association of French pharmacy students [Association Nationale des Etudiants en Pharmacie de France (ANEPF)]. The online survey was also sent to pharmacy students through social networks (Facebook) and though toxicology teachers [Groupement Associé des Enseignants de Toxicologie (GATOX)]. The number of pharmacy students was estimated at 18,000 according to the national numerus clausus for the admission to French pharmacy studies in 2016–2017 (Journal Oﬃciel de la République Française, 2017). Study data were collected and managed using REDCap electronic data capture tools hosted at CHU Clermont-Ferrand (Harris et al., 2009). REDCap (Research Electronic Data Capture) is a secure, web-based application designed to support data capture for research studies, providing: (1) an intuitive interface for validated data entry; (2) audit trails for tracking data manipulation and export procedures; (3) automated export procedures for seamless data downloads to common statistical packages; and (4) procedures for importing data from external sources. Participants could be included in the survey if they were 1st–6th year pharmacy students studying at a French pharmacy faculty. Exclusion criteria were deﬁned as follows: other university course (diﬀerent from pharmacy), pharmacy residents, young graduated pharmacists, age < 18 years. The study was designed to conform to the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for reporting observational studies (von Elm et al., 2007; Supplementary Table 2). The study was anonymous and registered with the local correspondent of the French Commission on Information Technology, Data Files and Civil Liberty (Commission Nationale de l’Informatique et des Eﬀorts have been done to address potential sources of bias. Selection bias should be limited because the survey has been sent to all the French Pharmacy students. However, there is no data available in France on age, sex and characteristics of these students, so it is diﬃcult to ensure comparability. Reporting bias for this topic can be present and diﬃcult to manage. Statistical Considerations Female (N = 2,575) 65.9% Age (N = 2,575) 22.0 ± 2.3 Median: 22, Q1: 20, Q3: 23 Study year (N = 2,575) 1st year 9.4% 2nd year 19.2% 3rd year 22.1% 4th year 17.9% 5th year 1.8% 6th year 9.6% Annual average mark (N = 2,496) <10/20 5.7% 10–12/20 43.2% 12–14/20 36.9% 14–16/20 12.6% >16/20 1.6% Study difﬁculty (VAS score: 0–100) (N = 2,468) 69.2 ± 17.0 Median: 70, Q1:62, Q3: 80 Attendance (VAS score: 0–100) (N = 2,498) 58.5 ± 35.2 Median: 69, Q1:23, Q3: 93 Year repetition (N = 2,551) 40.9% 1st year repetition (N = 1,025) 66.3% 2nd year repetition (N = 1,025) 18.5% 3rd year repetition (N = 1,025) 9.0% 4th year repetition (N = 1,025) 3.1% 5th year repetition (N = 1,025) 2.7% 6th year repetition (N = 1,025) 0.3% Tobacco consumption (daily) (N = 2,563) 18.7% e-cigarette consumption (daily) (N = 2,549) 1.8% Alcohol consumption (occasionally) (N = 2,156) 84.0% Hazardous alcohol consumption, male (N = 768) 8.9% Hazardous alcohol consumption, female (N = 1,388) 3.8% TABLE 1 \| Characteristics of the pharmacy students at the time of responding to the survey. Statistical Considerations Sample size was determined to achieve an accuracy of the conﬁdence interval of prevalence of psychoactive drugs use greater than ± 3%. More precisely, it was necessary to include more than 1068 subjects, with such assumption, for a two-sided type I error at 5%. Details of the psychotropic medications and illegal drugs used by the students during the last 3 months are presented in Table 2. Benzodiazepines and z drugs were the main psychotropic medications used (74.5%, 181/243) followed by opioid medications (29.2%, 71/243). Cannabis (cannabis herb and/or resin) was the main illegal drug used: 90.8% (503/554) of illegal drug users and 19.5% (503/2,575) of all the pharmacy students. MDMA use represented 3.5% of all the pharmacy students while cocaine was used by 2% (Table 2). y Statistical analysis was performed using Stata 13 (StataCorp, College Station, TX, United States). The tests were two- sided, with a type-I error set at α = 0.05. Continuous data were expressed as mean ± standard deviation or median and interquartile range according to statistical distribution. The assumption of normality was studied using the Shapiro–Wilk test. The quantitative data were compared between independent groups using ANOVA or the Kruskal–Wallis test when the assumptions of ANOVA were not met. The assumption of homoscedasticity was studied using the Bartlett test. When appropriate (omnibus p-value < 0.05), post hoc tests for multiple comparisons were performed to take into account corrections of type I errors: Tukey–Kramer test after ANOVA or Dunn’s test post Kruskal–Wallis. The comparisons between groups concerning categorical data were performed using Chi-squared or Fisher’s exact tests followed when appropriate by Marascuilo’s procedure. Multivariable analyses were carried out to adjust results on the age and gender of pharmacy students using regression models (multiple linear for quantitative dependent outcomes and logistic for dichotomous). As less than 5% of data were missing, handling of missing data was not apply. However, sensitivity analyses were conducted to guaranty that these missing data did not inﬂuence the results (data not shown). As proposed by several statisticians, we chose to report all the individual p-values without applying any mathematical correction for distinct tests comparing groups. Speciﬁc attention was given to the magnitude of diﬀerences and to clinical relevance (Rothman, 1990; Feise, 2002). TABLE 1 \| Characteristics of the pharmacy students at the time of responding to the survey. July 2018 \| Volume 9 \| Article 725 Psycho Drugs The mean stress score of the last 7 days was 53.8 ± 23.9 for all the students. Stress scores were signiﬁcantly higher for psychotropic medication users compared to non-users and illegal drug users (p < 0.05) (Table 5). This increase in the stress score was higher for alprazolam and/or bromazepam users than for non-users (66.7 ± 18.0 vs. 54.3 ± 23.3, p < 0.001). The mean fatigue score of the last 7 days was 62.0 ± 20.2 for all the students. Fatigue scores were signiﬁcantly lower for illegal drug users compared to non-users and self-medicated students (p < 0.05) (Table 5). Self-medicated students had higher levels of fatigue than others but this diﬀerence was not signiﬁcant. Among all the pharmacy students, 18.7% (480/2,563) smoked tobacco, 1.8% (45/2,549) used e-cigarettes and 84.0% (2,156/2,568) drank alcoholic beverages [9.0% of males (68/754) and 4.0% of females (53/1,340) drank hazardous levels of alcohol]. Tobacco smoking and alcohol consumption (even hazardous alcohol consumption) were associated with illegal drug use (p < 0.001) (Table 5). The proportion of females was diﬀerent between non-users, psychotropic medication users (medical prescription and self- medication) and illegal drug users (73.1% vs. 62.2% vs. 64.3% vs. 43.7%, respectively, p < 0.001). Interestingly, although the population surveyed was predominantly female, there were signiﬁcantly fewer females among the illegal drug users than non- users (p < 0.05), psychotropic medication users with medical prescriptions (p < 0.05) and psychotropic medication users with self-medication (p < 0.05). Annual average mark of the last year, attendance and perception of study diﬃculty were signiﬁcantly lower for illegal drug users than for non-users (Table 5). Table 3 shows the declared indications of the main psychotropic medications used (benzodiazepines: alprazolam According to HADS scoring at the time of the answer, 25.5% (657/2,575) and 24.2% (624/2,575) of students had suggestive and indicative scores of anxiety, and 6.7% (173/2,575) and 3.9% (101/2,575) had suggestive and indicative scores of depression, respectively. Students taking psychotropic medications had higher levels of anxiety and depression than non-users and illegal drug users. Students self-medicated with psychotropic medications had even higher levels of anxiety and depression than students taking psychotropic medications with a medical prescription (Figure 1). Anxiety (indicative and suggestive HADS scores) aﬀected 67.9% of psychotropic medication users (versus non-users, 47.9%, p < 0.001) and 78.0% of bromazepam and alprazolam users (versus non-users, p < 0.001). Use of Psychotropic Medications and Illegal Drugs During the last 3 months, 9.4% (243/2,575) of respondents had used psychotropic medications [6.1% (157/2,575) only July 2018 \| Volume 9 \| Article 725 Frontiers in Pharmacology \| www.frontiersin.org 4 Psychotropic Drugs and Pharmacy Students Balayssac et al. and bromazepam; Z-drugs: zolpidem; opioids: codeine and tramadol). Self-medication with these psychotropic medications ranged between 28.0% (tramadol) and 51.7% (codeine). Zolpidem and bromazepam were both used for self-medication by half of the respondents. Depending on the psychotropic medication, non-medical use was declared by 21.9% (alprazolam) and 44.0% (tramadol) of users. Alprazolam and bromazepam were the most misused medications by pharmacy students (quantitatively). Among benzodiazepines and z-drug users, 43.1% (78/181) were self-medicated and 40.3% (73/181) received a medical prescription. Non-medical use of benzodiazepines and Z-drugs was declared by 23.8% (43/181) of students using these medications. Among opioid users, 36.6% (26/71) were self- medicated and only 33.8% (24/71) had a medical prescription. Non-medical use of opioids was declared by 42.3% (30/71) of students using opioid medications. The age of the students was signiﬁcantly diﬀerent between non-users, psychotropic medication users (medical prescription and self-medication) and illegal drug users (21.9 ± 2.3 vs. 22.8 ± 2.4 vs. 23.3 ± 2.4 vs. 22.0 ± 2.2, respectively, p < 0.001). Students using psychotropic medications were signiﬁcantly older than non-users and illegal drug users (p < 0.05). Compared to non-users, the age diﬀerence was higher for alprazolam and bromazepam users (23.11 ± 2.48, p < 0.001) and for zolpidem users (23.27 ± 2.38, p = 0.001). When comparing the number of years of study, there was no diﬀerence between non-users and psychotropic medication (medical prescription) users. However, the proportions of self-medicated students were signiﬁcantly diﬀerent among the number of years of study and tended to increase over these years ([1st and 2nd year] 1.4% vs. [3rd and 4th year] 2.4% vs. [5th and 6th year] 4.4%, p = 0.001 and [1st and 2nd year] vs. [5th and 6th year], p < 0.05). Regarding illegal drugs, the proportions of users among the years of study were signiﬁcantly diﬀerent ([1st and 2nd year] 21.8% vs. [3rd and 4th year] 22.7% vs. [5th and 6th year] 18.1%, p = 0.047), but no signiﬁcant diﬀerence stood out between years taken individually. Table 4 presents the declared objectives of use for the main illegal drugs: cannabis (herb), cannabis (resin), MDMA and cocaine. Psycho Drugs In another large French study, the use of medically prescribed benzodiazepines was about 9.7% in 2013, 8.1% for anxiolytic benzodiazepines and 3.6% for hypnotic Z-drugs (18–44-years old, 69% women, N = 537,076) (Bénard-Laribière et al., 2016). In this last study, alprazolam, then zolpidem and bromazepam were the benzodiazepines most used (in our study by decreasing order: alprazolam, bromazepam, and zolpidem). Caution must be taken when interpreting these results because the age of the participants was not the same as those with Bénard-Laribière’s study, and benzodiazepine use increased with the age of the users (Bénard- Laribière et al., 2016; ANSM, 2017). The main mild opioid medications used in France in 2015 were tramadol, codeine and 25 years old (N = 580), showing that 11.5% of students had used psychotropic medications in the past year (females: 21.0% and males: 6.4%) (Beck et al., 2005). In another large French study, the use of medically prescribed benzodiazepines was about 9.7% in 2013, 8.1% for anxiolytic benzodiazepines and 3.6% for hypnotic Z-drugs (18–44-years old, 69% women, N = 537,076) (Bénard-Laribière et al., 2016). In this last study, alprazolam, then zolpidem and bromazepam were the benzodiazepines most used (in our study by decreasing order: alprazolam, bromazepam, and zolpidem). Caution must be taken when interpreting these results because the age of the participants was not the same as those with Bénard-Laribière’s study, and benzodiazepine use increased with the age of the users (Bénard- Laribière et al., 2016; ANSM, 2017). The main mild opioid medications used in France in 2015 were tramadol, codeine Compared to non-users, illegal drug users had lower annual average marks (p < 0.01). Illegal drug users had also the lowest level of attendance compared to non-users and psychotropic medication users (p < 0.001). Finally and more surprisingly, illegal drug users felt that pharmacy studies were less diﬃcult than non-users and psychotropic medication users (p < 0.001). In the multivariate analysis, the students using psychotropic drugs (self-medication and medical prescription) presented signiﬁcantly higher levels of stress, anxiety and depression compared to non-users (Figure 2). Conversely, the students using illegal drugs had lower levels of fatigue and anxiety compared to non-users (Figure 2). Students using illegal drugs had signiﬁcantly lower annual average marks than non-users (Figure 3). Psycho Drugs Attendance was also signiﬁcantly lower for students using psychotropic drugs or illegal drugs compared to non-users (p < 0.05), but the biggest decrease was for students using illegal drugs (p < 0.001). Perception of study diﬃculty was signiﬁcantly lower for students using illegal drugs compared to non-users (p < 0.001) (Figure 3). TABLE 4 \| Indication and type of use of the main illegal drugs. Cannabis (herb) Cannabis (resin) MDMA Cocaine Indication, n (%) N = 458 N = 99 N = 91 N = 52 Pleasure/recreational 421 (91.9) 88 (88.9) 88 (96.7) 47 (90.4) Get stone 102 (22.3) 27 (27.3) 26 (28.6) 16 (30.8) Self-medication 31 (6.8) 8 (8.1) 3 (3.3) 3 (5.8) Self-medication indication, n (%) N = 31 N = 8 N = 3 N = 3 Anxiety 15 (48.4) 5 (62.5) 1 (33.3) 1 (33.3) Exam related stress 7 (22.6) 2 (25.0) – 2 (66.7) Sleep disturbances 20 (64.5) 6 (75.0) 2 (66.7) 2 (66.7) Depression 14 (45.2) 3 (37.5) 1 (33.3) 1 (33.3) Pain 11 (35.5) – – 1 (33.3) Frequency of use, n (%) N = 458 N = 99 N = 89 N = 51 ≥1/3 months 227 (49.6) 43 (43.4) 65 (73.0) 41 (80.4) ≥1/month 91 (19.9) 21 (21.2) 21 (23.6) 7 (13.7) ≥1/week 102 (22.3) 23 (23.2) 3 (3.4) 3 (5.9) ≥1/day 38 (8.3) 12 (12.1) – – Among the 99 cannabis resin users, 54 also used cannabis herb. TABLE 4 \| Indication and type of use of the main illegal drugs. Psycho Drugs Similar trends were found for depression (indicative and suggestive HADS scores). Depression was found for 24.3% of psychotropic users (versus non-users, 9.2%, p < 0.001) and 33.0% of bromazepam and alprazolam users (versus non-users, p < 0.001). No diﬀerence for proportions of anxiety or depression stood out between zolpidem users and non-users. TABLE 2 \| List of the psychotropic medications and illegal drugs most used during the last 3 months. Psychotropic medications (N = 243) Illegal drugs (N = 554) Alprazolam 26.3% Cannabis (herb) 82.7% Bromazepam 17.7% Cannabis (resin) 17.9% Zolpidem 12.4% MDMA 16.4% Codeine 11.9% Cocaine 9.4% Tramadol 10.3% Hallucinogenic mushroom 4.3% Escitalopram 5.4% LSD 2.9% Zopiclone 5.4% Cathinones 0% Paroxetine 4.9% Crack/free base 0% Oxazepam 3.7% Heroin 0% Diazepam 3.3% Methamphetamine 0% Fluoxetine 2.1% Synthetic cannabinoids 0% Only the main psychotropic medications are presented. TABLE 2 \| List of the psychotropic medications and illegal drugs most used during the last 3 months. July 2018 \| Volume 9 \| Article 725 5 Psychotropic Drugs and Pharmacy Students Balayssac et al. TABLE 3 \| Indication, frequency of use of the main psychotropic self-medications. Alprazolam Bromazepam Zolpidem Codeine Tramadol Medical prescription, n/N (%) 30/64 (46.9) 11/43 (25.6) 7/30 (23.3) 10/29 (34.5) 10/25 (40.0) Self-medication, n/N (%) 24/64 (37.5) 22/43 (51.2) 15/30 (50.0) 15/29 (51.7) 7/25 (28.0) Non-medical use, n/N (%) 14/64 (21.9) 13/43 (30.2) 9/30 (30.0) 9/29 (31.0) 11/25 (44.0) Self-medication, n (%) N = 24 N = 22 N = 15 N = 15 N = 7 Indications Anxiety (yes/no) 19 (79.2) 16 (72.7) 4 (26.7) 2 (13.3) 1 (14.3) Insomnia (yes/no) 9 (37.5) 13 (59.1) 13 (86.7) 3 (20.0) 1 (14.3) Depression (yes/no) 6 (25.0) 2 (9.1) 1 (6.7) 1 (6.7) 1 (14.3) Pain (yes/no) 2 (8.3) 1 (4.6) – 10 (66.7) 5 (71.4) Frequency of use ≥1/3 months 10 (41.7) 9 (40.9) 10 (66.7) 5 (33.3) 2 (28.6) ≥1/month 9 (37.5) 10 (45.5) 1 (6.7) 5 (33.3) 4 (57.1) ≥1/week 4 (20.8) 2 (9.1) 4 (26.7) 3 (26.7) 1 (14.3) ≥1/day – 1 (4.6) – 1 (6.7) – TABLE 3 \| Indication, frequency of use of the main psychotropic self-medications. and 25 years old (N = 580), showing that 11.5% of students had used psychotropic medications in the past year (females: 21.0% and males: 6.4%) (Beck et al., 2005). Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION This study showed that among French pharmacy students, 9.4% used psychotropic medications during the last 3 months (benzodiazepines and Z-drugs: 7.0% of all pharmacy students; opioids: 2.7% of all pharmacy students), 21.5% used illegal drugs (19.5% of all pharmacy students used cannabis) and 3.3% both psychotropic medications and illegal drugs. These psychotropic medications were used in the case of a medical prescription by 49.0% of students, a self-medication by 42.4% and a non- medical intent by 26.3%. Stress scores of the last 7 days and proportions of anxiety and depression at the time of answer were higher for psychotropic medication users. Fatigue scores of the last 7 days and annual average marks of the last year, attendance and perception of study diﬃculty were lower for illegal drug users. Uses of psychotropic medications had been assessed in 2000 for a smaller number of French students aged between 18 July 2018 \| Volume 9 \| Article 725 6 Psychotropic Drugs and Pharmacy Students Balayssac et al. TABLE 5 \| Use of psychotropic medications and illegal drugs and tobacco, alcohol use, stress, fatigue, annual average mark, attendance, study difﬁculty, and year repetition. TABLE 5 \| Use of psychotropic medications and illegal drugs and tobacco, alcohol use, stress, fatigue, annual average mark, attendance, study difﬁculty, and year repetition. DISCUSSION This study highlighted the need for strengthening the screening of psychiatric disorders among pharmacy students and probably among all French students (e.g., medical, nurses, dentists, veterinarians. . .), in order to detect these undiagnosed patients and propose therapeutic care. This point has already been identiﬁed in the scientiﬁc literature and suggested that there are high levels of unmet need among University students (Fortney et al., 2016). Moreover, we recommend reinforcing information and preventive messages on psychotropic self- medication for pharmacy students and the necessity for these self-medicated students to see specialists (psychiatrists). This information could also be extended to other student groups (e.g., medical, nurses, dentists, veterinarians. . .) who might also have easy access to psychotropic drugs. For the Midwest United States college students from 2003 to 2013, between 21.7 and 25.8% had used in the past-year prescription medications (sleeping, sedative/anxiety, stimulant or pain medications) for medical use and 11.4–13.6% for non-medical use (McCabe et al., 2014). More precisely, 2.9– 4.0% of these students had used sleeping medications for medical reasons (2.0–2.6% non-medically), 3.0–4.5% used sedative/anxiety medications for medical reasons (1.8–3.0% non- medically) and 15.7–21.4% used pain medications (opioids) for medical reasons (4.0–9.3% non-medically) (McCabe et al., 2014). The use of prescription opioids in Lebanese college students, in a weighted lifetime, was about 25% medically (medical prescription) and 15% non-medically (Ghandour et al., 2013). In comparison, French pharmacy students seemed to use quantitatively fewer psychotropic medications, but proportionally more sedative/anxiety medications and less opioid medications than students in other countries. Self-medication with psychotropic medications, use of illegal drugs and alcohol abuse may also be associated with dual diagnosis, which is the co-occurrence of substance use disorders and mental disorders, well known among physicians (Braquehais et al., 2014). We hypothesize that these behaviors of self- medications with psychotropic medications, illegal drugs and alcohol abuse during university years may contribute to dual diagnosis in senior pharmacists. Self-medication with psychotropic medications was elevated (42.4%), even more elevated than medical prescriptions for bromazepam (51.2% vs. 25.6%), zolpidem (50.0% vs. 23.3%), and codeine (51.7% vs. 34.5%). This self-medication was mainly done following the authorized indications (i.e., anxiety and insomnia for benzodiazepines and Z-drugs; pain for opioids), but some respondents practiced self-medication without following these indications. For example, benzodiazepines were used for pain and opioids were used for anxiety, insomnia and depression. DISCUSSION Non-users Psychotropic medications Illegal drugs p-values Medical prescription Self- medication Tobacco, n/N (%) 199/1,856 (10.7) 8/81 (9.9) 9/70 (12.9) 253/533 (47.5) <0.001c,d,e e-cigarettes, n/N (%) 20/1,848 (1.1) 4/81 (4.9) 1/68 (1.5) 19/529 (3.6) <0.001c Alcohol, n/N (%) 1,494/1,860 (80.3) 66/82 (80.5) 54/69 (78.3) 519/534 (97.2) <0.001c,d,e Hazardous alcohol consumption, n/N (%) 41/1,494 (2.7) 2/66 (3.0) 1/54 (1.9) 69/519 (13.3) <0.001c,d,e Stress, mean ± sd 54.3 ± 23.3 62.3 ± 19.2 62.0 ± 19.2 49.9 ± 26.3 <0.001a,b,c,d,e Fatigue, mean ± sd 63.0 ± 19.4 63.8 ± 21.9 66.4 ± 18.9 58.0 ± 22.3 <0.001c,e Annual average mark, n (%) N = 1,812 N = 77 N = 69 N = 516 <12/20 844 (46.6) 39 (50.7) 29 (42.0) 294 (57.0) <0.001c 12–14/20 683 (37.7) 31 (40.3) 28 (40.6) 173 (33.5) NS >14/20 285 (15.7) 7 (9.1) 12 (17.4) 49 (9.5) <0.01c Attendance, mean ± sd 64.8 ± 33.7 55.4 ± 35.3 54.6 ± 33.8 38.9 ± 32.7 <0.001a,c,e Study difﬁculties, mean ± sd 70.6 ± 16.2 69.0 ± 16.1 68.3 ± 17.4 64.7 ± 19.1 <0.001c Year repetition, n/N (%) 720/1,846 (39.0) 40/82 (48.8) 34/69 (49.3) 239/532 (44.9) 0.02 aNon-users vs. psychotropic medications (medical prescription), p < 0.05. bNon-users vs. psychotropic medications (self-medication), p < 0.05. cNon-users vs. illegal drugs, p < 0.05. dPsychotropic medications (medical prescription) vs. illegal drugs, ePsychotropic medications (self-medication) vs. illegal drugs, p < 0.05. ons (medical prescription), p < 0.05. bNon-users vs. psychotropic medications (self-medication), p < 0.05. cNon-users vs. illegal ations (medical prescription) vs. illegal drugs, ePsychotropic medications (self-medication) vs. illegal drugs, p < 0.05. aNon-users vs. psychotropic medications (medical prescription), p < 0.05. bNon-users vs. psychotropic medications (self-medica drugs, p < 0.05. dPsychotropic medications (medical prescription) vs. illegal drugs, ePsychotropic medications (self-medication) vs combinations and opium combinations, while the strong opioids used were morphine (oral route) and oxycodone (with a very strong increase in use over the last few years +613%) (Hider- Mlynarz et al., 2018). In our study, codeine and tramadol were the main opioid drugs (1.1 and 1.0% of all pharmacy students). which is worrying since these behaviors may continue and increase over time. It is particularly worrying to see that students who self-medicated with psychotropic drugs had nearly the same disorders as students who received a medical prescription for psychotropic drugs. In this pharmacy student population, self- medication with psychotropic drugs seems to mask undiagnosed patients. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION Moreover, we hypothesize that as students get older with their years of study and become more like “senior pharmacists,” they increase their self-medication with psychotropic medications, Fortunately, both univariate and multivariate analyses did not show a strong negative impact of psychotropic medication use (medical prescription or self-medication) on academic achievement (annual average mark of the last year, repeating, study diﬃculties and attendance). These students could suﬀer from psychiatric (anxiety, depression), neurological (epilepsy, chronic pain) or rheumatological (chronic pain) diseases that July 2018 \| Volume 9 \| Article 725 7 Balayssac et al. Psychotropic Drugs and Pharmacy Students FIGURE 1 \| Association between use of psychotropic medications or illegal drugs and anxiety or depression. Scores of anxiety and depression were classiﬁed as normal (white), suggestive (gray) and indicative (black). Suggestive scores of anxiety: p = 0.003, p < 0.05: non-users vs. illegal drugs. Indicative scores of anxiety: p < 0.001, p < 0.05: non-users vs. medical prescription, p < 0.05: non-users vs. self-medication, p < 0.05: non-users vs. illegal drugs, p < 0.05: medical prescription vs. illegal drugs, p < 0.05: self-medication vs. illegal drugs. Suggestive scores of depression: p = 0.009, no difference between groups. Indicative scores of depression: p < 0.001, p < 0.05: self-medication vs. illegal drugs. FIGURE 2 \| Forrest plot of the multivariate analyses of the relation between the use of psychotropic medications and illegal drugs with comorbidities. The multivariate analyses show coefﬁcients for stress and fatigue scores (quantitative variables) and odd ratios for anxiety and depression (qualitative variables) related to the use of psychotropic medications and illegal drugs. may interfere negatively with their academic achievement due to students have nearly the same cannabis consumption as the FIGURE 1 \| Association between use of psychotropic medications or illegal drugs and anxiety or depression. Scores of anxiety and depression were classiﬁed as normal (white), suggestive (gray) and indicative (black). Suggestive scores of anxiety: p = 0.003, p < 0.05: non-users vs. illegal drugs. Indicative scores of anxiety: p < 0.001, p < 0.05: non-users vs. medical prescription, p < 0.05: non-users vs. self-medication, p < 0.05: non-users vs. illegal drugs, p < 0.05: medical prescription vs. illegal drugs, p < 0.05: self-medication vs. illegal drugs. Suggestive scores of depression: p = 0.009, no difference between groups. Indicative scores of depression: p < 0.001, p < 0.05: self-medication vs. illegal drugs. DISCUSSION FIGURE 1 \| Association between use of psychotropic medications or illegal drugs and anxiety or depression. Scores of anxiety and depression were classiﬁed as normal (white), suggestive (gray) and indicative (black). Suggestive scores of anxiety: p = 0.003, p < 0.05: non-users vs. illegal drugs. Indicative scores of anxiety: p < 0.001, p < 0.05: non-users vs. medical prescription, p < 0.05: non-users vs. self-medication, p < 0.05: non-users vs. illegal drugs, p < 0.05: medical prescription vs. illegal drugs, p < 0.05: self-medication vs. illegal drugs. Suggestive scores of depression: p = 0.009, no difference between groups. Indicative scores of depression: p < 0.001, p < 0.05: self-medication vs. illegal drugs. FIGURE 2 \| Forrest plot of the multivariate analyses of the relation between the use of psychotropic medications and illegal drugs with comorbidities. The multivariate analyses show coefﬁcients for stress and fatigue scores (quantitative variables) and odd ratios for anxiety and depression (qualitative variables) related to the use of psychotropic medications and illegal drugs. FIGURE 2 \| Forrest plot of the multivariate analyses of the relation between the use of psychotropic medications and illegal drugs with comorbidities. The multivariate analyses show coefﬁcients for stress and fatigue scores (quantitative variables) and odd ratios for anxiety and depression (qualitative variables) related to the use of psychotropic medications and illegal drugs. may interfere negatively with their academic achievement due to disease relapses, the need to rest at home and hospitalizations. students have nearly the same cannabis consumption as the French population aged between 18 and 25 years old (19.5% for all pharmacy students during the last 3 months). Data from Ghandour et al. (2013) regarding a Lebanese college, showed that in a weighted lifetime 20.4% of students used cannabis herb, 2.8% MDMA and 1.6% cocaine/crack, which was very close to our study, in which 3.5% of French pharmacy students had used MDMA in the last 3 months and 2.0% cocaine. may interfere negatively with their academic achievement due to disease relapses, the need to rest at home and hospitalizations. In 2000 in France, 46.5% of students aged between 18 and 25 years old (N = 580) had experimented with cannabis at least once (males: 59.2% and females: 35.7%) and 29.7% at least once in the 12 past months (males: 38.3% and females: 22.3%) (Beck et al., 2005). DISCUSSION In another American pharmacy college, about 86.5% of students had drunk alcohol during the past year and about 23.2% had hazardous or harmful alcohol use (AUDIT questionnaire ≥8/40) (Oliver et al., 2014). At the University of Leeds (United Kingdom), 86% of medical students declared they drank alcohol and 52.6% of the men and 50.6% of the women had a hazardous alcohol consumption (21 units for men and 14 units for women per week) (Pickard et al., 2000). In conclusion, the proﬁle of French Pharmacy students regarding tobacco and alcohol consumption (20 and 85%, respectively) is very close to that of the French population in general, but they seem to have less hazardous alcohol consumption than Anglo-Saxon college students. Care is needed when comparing the results of the present study and those of the scientiﬁc literature because consumptions of tobacco, alcohol, psychotropic medications and illegal drugs are assessed diﬀerently regarding the quantities, frequencies, durations and years studied. The other surprising result is that illegal drug users had lower levels of stress (during the last 7 days), fatigue (during the last 7 days), anxiety and depression (at the time on the answer) than non-users and users of psychotropic medications. However, illegal drug users had signiﬁcantly higher tobacco and alcohol consumptions (even hazardous alcohol consumption). Illegal drug users had a very low level of attendance compared to non-users and users of psychotropic medications. These pharmacy students found their studies less diﬃcult than did the other students. Year repetition was slightly more elevated in this population without signiﬁcance, but the annual average mark was lower compared to non-users (57% of low annual average mark <12/20 and 9.5% Annual average mark >14/20). Admittedly, French Pharmacy studies are diﬃcult and exhausting for students. This point was related to the high level of anxiety (24.2% indicative score of HADS anxiety), which was nearly twice the prevalence of anxiety (11.7%) in college students from the WHO World Mental Health Surveys of college students from 21 countries and assessed with the Composite International Diagnostic Interview (Auerbach et al., 2016). DISCUSSION In France in 2014, 17% of people aged between 18 and 25 years old had smoked cannabis during the last month, of whom 8% were regular users (at least 10 uses during the last 30 days) and 4% daily users (OFDT, 2017b). French pharmacy In 2000 in France, 46.5% of students aged between 18 and 25 years old (N = 580) had experimented with cannabis at least once (males: 59.2% and females: 35.7%) and 29.7% at least once in the 12 past months (males: 38.3% and females: 22.3%) (Beck et al., 2005). In France in 2014, 17% of people aged between 18 and 25 years old had smoked cannabis during the last month, of whom 8% were regular users (at least 10 uses during the last 30 days) and 4% daily users (OFDT, 2017b). French pharmacy The proportion of smokers and e-cigarette users seems to be low in French pharmacy students (18.7 and 1.8%, respectively). July 2018 \| Volume 9 \| Article 725 Frontiers in Pharmacology \| www.frontiersin.org 8 Psychotropic Drugs and Pharmacy Students Balayssac et al. FIGURE 3 \| Forrest plot of the multivariate analyses of the relation between the use of psychotropic medications and illegal drugs with academic success. The multivariate analyses show coefﬁcients for annual average mark (qualitative variables) and odd ratios for attendance and study difﬁculty (quantitative variables) related to the use of psychotropic medications and illegal drugs. FIGURE 3 \| Forrest plot of the multivariate analyses of the relation between the use of psychotropic medications and illegal drugs with academic success. The multivariate analyses show coefﬁcients for annual average mark (qualitative variables) and odd ratios for attendance and study difﬁculty (quantitative variables) related to the use of psychotropic medications and illegal drugs. In 2016, 29.4% of French citizens aged 18–75 years smoked cigarettes daily and 2.5% e-cigarettes (OFDT, 2017c). About 21– 23% of French students are daily smokers (Tavolacci et al., 2013; Gignon et al., 2015) while this ﬁgure is 22% for European students (Beck et al., 2014). The alcohol consumption of French pharmacy students was about 84.0% and hazardous alcohol consumption was 9.0% for males and 4.0% for females. Alcohol consumption by French college students is about 80.6–97% (Sommet et al., 2012; Gignon et al., 2015). Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION This alcohol consumption was very close to French citizens aged 18–75 years of whom 87% had drunk alcohol at least once in the last 12 months in 2014 (OFDT, 2017a). In three American pharmacy colleges, 82.8% of students reported they had consumed alcohol in the past year and 25.4% consumed tobacco (Baldwin et al., 2011). In another American pharmacy college, about 86.5% of students had drunk alcohol during the past year and about 23.2% had hazardous or harmful alcohol use (AUDIT questionnaire ≥8/40) (Oliver et al., 2014). At the University of Leeds (United Kingdom), 86% of medical students declared they drank alcohol and 52.6% of the men and 50.6% of the women had a hazardous alcohol consumption (21 units for men and 14 units for women per week) (Pickard et al., 2000). In conclusion, the proﬁle of French Pharmacy students regarding tobacco and alcohol consumption (20 and 85%, respectively) is very close to that of the French population in general, but they seem to have less hazardous alcohol consumption than Anglo-Saxon college students. Care is needed when comparing the results of the present study and those of the scientiﬁc literature because consumptions of tobacco, alcohol, psychotropic medications and illegal drugs are assessed diﬀerently regarding the quantities, frequencies, durations and years studied. psychotropic medications and illegal drugs have not been investigated in depth, but it is known that the non-medical use of prescription drugs (any, stimulant, sedative and opioid drugs) is signiﬁcantly associated with psychiatric disorders and suicidal behavior among adolescents (Guo et al., 2016). In 2016, 29.4% of French citizens aged 18–75 years smoked cigarettes daily and 2.5% e-cigarettes (OFDT, 2017c). About 21– 23% of French students are daily smokers (Tavolacci et al., 2013; Gignon et al., 2015) while this ﬁgure is 22% for European students (Beck et al., 2014). The alcohol consumption of French pharmacy students was about 84.0% and hazardous alcohol consumption was 9.0% for males and 4.0% for females. Alcohol consumption by French college students is about 80.6–97% (Sommet et al., 2012; Gignon et al., 2015). This alcohol consumption was very close to French citizens aged 18–75 years of whom 87% had drunk alcohol at least once in the last 12 months in 2014 (OFDT, 2017a). In three American pharmacy colleges, 82.8% of students reported they had consumed alcohol in the past year and 25.4% consumed tobacco (Baldwin et al., 2011). DISCUSSION This high level of anxiety was, however, close to the level of anxiety (21.6%, full Composite International Diagnostic Interview Short Form, CIDI-SF) found in French college students in 2005 (Kovess- Masfety et al., 2016) and found in medical, pharmacy and dentistry students from Lebanon (21.8% of severe and extremely severe anxiety assessed with the Depression Anxiety Stress Scales, DASS 21) (Younes et al., 2016). Levels of depression seemed to be lower for French Pharmacy students (3.9% indicative score of HADS for depression) compared to the WHO World The misuse of psychotropic medications for non-medical use was also elevated and depended on the drug (alprazolam: 30.2%/0.5% and tramadol: 44.0%/0.4% for psychotropic medication users and for all pharmacy students, respectively). The psychiatric consequences associated with the use of July 2018 \| Volume 9 \| Article 725 9 Psychotropic Drugs and Pharmacy Students Balayssac et al. Mental Health Surveys (6.0%, mood disorders) and the Lebanese students of medicine, pharmacy and dentistry (8%, severe and extremely severe depression, DASS21) (Younes et al., 2016), and French college students (8.5%, full CIDI-SF) (Kovess-Masfety et al., 2016). Mental Health Surveys (6.0%, mood disorders) and the Lebanese students of medicine, pharmacy and dentistry (8%, severe and extremely severe depression, DASS21) (Younes et al., 2016), and French college students (8.5%, full CIDI-SF) (Kovess-Masfety et al., 2016). general French population. However, in comparison to other students in other countries, the use of psychotropic medications by French pharmacy students seemed to be lower, but with a proportionally higher use of anxiety/sedative medications and a lower use of opioid medications. Compared to a French sample of community pharmacists (N = 1,322 and age = 45.1 ± 10.6) studied for burnout and work- related stress in community pharmacies, senior pharmacists seemed to have a higher consumption of illicit drugs (24.5%) than pharmacy students (21.5%), whereas tobacco consumption (14.0%) and alcohol consumption (72.9%) tended to be lower than that of pharmacy students (18.7 and 84.0%, respectively) (Balayssac et al., 2017a). Scores of stress and fatigue remained very close between senior pharmacists and pharmacy students (stress: 52.31 ± 27.23 vs. 53.8 ± 23.9 and fatigue 59.6 ± 24.6 vs. 62.0 ± 20.2, respectively) (Balayssac et al., 2017b). However, proportions of indicative scores of anxiety and depression increased dramatically in senior pharmacists compared to students (42.4% vs. 24.2% and 15.7% vs. 3.9%, respectively) (Balayssac et al., 2017a). ACKNOWLEDGMENTS The authors thank the respondents for their participation. They thank the GATOX and the ANEPF for their assistance in sending the questionnaire to French pharmacy students. They also thank Accent Europe for its assistance in reading the manuscript. CONCLUSION Regarding the main objective, we can conclude that the use of psychotropic medications and illegal drugs by French pharmacy students were similar to, but slightly lower than that of, the The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2018.00725/full#supplementary-material DISCUSSION Besides the answer to the main objective and the prevalence of psychotropic medications and drug use by pharmacy students, two problems became clear. First, we think that pharmacy students using self-medication escape from a medical support and probably hide (consciously or unconsciously) their psychiatric disease. Second, illegal drugs have a negative impact on the academic success of pharmacy students. Other research could be engaged after this study. First, it would be very interesting to extend this study to medical students and to all students, in view to seeking an interaction between the type of study and the use of psychotropic medications and illegal drugs. Also, in the case of self-medication and non-medical use, we do not know how students get their medications: are they obtained through medical prescription or from pharmacy stocks? Therefore, it would be interesting to explore how pharmacy students obtain their medications. Academic doping could also be explored. Academic doping consists in taking medications or drugs in order to enhance cognitive capacities and academic success (Garasic and Lavazza, 2016). We can presume that pharmacy and medical studies, which are hard and selective, may be a strong determinant for academic doping. AUTHOR CONTRIBUTIONS DB, BP, MD, BV, and NA contributed to the design of the manuscript and the acquisition of data. LV and PC contributed to the acquisition of data. DB, BP, MD, LV, PC, AC, BV, and NA participated in drafting and revising the manuscript. All authors approved the ﬁnal version of the manuscript for submission. Study Limitations The rate of response to the survey was low (14%). This low response rate could have been improved with a longer survey duration. Students of the 1st and the 6th year of pharmacy studies were less represented. During the 1st year of study, the students have a lot of work and competitive exams, so they would probably be less involved in “everyday life in the university.” Students of the 6th year performed an internship of 6 months (community pharmacy or pharmaceutical industry) during the survey schedule. Consequently, students from the 1st and the 6th years would be more diﬃcult to recruit. Selection bias seems to be limited because psychotropic medication and illegal drug users did not seem to be over-represented in comparison to the scientiﬁc literature. It is also impossible to know if the population of the study is representative to the French pharmacy students, because there is no data available. The survey was sent by email to the students, which was a very good way to contact them, because they are all used to emails and digital environments. Each pharmacy faculty has digital work environment which allows students and professors to share lessons and homework. However, it was impossible to check that a student do not respond twice or more times. ANSM (2017). Etat des Lieux de la Consommation des Benzodiazépines - Point d’Information - ANSM?: Agence Nationale de Sécurité du Médicament et des Produits de Santé. Available at: http://ansm.sante.fr/S-informer/Points-d- information-Points-d-information/Etat-des-lieux-de-la-consommation-des- benzodiazepines-Point-d-Information [accessed October 16, 2017]. ANSM (2017). 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https://openalex.org/W2131078192	https://bmcecolevol.biomedcentral.com/counter/pdf/10.1186/1471-2148-11-71	English	null	Position and sequence conservation in Amniota of polymorphic enhancer HS1.2 within the palindrome of IgH 3'Regulatory Region	BMC evolutionary biology	2,011	cc-by	10,285	Abstract Background: The Immunoglobulin heavy chain (IgH) 3’ Regulatory Region (3’RR), located at the 3’ of the constant alpha gene, plays a crucial role in immunoglobulin production. In humans, there are 2 copies of the 3’RR, each composed of 4 main elements: 3 enhancers and a 20 bp tandem repeat. The single mouse 3’RR differs from the two human ones for the presence of 4 more regulative elements with the double copy of one enhancer at the border of a palindromic region. Results: We compared the 3’RR organization in genomes of vertebrates to depict the evolutionary history of the region and highlight its shared features. We found that in the 8 species in which the whole region was included in a fully assembled contig (mouse, rat, dog, rabbit, panda, orangutan, chimpanzee, and human), the shared elements showed synteny and a highly conserved sequence, thus suggesting a strong evolutionary constraint. In these species, the wide 3’RR (~30 kb in human) bears a large palindromic sequence, consisting in two ~3 kb complementary branches spaced by a ~3 kb sequence always including the HS1.2 enhancer. In mouse and rat, HS3 is involved by the palindrome so that one copy of the enhancer is present on each side. A second relevant feature of our present work concerns human polymorphism of the HS1.2 enhancer, associated to immune diseases in our species. We detected a similar polymorphism in all the studied Catarrhini (a primate parvorder). The polymorphism consists of multiple copies of a 40 bp element up to 12 in chimpanzees, 8 in baboons, 6 in macaque, 5 in gibbons, 4 in humans and orangutan, separated by stretches of Cytosine. We show specific binding of this element to nuclear factors. Conclusions: The nucleotide sequence of the palindrome is not conserved among evolutionary distant species, suggesting pressures for the maintenance of two self-matching regions driving a three-dimensional structure despite of the inter-specific divergence at sequence level. The information about the conservation of the palindromic structure and the settling in primates of the polymorphic feature of HS1.2 show the relevance of these structures in the control and modulation of the Ig production through the formation of possible three-dimensional structures. © 2011 D’Addabbo 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. Position and sequence conservation in Amniota of polymorphic enhancer HS1.2 within the palindrome of IgH 3’Regulatory Region Pietro D’Addabbo1, Moira Scascitelli2, Vincenzo Giambra3, Mariano Rocchi1, Domenico Frezza4 * Correspondence: frezza@uniroma2.it 4Department of Biology, University of Roma Tor Vergata, Viale della ricerca scientifica. 00133 Roma, Italy Full list of author information is available at the end of the article D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Open Access Background importance in producing diversity [1], and constituted a crucial step in B cell maturation [2]. The region involved by the somatic rearrangements, allowing the class switch, is the Immunoglobulin heavy chain (IgH) locus. This domain is in a single copy in the genome of most extant species (as example, see the mouse locus in Figure 1A). Hominoidea (human, chimpanzee, gorilla and gibbon) are an exception, because the constant genes of the IgH locus underwent duplication in their common ancestor [3]. Studies in humans have shown that the duplication of the IgH locus included the The immunoglobulin genes appeared, during evolution, in vertebrates. Because of their increasing physiological relevance, the evolution of these genes in fish, amphi- bian, birds and mammals witnessed several series of duplications that ended in adding copies and complexity to these genes. The class switch, in particular, was of * Correspondence: frezza@uniroma2.it 4Department of Biology, University of Roma Tor Vergata, Viale della ricerca scientifica. 00133 Roma, Italy Full list of author information is available at the end of the article Page 2 of 12 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Figure 1 Schematic map of the mouse and human IgH genetic cluster. The IgH cluster and the closest genes (dark blue) are highly conserved in the mouse and human chromosomes. The same transcription direction was also detected in both species, with a “telomere towards centromere” transcription versus. The mouse IgH cluster harbors 8 constant genes and one copy of the 3’ Regulatory Region (3’RR) with 7 enhancers, 3 of them (HS5, HS6, HS7) with possible insulator function at the 3’ boundary of the cluster. The human IgH region has some differences for the constant genes since a wide duplication of gamma, epsilon and alpha genes occurred in the ancestor of Hominoidea. This duplication included also the 3’RR at the 3’ of the constant alpha gene. Both the two copies of the 3’RR in humans have just three enhancers. In fact, the human loci do not harbor both the duplicated HS3.B enhancer and the insulators present in the mouse locus. enhancers [15]. Binding sites variation affecting the enhancers sequence can lead to different epigenetic changes and bring cells to differently act. Background In our recently performed population studies we found, in fact, that some of the 3’RR enhancer HS1.2 variants (Figure 2) were associated to a higher risk for autoimmune dis- eases onset and other immune-disorders as IgA defect, systemic sclerodermia, Rheumatic arthritis, Psoriasis and Celiac disease [16-20]. We hypothesized that the cause was a change of a binding consensus for NF-B and other transcription factors as “in silico“ predicted or experimentally determined [16,21]. In humans the pre- sence of an allele with the NF-B consensus site was associated to increased haematic concentration of IgM, suggesting a contribution to the mechanism of class switch [16,17]. Figure 1 Schematic map of the mouse and human IgH genetic Figure 1 Schematic map of the mouse and human IgH genetic cluster. The IgH cluster and the closest genes (dark blue) are highly conserved in the mouse and human chromosomes. The same transcription direction was also detected in both species, with a “telomere towards centromere” transcription versus. The mouse IgH cluster harbors 8 constant genes and one copy of the 3’ Regulatory Region (3’RR) with 7 enhancers, 3 of them (HS5, HS6, HS7) with possible insulator function at the 3’ boundary of the cluster. The human IgH region has some differences for the constant genes since a wide duplication of gamma, epsilon and alpha genes occurred in the ancestor of Hominoidea. This duplication included also the 3’RR at the 3’ of the constant alpha gene. Both the two copies of the 3’RR in humans have just three enhancers. In fact, the human loci do not harbor both the duplicated HS3.B enhancer and the insulators present in the mouse locus. Little is known on the presence and organization of the 3’RR in Amniota other than human and mouse [22,23]. Comparative studies of this regulatory region can provide hypotheses on which elements are crucial with respect to their function [24]. To fill the gap, we investigated the genomic organization of this region tak- ing advantage of the sequence data present in GenBank and Trace Archive (see Methods below). The most rele- vant achievement of the analysis was the discovering that the palindrome surrounding the HS1.2 enhancer is present in every mammal species for which enough sequence data were available. This finding has important implication on the understanding of HS1.2 functioning. Background In addition, our data supports the view that HS1.2 poly- morphisms are widely spread in the primate parvorder of Catarrhini (Cercopithecoidea and Hominoidea). The improvement on the comparative studies on the non coding genome is a relevant task for new insight in the epigenetic and mechanisms of genome regulation [25]. Regulatory Region (3’RR) located immediately down- stream of the constant alpha exons [2]. Portions of the 3’RR were first cloned in 1990 and 1991 [4,5] but only later were fully assembled as a complete contig sequence because they are repeats-rich unstable regions, moreover containing palindromic sequences [6,7]. In humans, the 2 copies of this 3’ Regulatory Region (3’RR) have been reported as 3’RR1 and 3’RR2 (Figure 1B) [8]. Each human 3’RR copy harbors three different enhancers. The mouse and rat 3’RR possess 4 more boundary regu- latory regions instead. Their existence in other organ- isms may be hypothesized but have not been demonstrated yet [2] (Figure 1A). 3’RR genomic organization (B) The sequence alignment of four alleles of HS1.2 enhancer sequences colored according to the schematic elements reported in the upper part. The binding for the Oct1, Sp1 and NF-B factors are reported on the sequence as determined by EMSA experiments. Figure 2 The polymorphisms of the human enhancer HS1.2. (A) The schematic representation of the human alleles shows the different invariant and variable elements. Up to now 6 different alleles are reported in GenBank, each one identified for the number of copies (1, 2, 3, 4) of the 40 bp element (40 mer, in yellow). More variability was added by the presence of either a short stretch of cytosines (18 bp, in green) or a different larger element (29 bp, in red) between the enhancer core (in fuchsia) and the first 40 mer. The 3’ end of the enhancer (in light blue) also is highly conserved. The green stretches of cytosines parting the 40 mer ranged between 12 and 18 bp. (B) The sequence alignment of four alleles of HS1.2 enhancer sequences colored according to the schematic elements reported in the upper part. The binding for the Oct1, Sp1 and NF-B factors are reported on the sequence as determined by EMSA experiments. Figure 2 The polymorphisms of the human enhancer HS1.2. (A) The schematic representation of the human alleles shows the different invariant and variable elements. Up to now 6 different alleles are reported in GenBank, each one identified for the number of copies (1, 2, 3, 4) of the 40 bp element (40 mer, in yellow). More variability was added by the presence of either a short stretch of cytosines (18 bp, in green) or a different larger element (29 bp, in red) between the enhancer core (in fuchsia) and the first 40 mer. The 3’ end of the enhancer (in light blue) also is highly conserved. The green stretches of cytosines parting the 40 mer ranged between 12 and 18 bp. (B) The sequence alignment of four alleles of HS1.2 enhancer sequences colored according to the schematic elements reported in the upper part. The binding for the Oct1, Sp1 and NF-B factors are reported on the sequence as determined by EMSA experiments. chimpanzee, and orangutan. HS3 and the 20 bp repeat, were present in 12 mammals, but were undetected in cow, elephant, opossum, and platypus. 3’RR genomic organization The region delimited by the alpha exons and HS3.A enhancer and encompassing the 20 bp repeat, appeared to be highly conserved in placental mammals. We remark that, at the contrary, the Alpha marker remains entirely unde- tected in chicken and pig, because the similarity versus rodent IgA is low even at the level of peptide sequence. hgGateway?org=Mouse report graphical representations of Lastz comparison http://www.bx.psu.edu/miller_lab/ between mouse and each one of 19 Amniota genomes (rat, guinea pig, rabbit, human, chimp, orangutan, rhe- sus, marmoset, panda, dog, cat, horse, elephant, cow, pig, opossum, platypus, lizard, chicken). Some of these assemblies, human and mouse in particular, are very accurate. At the contrary some others are based on rela- tively low sequence coverage, with several unresolved gaps. This peculiar consideration has to be kept in mind when dealing with negative results of sequences com- parison among genomes drafts. As expected, the search in the Neanderthal genome [27] by inspection of related track in the UCSC human genome browser demonstrated that HS3, HS1.2 and HS4 were also present in the genome of our extinct relative (data not shown). Results of our search for the mouse 3’RR main ele- ments in Amniota genomes are summarized in Figure 3. The full set of elements present in mouse (IgH alpha exons, 20 bp tandem repeat and 7 enhancers) was detected only in the rat genome. The HS5, HS6, and HS7 were always absent in all the remaining species. The HS3, HS1.2, and HS4 set was detected with certainty in mouse, rat, dog, rabbit, panda, human, 3’RR genomic organization The mouse 3’RR region contains 7 enhancers (HS3.A- HS1.2-HS3.B-HS4-HS5-HS6-HS7), while human has only three enhancers (Figure 1). For this reason the mouse was used as the reference genome for the preli- minary analysis. The IgA class exons are the tran- scripted sequence closest to the 3’RR, so we included a portion of this DNA in our analysis. It is to keep in mind that the IgA was the last class to appear during the evolution of IgH, because it is just shared among Amniota species (reptiles, bird and mammals) [26]. Finally, we surveyed also a satellite repeat. This is a con- served stretch of DNA (812 bp in human) composed of tandemly repeated 20 bp element and located inside the 3’UTR of the IgA gene, close to the 3’RR enhancer. The 3’RR has a crucial role in recruiting transcription factors for the initiation of germ line transcription of the constant genes to induce IgH switch [9]. The role of the 3’RR enhancers was studied with mice transgenic for the c-myc translocation, showing the active role of HS3. B and HS4 in peripheral B-cell lymphomas progression and not in pro-B lymphomas [10,11]. Relevant studies demonstrate by chromosome conformation capture techniques the presence of a three-dimensional structure originated by a loop among Regulatory Regions during class switch recombination [12]. New studies on 3’RR transgenic deleted mice report impairment of class switch and Ig expression [13,14]. Activation of the mouse 3’RR begins with selective demethylation of The “Comparative Genomics” tracks of the UCSC mouse genome browser http://genome.ucsc.edu/cgi-bin/ D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Page 3 of 12 Figure 2 The polymorphisms of the human enhancer HS1.2. (A) The schematic representation of the human alleles shows the different invariant and variable elements. Up to now 6 different alleles are reported in GenBank, each one identified for the number of copies (1, 2, 3, 4) of the 40 bp element (40 mer, in yellow). More variability was added by the presence of either a short stretch of cytosines (18 bp, in green) or a different larger element (29 bp, in red) between the enhancer core (in fuchsia) and the first 40 mer. The 3’ end of the enhancer (in light blue) also is highly conserved. The green stretches of cytosines parting the 40 mer ranged between 12 and 18 bp. Dot plot analysis of the 3’RR The black blocks in the “Mouse main elements” section localize the positions of the chosen tags, mapped by the BLAT tool of the site. The colored blocks in the “Comparative Genomics” section show the most similar region in each genome of the species listed, versus the mouse genome. The color of each block represents a portion of a chromosome (see the chromosome color legend below the graphic) of the species similar to that portion of the mouse chromosome 12. The “RepeatMasker” and “Tandem Repeat Finder” sections show the position of repeats identified by the two homonymous algorithms in this portion of mouse genome. region chr12:114,459,657-114,497,890 of the UCSC Mouse Genome Browser (mm9 draft). The comparative genomic tracks were inspected and the graphic was edited as in this figure. The species common names are listed in the first column on the left. The black blocks in the “Mouse main elements” section localize the positions of the chosen tags, mapped by the BLAT tool of the site. The colored blocks in the “Comparative Genomics” section show the most similar region in each genome of the species listed, versus the mouse genome. The color of each block represents a portion of a chromosome (see the chromosome color legend below the graphic) of the species similar to that portion of the mouse chromosome 12. The “RepeatMasker” and “Tandem Repeat Finder” sections show the position of repeats identified by the two homonymous algorithms in this portion of mouse genome. match, even though it is expected. These missing sequences, however, can be present in the shot-gun sequences databases, as unassembled short sequences. We searched in GenBank genome-related databases, by BLAST, for the presence of murine (data not shown) and human IgH alpha exons (Table 1). Querying for this transcripted sequence, we tested the method limits. All the 35 positive species were mammals (8 primates). Then we BLASTed the murine (data not shown) and human (Table 1) enhancers against the same positive databases. The analysis showed that at least one 3’RR enhancer was present in 23 species, while all the three enhancers were present in only 8 species (in bold in Table 1), apart from human. The negative findings may be ascribed either to the non-completeness of the avail- able genomic drafts/wgs/htgs databases or to an actual sequences divergence. Dot plot analysis of the 3’RR It is worth noting that the longer map distance between the 3’RR features and the IgA gene, the less species detected (Table 1). Finally, the analysis confirmed that the mouse HS5, HS6, and HS7 were detectable by sequence similarity only in rat (data not shown). match, even though it is expected. These missing sequences, however, can be present in the shot-gun sequences databases, as unassembled short sequences. We searched in GenBank genome-related databases, by BLAST, for the presence of murine (data not shown) and human IgH alpha exons (Table 1). Querying for this transcripted sequence, we tested the method limits. All the 35 positive species were mammals (8 primates). Then we BLASTed the murine (data not shown) and human (Table 1) enhancers against the same positive databases. The analysis showed that at least one 3’RR enhancer was present in 23 species, while all the three enhancers were present in only 8 species (in bold in Table 1), apart from human. The negative findings may be ascribed either to the non-completeness of the avail- able genomic drafts/wgs/htgs databases or to an actual sequences divergence. It is worth noting that the longer map distance between the 3’RR features and the IgA gene, the less species detected (Table 1). Finally, the analysis confirmed that the mouse HS5, HS6, and HS7 were detectable by sequence similarity only in rat (data not shown). Dot plot analysis of the 3’RR Dot plot analysis of the 3’RR It has been already reported, in man and mouse, that each HS1.2 enhancer is flanked, at some distance on both sides, by a 3 kb segment, and that these segments are in opposite orientation (palindromic), as evident from the dot plot analysis reported in Figure 4 (palin- dromic sequence in light blue). Very interestingly, we found that this organization is shared by 8 species in which the whole region was included in a fully assembled contig. The human versus non-primate dot- plots are reported in Figure 5. It is worth noting that, while the similarity between the two components of the same palindrome is always very high (94% in human, Figure 4), the sequence itself almost completely varied among species (Figure 5). Interestingly, HS1.2 always lies in the center of the palindrome. In the human 3’ RR1, the region internal to the two components of the palindrome is inversely oriented with respect to the cor- responding sequence of 3’RR2, as shown by the second- ary diagonal line present at the core of the light blue frame (Figure 4). In addition, sequence comparisons of the human 3’RRs with non-primate mammals harboring a single copy of 3’RR (panda, rabbit, mouse, and dog) showed that in 3 of them, with the exception of the mouse, the orientation of the region internal to the palindrome (containing the HS1.2) was identical to the human 3’RR2 (Figure 5). This finding suggests that the 3’RR2 is ancestral with respect to the 3’RR1. The mouse showed an opposite orientation of the region internal to the palindrome. Moreover the mouse palindrome is lar- ger than the human one, including the HS3 and the 20 bp repeat, thus originating HS3.A and HS3.B (Figure 5). Very likely, an inversion event was triggered by the palindrome both in the mouse 3’RR and in the human 3’RR1. Figure 3 Contig sequence alignment of the 3’RR of vertebrate species with completed genome sequence. This figure shows the Figure 3 Contig sequence alignment of the 3 RR of vertebrate species with completed genome sequence. This figure shows the region chr12:114,459,657-114,497,890 of the UCSC Mouse Genome Browser (mm9 draft). The comparative genomic tracks were inspected and the graphic was edited as in this figure. The species common names are listed in the first column on the left. Enhancers in the sequenced species Figure 3 shows comparisons among assembled genome drafts. If the sequence of a specific region is still missing in the genome draft of a particular species, then the comparison versus the mouse genome will not find any D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Page 4 of 12 Page 4 of 12 Figure 3 Contig sequence alignment of the 3’RR of vertebrate species with completed genome sequence. This figure shows the region chr12:114,459,657-114,497,890 of the UCSC Mouse Genome Browser (mm9 draft). The comparative genomic tracks were inspected and the graphic was edited as in this figure. The species common names are listed in the first column on the left. The black blocks in the “Mouse main elements” section localize the positions of the chosen tags, mapped by the BLAT tool of the site. The colored blocks in the “Comparative Genomics” section show the most similar region in each genome of the species listed, versus the mouse genome. The color of each block represents a portion of a chromosome (see the chromosome color legend below the graphic) of the species similar to that portion of the mouse chromosome 12. The “RepeatMasker” and “Tandem Repeat Finder” sections show the position of repeats identified by the two homonymous algorithms in this portion of mouse genome. HS1.2 enhancer in Trace Archive While no polymorphisms have been reported for the enhancers HS3 and HS4 [28], the 2 human HS1.2 copies share a set of variant forms (Figure 2) [21,29]. The main polymorphic feature of human HS1.2 consists of a tan- dem repeated pair of elements, i.e. a 40 bp sequence (40 mer, yellow boxes in Figure 2) and a ~15 bp cytosine- rich stretch (green boxes), that can or cannot be sepa- rated from the enhancer core (purple boxes) by a 29 bp sequence (red boxes). The HS1.2 human variants with more copies of the 40 mer showed an increasing effect on the transcription of a reporter gene in transfected cells [30]. In mouse there is just one copy of HS1.2 that constantly harbors a single copy of the 40 mer. The Trace Archive databases of primate species sequences was searched by BLAST using the human HS1.2 sequence as query, to investigate the evolutionary history of this enhancer and to search for potential D’Addabbo et al. HS1.2 enhancer in Trace Archive BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Page 5 of 12 Table 1 List of species, other than human, identified by BLAST in wgs and htgs databases Alpha repeat HS3 HS1,2 HS4 MI QC MI QC MI QC MI QC MI QC Gorilla gorilla - Gorilla (P) 94% 100% 93% 100% 97% 100% 94% 78% 98% 100% Pongo abelii - Orangutan (P) 94% 62% 88% 99% 92% 100% 92% 100% 95% 99% Pan troglodytes - Chimpanzee (P) 93% 87% 87% 100% 99% 100% 95% 100% - - Macaca mulatta - Macaque (P) 89% 100% 82% 100% 93% 100% 90% 80% - - Felis catus - Cat 88% 75% 76% 100% 77% 66% 76% 46% 69% 100% Callithrix jacchus - Marmoset (P) 85% 99% 67% 99% 85% 100% 89% 78% - - Sorex araneus - European shrew 81% 71% 70% 52% - - 76% 26% - - Otolemur garnettii - Galago (P) 79% 36% 74% 99% - - - - - - Tarsius syrichta - Tarsier (P) 76% 43% 64% 92% 82% 88% - - - - Echinops telfairi - Madagascar hedgehog 75% 42% - - 67% 77% 81% 30% - - Choloepus hoffmanni - Sloth 74% 30% - - - - - - - - Tursiops truncatus - Dolphin 73% 98% 76% 99% 77% 84% 73% 45% - - Canis familiaris - Dog 72% 99% 75% 99% 74% 90% 76% 47% 79% 44% Microcebus murinus - Lemur (P) 72% 57% 67% 98% 81% 84% - - - - Ovis aries - Sheep 72% 43% - - 76% 75% 77% 36% - - Ailuropoda melanoleuca - Panda 71% 99% 79% 99% 75% 82% 75% 86% 70% 83% Pteropus vampyrus - Large flying fox 71% 99% 67% 61% 78% 84% - - 66% 88% Bos taurus - Cattle 70% 79% 82% 99% 77% 84% - - - - Ornithorhynchus anatinus - Platypus 70% 31% 66% 58% - - - - - - Myotis lucifugus - Little brown bat 70% 21% 69% 86% - - 69% 43% - - Equus caballus - Horse 69% 78% 78% 99% 80% 77% - - - - Spermophilus tridecemlineatus - Squirrel 69% 30% 66% 71% - - - - - - Oryctolagus cuniculus - Rabbit 68% 99% 76% 99% 66% 79% 66% 52% 70% 89% Rattus norvegicus - Brown rat 68% 99% 65% 98% 78% 84% 76% 34% 75% 49% Mus musculus - Mouse 67% 98% 70% 99% 72% 76% 77% 40% 74% 46% Procavia capensis - Hyrax 67% 97% - - - - - - 75% 50% Tupaia belangeri - Tree shrew 67% 26% 64% 99% - - - - - - Macropus eugenii - Wallaby 66% 28% - - - - - - - - Ochotona princeps - Pika 65% 99% 71% 99% - - - - - - Cavia porcellus - Guinea pig 65% 98% 67% 99% 75% 82% 74% 42% - - Monodelphis domestica - Opossum 65% 45% 65% 80% - - - - - - Erinaceus europaeus - European hedgehog 63% 64% 62% 91% 72% 68% - - - - Dasypus novemcinctus - Armadillo 62% 29% 71% 99% - - - - - - Dipodomys ordii - Kangaroo rat 61% 34% 65% 99% - - - - - - Loxodonta africana - Elephant 61% 29% 64% 98% - - - - - - Total 35 30 21 17 10 MI, Max Identity (i.e. HS1.2 enhancer in Trace Archive the higher value among the identity values of the BLAST output); QC, Query Coverage (i.e. portion of the query sequence that was recognized by some subject sequence of that species); P, Primate. Alpha, [GenBank:NC_000014:106053245-106054732] (1488 bp); repeat, [GenBank: NC_000014:106048991-106049802] (812 bp); HS3, [GenBank:NC_000014:106048351-106048676] (326 bp); HS1,2, [GenBank:NC_000014:106041545-106042009] (465 bp); HS4, [GenBank:NC_000014:106032614-106032974] (361 bp). Table 1 List of species, other than human, identified by BLAST in wgs and htgs databases MI, Max Identity (i.e. the higher value among the identity values of the BLAST output); QC, Query Coverage (i.e. portion of the query sequence that was recognized by some subject sequence of that species); P, Primate. Alpha, [GenBank:NC_000014:106053245-106054732] (1488 bp); repeat, [GenBank: NC_000014:106048991-106049802] (812 bp); HS3, [GenBank:NC_000014:106048351-106048676] (326 bp); HS1,2, [GenBank:NC_000014:106041545-106042009] (465 bp); HS4, [GenBank:NC_000014:106032614-106032974] (361 bp). primates, was entirely detected only in panda when searched in non-primate mammals. polymorphisms. Figure 6 summarizes the obtained results, along with all the available data from previous works [21] and from our previously unpublished sequencing data. This figure shows the organization of the HS1.2 in the dif- ferent species. The highly conserved core of the enhancer (113 bp, purple) is constantly flanked, in the 11 non- primate mammalian species (olive green background), by a partial 29 mer stretch (red element). The 102 bp term- inal element (blue in Figure 6), constantly found in The most interesting finding of this analysis is the presence of a variable number of copies of the 40 mer (yellow in Figure 6) in all Catarrhini parvorder species, i.e. in both Hominoidea and Cercopithecoidea superfa- milies. On the contrary, the duplication of the whole locus of the constant genes was found only in Hominoi- dea [3]. This observation strongly suggests that the D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Page 6 of 12 Figure 4 Dot plot of the human 3’RR-2 against itself and 3’RR-1. The two human 3’RR are compared to show the almost identical sequence and the palindromic structure encompassing the enhancer HS1.2 (light blue frame). Each portion of the palindrome is extended for ~3 kb. The black squares of spots indicate the presence of short stretches of DNA satellite tandem repeat. The two HS1.2 enhancers have inverted orientation in the two 3’RR as a result of the inverted orientation of the sequence internal to the palindrome. The orientation of the other enhancers is conserved and also the distances. Discussion In the present article we have compared the genomic structure of the 3’RR domain of the IgH gene cluster in various species. We have confirmed that in all the ana- lyzed species the 3’RR elements order is largely main- tained. Two main results were also achieved: (i) a palindromic structured sequence flanks each HS1.2 enhancer; (ii) HS1.2 is polymorphic in all analyzed Cat- arrhini species and therefore rose before of the IgH locus duplication. HS1.2 Transcription Factor Binding Sites (TFBS) p g ( ) All the HS1.2 forms found in the different species (Figure 6) were searched for transcription factor binding sites, using Alibaba2 software. Relevant results are sum- marized in Additional file 1 (full list in Additional file 2). Four TFBS (C/EBPalp, AP-2alpha, SP1, Oct1) are present in all the analyzed species; moreover, the NF variants are almost ubiquitous. The Additional file 1 clearly indicates that, while the number of C/EBPalp and Oct1 TFBS is substantially constant in different HS1.2 forms, the num- ber of AP-2alpha and SP1 TFBS is proportional to the copies of the 40 mer present in that specific HS1.2 form. Note that c-myc containing 40 mers appears only in the Catarrhini HS1.2 forms and in dog. HS1.2 enhancer in Trace Archive The 20 bp repeat in dark blue appears to be larger in the 3’RR1. Figure 4 Dot plot of the human 3’RR-2 against itself and 3’RR-1. The two human 3’RR are compared to show the almost identical sequence and the palindromic structure encompassing the enhancer HS1.2 (light blue frame). Each portion of the palindrome is extended for ~3 kb. The black squares of spots indicate the presence of short stretches of DNA satellite tandem repeat. The two HS1.2 enhancers have inverted orientation in the two 3’RR as a result of the inverted orientation of the sequence internal to the palindrome. The orientation of the other enhancers is conserved and also the distances. The 20 bp repeat in dark blue appears to be larger in the 3’RR1. human, gorilla, orangutan, mouse, rat, rabbit, panda, dog and cat (see Additional file 3). The phylogenetic analysis obtained with the maximum likelihood method for C-alpha, HS3, HS1.2 and HS4 (Figure 7A, B, C and 7D respectively), showed in all cases a similar variation from the standard reconstruction of mammals’ phylo- geny. Rodents and lagomorphs diverged from each other and from primates and carnivores, confirming at the nucleotide level the hypothesis of different evolutionary routes taken from the different groups, as shown after structural analysis. The concordance between the coding region (C-alpha) and the three enhancers in the observed divergence furthermore indicates that similar forces shaped the evolution of the whole 3’ regulatory region, suggesting potential functional constrains also for the non coding sections. emergence of the polymorphism occurred earlier with respect to the duplication. The number of the 40 mer varies from the 12 copies found in chimpanzee HS1.2, to a single one, as detected in some alleles in human, chimpanzee, and gorilla and in all the non-primate mammals. An additional variability found in Hominoi- dea is the occasional absence of the 29 mer, replaced by an 18 bp stretch of cytosine (green in Figure 6). Phylogenetic analysis The four structures clustered in the 3’RR were analyzed for their sequence variation in 9 species comprising D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Page 7 of 12 Figure 5 Dot plot of the human 3’RR-2 against 4 species. The comparison of the human regulatory region against the homologous sequences of Ailuropoda melanoleuca (panda), Canis familiaris (dog), Mus musculus (mouse) and Oryctolagus cuniculus (rabbit) shows the conservation of the enhancer positions and the high similarity in the sequence spanning from the alpha gene to the other extreme enhancer HS4. In the mouse the two copies of the enhancer HS3 A and B have in the neighbor region also the 20 bp repeat. In the mouse palindrome (light blue frame) are shown by the black segments with inverted orientation three copies of a region that is in two copies in humans. The wider extension of the palindrome in mouse includes the two HS3 enhancers on the boundaries. The same structures are conserved along the homologues 3’RR in these species and the palindrome is present in the cluster of all species as evidenced by the pale blue rectangle with the central region represented by the HS1.2 enhancer. The black lines depict the internal repeats present in more than one copy. The lines when parallel to the major diagonal have the same orientation, when are perpendicular have inverted orientation, as in the palindrome sequence. Figure 5 Dot plot of the human 3’RR-2 against 4 species. The comparison of the human regulatory region against the homologous sequences of Ailuropoda melanoleuca (panda), Canis familiaris (dog), Mus musculus (mouse) and Oryctolagus cuniculus (rabbit) shows the conservation of the enhancer positions and the high similarity in the sequence spanning from the alpha gene to the other extreme enhancer HS4. In the mouse the two copies of the enhancer HS3 A and B have in the neighbor region also the 20 bp repeat. In the mouse palindrome (light blue frame) are shown by the black segments with inverted orientation three copies of a region that is in two copies in humans. The wider extension of the palindrome in mouse includes the two HS3 enhancers on the boundaries. The same structures are conserved along the homologues 3’RR in these species and the palindrome is present in the cluster of all species as evidenced by the pale blue rectangle with the central region represented by the HS1.2 enhancer. Phylogenetic analysis The black lines depict the internal repeats present in more than one copy. The lines when parallel to the major diagonal have the same orientation, when are perpendicular have inverted orientation, as in the palindrome sequence. D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Page 8 of 12 Figure 6 Representation of the HS1.2 variants in 20 different species. The alignment of the HS1.2 enhancer clones of the different species is shown by schematic representation. The conserved elements from the 5’ towards the 3’ are: the core constant part of the enhancer (fuchsia), the 29 mer sequence (red), the 40 mer (yellow), the stretch of cytosines (green), and the 3’ end region (light blue) (see also Figure 2). The number of 40 mer copies appears variable in most of the primates. In these species the alleles with different number of the 40 mer can harbor either the 29 mer or the cytosine stretch between the core and the first 40 mer. Alleles with the 29 mer are present in the major part of the species. We found only clones with the cytosine stretch at the place of the 29 mer just in Gorilla. The identification number (ID) of each sequence is reported in the second column: the ID including a letter can be found in GenBank http://www.ncbi.nlm.nih.gov/Entrez/, the others in the Trace Archive database http://www.ncbi.nlm.nih.gov/Traces/trace.cgi. The bold characters are used to indicate sequences obtained in our laboratory. Figure 6 Representation of the HS1.2 variants in 20 different species. The alignment of the HS1.2 enhancer clones of the different species is shown by schematic representation. The conserved elements from the 5’ towards the 3’ are: the core constant part of the enhancer (fuchsia), the 29 mer sequence (red), the 40 mer (yellow), the stretch of cytosines (green), and the 3’ end region (light blue) (see also Figure 2). The number of 40 mer copies appears variable in most of the primates. In these species the alleles with different number of the 40 mer can harbor either the 29 mer or the cytosine stretch between the core and the first 40 mer. Alleles with the 29 mer are present in the major part of the species. We found only clones with the cytosine stretch at the place of the 29 mer just in Gorilla. Phylogenetic analysis The identification number (ID) of each sequence is reported in the second column: the ID including a letter can be found in GenBank http://www.ncbi.nlm.nih.gov/Entrez/, the others in the Trace Archive database http://www.ncbi.nlm.nih.gov/Traces/trace.cgi. The bold characters are used to indicate sequences obtained in our laboratory. Palindromic sequences flanking the HS1.2 could be involved in rearrangements and translocation effect as in c-myc relocation [31]. An exchange invol- ving the stem may result in the inversion of the loop region, changing the HS1.2 orientation. We can hypothesize that an inversion occurred at least two times since the divergence between Homo sapiens and Mus musculus (as suggested by dot plot analysis, see Figure 5 and above in Results). The inversions limits at least partially spanned the two palindromic regions, sug- gesting a cause/effect relationship. The palindrome could facilitate the inversion event, and the latter could contribute to perpetuate the palindrome. The most relevant result of our analysis was the finding that each HS1.2 enhancer, in all the examined species, is flanked by two 3 kb segments forming a palindromic structure (see Figures 4 and 5). Impressively, while the similarity of each pair of segments is extremely high, the similarity of the palindromic sequences among the dif- ferent species is strikingly low. These findings suggest that the evolutionary pressure was much higher in maintaining the palindromic structure rather than the sequence conservation. As a consequence, it can be con- cluded that the palindrome plays a conformational role in the 3’RR functioning. The fact that the HS1.2 enhan- cer is constantly placed in the middle of the sequence spacing the two inverted elements, further supports the crucial role of the conformation of the region. We hypothesize that the palindrome triggers the formation of a hairpin structure externally exposing the HS1.2 enhancer (Figure 8). The orientation of HS1.2 is there- fore irrelevant for the enhancer function. Indeed, it was found in different orientation in different species and also in different orientation in the two 3’RR human domains. Moreover, the opposite orientations of the two HS1.2 in human add support to the actual formation of the hairpin in vivo. The paired inverted sequences could form the stem of the hairpin. This is a fragile site that HS1.2 polymorphisms l f Population genetics of HS1.2 polymorphisms is available only in humans, for which six distinct variants have been sequenced (AY530201, AY530200, AJ544220, AJ544219, AJ544218 [21]; HM756255, our previously unpublished data). The human variants result from (i) a variable number of the 40 mer and its flanking cytosine- rich box (yellow and green, respectively, in Figure 6); (ii) the sequence connecting the constant core of the HS1.2 (purple in Figure 6) to the stretches of 40 mer repeats, that is constituted by a 18 bp cytosine-rich box or by a 29 mer (green and red, respectively, in Figure 6). HS1.2 polymorphisms have been detected also in 6 out of 8 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Page 9 of 12 Figure 7 Phylogenetic analysis of the C-alpha gene, HS3, HS1.2 and HS4 enhancers. Unrooted phylogenetic trees for C-Alpha (A), HS3 (B), HS1.2 (C) and HS4 (D). Branch length is scaled according to the evolutionary distance, shown as the number of base substitutions per site. The percentage of 100 replicate trees in which the taxa clustered together after the bootstrap analysis is shown at the root of the branches when significant (i.e. when higher than 50%). As examples, the value ‘100’ at the base of the separation between the taxa Mus musculus and Rattus norvegicus in panel D means that the two taxa were clustered together in all the replicate trees, while ‘72’ between Homo sapiens and Gorilla gorilla in the same panel means that 72% of the 100 replicate trees clustered these two groups together. Figure 7 Phylogenetic analysis of the C-alpha gene, HS3, HS1.2 and HS4 enhancers. Unrooted phylogenetic trees for C-Alpha (A), HS3 (B), HS1.2 (C) and HS4 (D). Branch length is scaled according to the evolutionary distance, shown as the number of base substitutions per site. The percentage of 100 replicate trees in which the taxa clustered together after the bootstrap analysis is shown at the root of the branches when significant (i.e. when higher than 50%). As examples, the value ‘100’ at the base of the separation between the taxa Mus musculus and Rattus norvegicus in panel D means that the two taxa were clustered together in all the replicate trees, while ‘72’ between Homo sapiens and Gorilla gorilla in the same panel means that 72% of the 100 replicate trees clustered these two groups together. HS1.2 polymorphisms l f non-human primates for whom this enhancer was iden- tified in genomic databases (Figure 6). We acknowledge that the number of individuals for each species present in these archives is not known, as well as the sequence coverage. We can suppose that very few individuals, maybe a single one, are present in GenBank or Trace Archive. The variants we have detected are, therefore, very likely just the most frequent ones of each species. Nevertheless, it is worth noting that the two Platyrrhini (marmoset and titi) share the same HS1.2 form of the panda. We then hypothesize that this shared form of the enhancer was also at the base of all the Catarrhini variants. has the central position in the palindromic structure, on top of the hairpin (Figure 8). We hypothesize that it can influence the modulation of the Ig switch through an interaction between the extruded enhancer and peptidic factors. The resulting molecular complexes may affect the mobility of the entire 3’RR and finally the formation of loops joining different constant and variable Ig por- tions. It could be interesting to investigate the role of the variants we have found in differently modulating the Ig switch and production in different species, especially in animal models such as macaque and mouse. Database searches for enhancers Database searches for enhancers We searched for clones with the mouse (see previous section) and human version of the Alpha portion, repeat and enhancers (Alpha, GenBank:NC_000014:1060 53245-106054732, 1488 bp; repeat, GenBank: NC_000014:106048991-106049802, 812 bp; HS3, Gen- Bank:NC_000014:106048351-106048676, 326 bp; HS1,2, GenBank:NC_000014:106041545-106042009, 465 bp; HS4, GenBank:NC_000014:106032614-106032974, 361 bp), using the nucleotide version of BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/ Blast.cgi). The asked databases were from the “Others” group (i.e. limiting results to non-human and non- mouse records), further specifying in the Taxonomy field to exclude “Homo (taxid:9605)”. The three checked databases were: refseq_genomic (fully-sequenced gen- ome entries from NCBI’s Reference Sequence project); wgs (whole genome shotgun sequence); htgs (high throughput genomic sequences). To allow a better matching among so divergent species, especially among their 3’end tails, we chose a non-standard set of para- meters: Match/Mismatch Scores 1/-1; Existence/Exten- sion Gap Costs 0/2. Then we chose from the BLAST output the relevant sequences (see Table 1 for the list of species with sequences similar to the human queries) by empirically setting the “Expect” threshold-value to 1e-05. aimed to preserve the 3’RR three-dimensional structure for the conservation of the regulatory function necessary for class switch recombination [12]. Conclusions Figure 8 Structural model of the 3’RR palindrome-driven internal hairpin. This figure shows (A) the linear map of human 3’RR2 and (B) the hairpin loop shape that this sequence can putatively assumes. The whole HS1.2 enhancer (i.e. core, 29 mer, 40 mers and 3’end sequence) is here sketched as orange box. We did not show the HS1.2 orientation in this graphic because it differs between the two copies of the 3’RR in human, and among the 3’RR of the species in Figure 5. The hypothetical hairpin structure is independent from the orientation of HS1.2. Conserved synteny screening We used the following list of mouse sequence tags from [GenBank:NC_000078.5]: a 20 bp tandem repeat (114493451-114493793), an alpha locus portion (114496952-114497890) and seven enhancers, that is HS1.2 (114483000-114483159), HS3.A (114470545- 114470747), HS3.B (114492057-114492261), HS4 (114466607-114466729), and HS5, HS6 and HS7. Those last three were localized by their primers sequences, listed in previous paper [33]. These sequences were aligned by BLAT (BLAST-Like Alignment Tool, http://genome.ucsc.edu/cgi-bin/hgBlat) versus the 2007 release of the Mus musculus (mouse) genome, to identify the limits of the mouse 3’RR contig (chr12:114,459,657-114,497,890 in mmu9 draft; size 38234 bp)(Figure 3). This region was used to investigate the Amniota species of which the sequenced genome was available in the “Comparative Genomics” group of tracks at the UCSC mouse genome browser site http:// genome.ucsc.edu/cgi-bin/hgGateway?db=mm9. So the list of the species we checked was in the order: rat (Rat- tus norvegicus, rn4), Guinea pig (Cavia porcellus, cav- Por3), man (Homo sapiens, hg19), chimpanzee (Pan troglodytes, panTro2), orangutan (Pongo abelii, ponAbe2), macaque (Macaca mulatta, rheMac2), mar- moset (Callithrix jacchus, calJac3), dog (Canis familiaris, canFam2), panda (Ailuropoda melanoleuca, ailMel1), Conclusions IgA and 3’RR have relevance in response to infections and in diseases [3,16-18,20,32]. The more remarkable polymorphism found in 3’RR lies within the HS1.2 that We remark that both coding sequences and wide non- coding regulatory regions have undergone to some evo- lutionary pressure, and that part of this pressure was D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 Page 10 of 12 horse (Equus caballus, equCab2), cow (Bos taurus, bos- Tau4), rabbit (Oryctolagus cuniculus, oryCun2), elephant (Loxodonta africana, loxAfr3), opossum (Monodelphis domestica, monDom5), platypus (Ornithorhynchus ana- tinus, ornAna1), lizard (Anolis carolinensis, anoCar1), chicken (Gallus gallus, galGal3). We reported in par- enthesis the scientific name and the genome draft code. Figure 8 Structural model of the 3’RR palindrome-driven internal hairpin. This figure shows (A) the linear map of human 3’RR2 and (B) the hairpin loop shape that this sequence can putatively assumes. The whole HS1.2 enhancer (i.e. core, 29 mer, 40 mers and 3’end sequence) is here sketched as orange box. We did not show the HS1.2 orientation in this graphic because it differs between the two copies of the 3’RR in human, and among the 3’RR of the species in Figure 5. The hypothetical hairpin structure is independent from the orientation of HS1.2. horse (Equus caballus, equCab2), cow (Bos taurus, bos- Tau4), rabbit (Oryctolagus cuniculus, oryCun2), elephant (Loxodonta africana, loxAfr3), opossum (Monodelphis domestica, monDom5), platypus (Ornithorhynchus ana- tinus, ornAna1), lizard (Anolis carolinensis, anoCar1), chicken (Gallus gallus, galGal3). We reported in par- enthesis the scientific name and the genome draft code. Inspecting the homologous human region in the UCSC human genome browser http://genome.ucsc.edu/ cgi-bin/hgTracks?db=hg18, we searched the tracks related to the Neanderthal genome [27] for the pre- sence of HS3, HS1.2 and HS4. Figure 8 Structural model of the 3’RR palindrome-driven Figure 8 Structural model of the 3’RR palindrome-driven internal hairpin. This figure shows (A) the linear map of human 3’RR2 and (B) the hairpin loop shape that this sequence can putatively assumes. The whole HS1.2 enhancer (i.e. core, 29 mer, 40 mers and 3’end sequence) is here sketched as orange box. We did not show the HS1.2 orientation in this graphic because it differs between the two copies of the 3’RR in human, and among the 3’RR of the species in Figure 5. The hypothetical hairpin structure is independent from the orientation of HS1.2. Database hunting for HS1.2 polymorphisms Database hunting for HS1.2 polymorphisms The search for HS1.2 polymorphisms was refined using the specialized alignment tool Trace-Archive BLAST http://blast.ncbi.nlm.nih.gov/Blast.cgi?BLAST_SPEC=- TraceArchive&BLAST_PROGRAMS=megaBlast&PA- GE_TYPE=BlastSearch. The Trace Archive http://www. ncbi.nlm.nih.gov/Traces/trace.cgi is a repository of sequencing data from gel/capillary platforms, parti- tioned by genome and sequencing methods. There is a daily-growing amount of available reads from thou- sands of WGS projects. We performed a BLAST search on every available database referring to species identified in the previous analysis for the presence of this enhancer and to the 24 primates available in the Archive (Aotus nancymaae, Ateles geoffroyi, Callicebus moloch, Callithrix jacchus, Cercopithecus aethiops, Colobus guereza, Eulemur macaco, Gorilla gorilla, Homo sapiens, Hylobates concolor, Lemur catta, Macaca fuscata, Macaca mulatta, Microcebus murinus, Nomascus leucogenys, Otolemur garnettii, Pan panis- cus, Pan troglodytes, Papio anubis, Papio cynocephalus, Papio hamadryas, Pongo pygmaeus abelii, Saimiri boli- viensis, Tarsius syrichta). The sequences detected were selected for HS1.2 completeness and multi-aligned by use of ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/ index.html [35]) and MAFFT (http://align.bmr.kyushu- u.ac.jp/mafft/software/ [36]) and by use of Seaview http://pbil.univ-lyon1.fr/software/seaview.html to man- ual revise the ClustalW/MAFFT outputs. See Figure 6 for a graphical representation of the detected HS1.2 variants. Transfac analysis for transcription factors The search for the transcription factor consensus was performed on the variant sequences of HS1.2 by the software AliBaba2.1 http://www.gene-regulation.com/ pub/programs/alibaba2/index.html. Additional file 1 lists the transcription factors detected at least in ten loci. The full list can be inspected as Additional file 2. Additional files Note: in the three additional files, the Accessions including at least one letter are picked from GenBank http://www.ncbi.nlm.nih.gov/Entrez/, the others includ- ing just numbers from the Trace Archive database http://www.ncbi.nlm.nih.gov/Traces/trace.cgi. List of nonstandard abbreviations IgH: Immunoglobulin heavy chain; 3’RR: 3’ Regulatory Region; TFBS: Transcription Factor Binding Sites. Additional material Additional file 1: Identified Transcription Factor Binding Sites (listed if found more than 10 time). Additional file 2: Full list of identified Transcription Factor Binding Sites. Additional file 3: List of sequences used for phylogenetics analysis in figure 8. Additional file 1: Identified Transcription Factor Binding Sites (listed if found more than 10 time). Additional file 2: Full list of identified Transcription Factor Binding Sites. Additional file 3: List of sequences used for phylogenetics analysis in figure 8. Additional file 3: List of sequences used for phylogenetics analysis in figure 8. Author details 1 f 1Department of Genetics and Microbiology, University of Bari, Bari, Italy. 2Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada. 3Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada. 4Department of Biology, University of Roma Tor Vergata, Viale della ricerca scientifica. 00133 Roma, Italy. Acknowledgements h k We thank Giovanna Carignani for her suggestions and discussion on the results about evolution, Marcello Brancato, Pasquale Mancini and Arteo Ciancarelli for administration of founds. Part of the work was supported by the MIUR PRIN N.20073RH73W003 (D. Frezza). We are also grateful to Lynell Bates, Francesca Antonacci and Giuliana Giannuzzi for critical reading and English improvement of the manuscript. Authors’ contributions PD’A was the main researcher and performed comparative genomics analysis, database searches, dot plot analysis and data interpretation and participated in manuscript writing and editing. MS carried out phylogenetic analysis, data interpretation, and participated in manuscript editing. VG sequenced the DNA samples, investigated the presence of Transcription Factor Binding Site, and participated in manuscript editing. MR participated in planning the experiment, in data interpretation, and in manuscript writing and editing. DF ideated the work and planned the experiments, and participated in manuscript writing and editing. All authors read and approved the final manuscript. Dot plot analysis p y We identified 2 genomic contigs of Homo sapiens (3’RR1, [GenBank:NC_000014.8:106152458-106175002], and 3’RR2, [GenBank:NC_000014.8:106032614-10605 4732]), and one contig of Mus musculus [GenBank: NC_000078.5:114466607-114497890], Oryctolagus cuni- culus [GenBank:AY386698.1:7160-21816] and Canis lupus familiaris [GenBank:AC187024.23:146314- 165755]. Finally, we assembled a contig of Ailuropoda melanoleuca, sewing the scaffold3005_4 [GenBank: ACTA01092430.1:1574-6589] and the scaffold3005_5 [GenBank:ACTA01100430.1:1-7968] by a stretch of 600 N. We performed a series of pairwise comparisons among these six genomic contigs, by use of two dot plot Page 11 of 12 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 inspected. The best-fitting substitution model was selected using ModelGenerator [38], under the Akaike information criterion (AIC1), as implemented in Multi- Phyl online [39]. The following models were integrated in the phylogenetic analysis: GTR + I + G for C-alpha; HKY + I for HS3 and HS4; HKY + G for HS1.2. analysis softwares: Gepard http://mips.gsf.de/services/ analysis/gepard [34] for the human versus human dot plots (Figure 4), and Blast2seq http://www.ncbi.nlm.nih. gov/blast/bl2seq/wblast2.cgi for the human versus others species dot plots (Figure 5). Graphics were edited using Adobe Illustrator. An unrooted tree was constructed using the maximum likelihood method applied to nucleotides, as implemen- ted in Garli version 0.96 http://www.bio.utexas.edu/ faculty/antisense/garli/Garli.html, with bootstrap percen- tages obtained as a consensus after 100 replicates. 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Multiple alignments of the sequences were obtained with Opal [37] and the results were manually Received: 30 November 2010 Accepted: 15 March 2011 Published: 15 March 2011 Page 12 of 12 Page 12 of 12 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 D’Addabbo et al. BMC Evolutionary Biology 2011, 11:71 http://www.biomedcentral.com/1471-2148/11/71 References 21. Giambra V, Fruscalzo A, Giufre M, Martinez-Labarga C, Favaro M, Rocchi M, Frezza D: Evolution of human IgH3’EC duplicated structures: both enhancers HS1,2 are polymorphic with variation of transcription factor’s consensus sites. Gene 2005, 346:105-114. 1. Stavnezer J, Amemiya CT: Evolution of isotype switching. Semin Immunol 2004, 16(4):257-275. 2. Cognè M, Birshtein BK: Regulation of Class Switch Recombination. In Molecular biology of B cells. Edited by: Honjo T, Alt FW, Neuberger MS. New York: Elsevier; 2004:289-305. 22. 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Lieberson R, Giannini SL, Birshtein BK, Eckhardt LA: An enhancer at the 3’ end of the mouse immunoglobulin heavy chain locus. Nucleic acids research 1991, 19(4):933-937. 25. Meireles-Filho AC, Stark A: Comparative genomics of gene regulation- conservation and divergence of cis-regulatory information. Curr Opin Genet Dev 2009, 19(6):565-570. 6. Chauveau C, Cogne M: Palindromic structure of the IgH 3’locus control region. Nature genetics 1996, 14(1):15-16. 6. Chauveau C, Cogne M: Palindromic structure of the IgH 3’locus control region. Nature genetics 1996, 14(1):15-16. 26. Gambon-Deza F, Sanchez-Espinel C, Magadan-Mompo S: The immunoglobulin heavy chain locus in the platypus (Ornithorhynchus anatinus). Mol Immunol 2009, 46(13):2515-2523. 7. Saleque S, Singh M, Little RD, Giannini SL, Michaelson JS, Birshtein BK: Dyad symmetry within the mouse 3’ IgH regulatory region includes two virtually identical enhancers (C alpha3’E and hs3). Journal of immunology 1997, 158(10):4780-4787. 27. Green RE, Krause J, Briggs AW, Maricic T, Stenzel U, Kircher M, Patterson N, Li H, Zhai W, Fritz MH, et al: A draft sequence of the Neandertal genome. Science 2010, 328(5979):710-722. 8. References BMC Evol Biol 2006, 6:29. 18. Tolusso B, Frezza D, Mattioli C, Fedele AL, Bosello S, Faustini F, Peluso G, Giambra V, Pietrapertosa D, Morelli A, et al: Allele 2 of the HS1,2A enhancer of the Ig regulatory region associates with rheumatoid arthritis. Ann Rheum Dis 2009, 68(3):416-419. 39. Keane TM, Naughton TJ, McInerney JO: MultiPhyl: a high-throughput phylogenomics webserver using distributed computing. Nucleic Acids Res 2007, 35(Web Server):W33-37. doi:10.1186/1471-2148-11-71 Cite this article as: D’Addabbo et al.: Position and sequence conservation in Amniota of polymorphic enhancer HS1.2 within the palindrome of IgH 3’Regulatory Region. BMC Evolutionary Biology 2011 11:71. doi:10.1186/1471-2148-11-71 Cite this article as: D’Addabbo et al.: Position and sequence conservation in Amniota of polymorphic enhancer HS1.2 within the palindrome of IgH 3’Regulatory Region. BMC Evolutionary Biology 2011 11:71. 19. Cianci R, Giambra V, Mattioli C, Esposito M, Cammarota G, Scibilia G, Magazzu G, Orlando A, Sandri G, Bianchi L, et al: Increased frequency of Ig heavy-chain HS1,2-A enhancer *2 allele in dermatitis herpetiformis, plaque psoriasis, and psoriatic arthritis. J Invest Dermatol 2008, 128(8):1920-1924. 20. Frezza D, Giambra V, Cianci R, Fruscalzo A, Giufre M, Cammarota G, Martinez-Labarga C, Rickards O, Scibilia G, Sferlazzas C, et al: Increased frequency of the immunoglobulin enhancer HS1,2 allele 2 in coeliac disease. Scand J Gastroenterol 2004, 39(11):1083-1087.
https://openalex.org/W4390352322	https://journals.stratapersada.com/index.php/slr/article/download/94/39	English	null	Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious Court	Strata Law Review	2,023	cc-by	6,313	Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious Court Fadli Daud Abdullaha, Tajul Arifinb, Abdalc a UIN Sunan Gunung Djati, Indonesia. e-mail: fadli.daud31@gmail.com bUIN Sunan Gunung Djati, Indonesia. e-mail: tajularifin64@uinsgd.ac.id cUIN Sunan Gunung Djati, Indonesia. e-mail: abdal@uinsgd.ac.id Article History journals.stratapersada.com/index.php/slr journals.stratapersada.com/index.php/slr Strata Law Review, 2023, Vol. 1, No. 2, 118–127 E-ISSN: 2986-8114 \| P-ISSN: 2987-3185 \| doi: 10.59631/slr.v1i2.94 Advance Access publication September 22, 2023 © The Author(s) 2023. Published by CV. Strata Persada Academia. This is an Open Access article distributed under the terms of the Creative Common Attribution 4.0 International (CC BY 4.0) Strata Law Review, 2023, Vol. 1, No. 2, 118–127 E-ISSN: 2986-8114 \| P-ISSN: 2987-3185 doi: 10.59631/slr.v1i2.94 Advance Access publication September 22, 2023 journals.stratapersada.com/index.php/slr Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious Court Fadli Daud Abdullaha, Tajul Arifinb, Abdalc a UIN Sunan Gunung Djati, Indonesia. e-mail: fadli.daud31@gmail.com bUIN Sunan Gunung Djati, Indonesia. e-mail: tajularifin64@uinsgd.ac.id cUIN Sunan Gunung Djati, Indonesia. e-mail: abdal@uinsgd.ac.id Article History Submitted Revised Accepted 2023-09-12 2023-09-19 2023-09-22 Abstract This research explores the settlement of Murabahah disputes through sociological and Strata Law Review, 2023, Vol. 1, No. 2, 118–127 E-ISSN: 2986-8114 \| P-ISSN: 2987-3185 doi: 10.59631/slr.v1i2.94 Advance Access publication September 22, 2023 journals.stratapersada.com/index.php/slr Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious Court Fadli Daud Abdullaha, Tajul Arifinb, Abdalc a UIN Sunan Gunung Djati, Indonesia. e-mail: fadli.daud31@gmail.com bUIN Sunan Gunung Djati, Indonesia. e-mail: tajularifin64@uinsgd.ac.id cUIN Sunan Gunung Djati, Indonesia. e-mail: abdal@uinsgd.ac.id Article History Submitted Revised Accepted 2023-09-12 2023-09-19 2023-09-22 Abstract This research explores the settlement of Murabahah disputes through sociological and 1 Azharsyah Ibrahim and Arinal Rahmati, “Analisis Solutif Penyelesaian Pembiayaan Bermasalah di Bank Syariah: Kajian pada Produk Murabahah di Bank Muamalat Indonesia Banda Aceh,” Jurnal Kajian Ekonomi dan Bisnis Islam 10, no. 1 (2017): 75. 2 Mohd Winario, “Masalah Hukum Islam Perspektif Sosiologi Antropologi Hukum,” Jurnal Al Himayah 1, no. 2 (2017): 269. 3 Erna Damayanti, “Aplikasi Murabahah pada Lembaga Keuangan Syariah,” el-Jizya : Jurnal Ekonomi Islam 5, no. 2 (November 1, 2018): 215. 4 Yeni Triana et al., “Wanprestasi Akad Murabahah Nasabah terhadap Bank Syariah,” Journal Of Social Science Research 3, no. 2 (2023): 6. 5 Tajul Arifin, Antropologi Hukum Islam, (Pusat Penelitian dan Penerbitan UIN Sunan Guung Djati Bandung, 2016), 9. Abstract This research explores the settlement of Murabahah disputes through sociological and anthropological studies of Islamic economic law. The focal point of this analysis is Case Number 1/Pdt.G.S/2020/Pa.Cn, which is a lawsuit related to the Murabahah transaction heard at the Cirebon Religious Court. This study utilized qualitative research methods, which were collected through secondary data. The result of this study is that the decision-making process in resolving Murabahah disputes at the Cirebon Religious Court significantly reflects the socio- anthropological dimensions of Islamic economic law. This includes recognizing the social and economic impacts of sharia transactions, particularly the large material losses resulting from defaults. This underscores the importance of Sharia norms, ethical considerations in transactions, and social responsibility in ensuring fairness in the Sharia economy. In addition, it recognizes the challenges faced in reaching solutions within the framework of Sharia economics and emphasizes the importance of collaboration among the parties involved in the process. Ultimately, the implications of this judgment highlight how social and cultural elements in Sharia-compliant societies influence economic transactions and provide a comprehensive understanding of the dynamics of Sharia economic law in a practical context and its implications for case resolution in Religious Courts. Keywords: Murabaha disputes; sharia economic law; sociology anthropology © The Author(s) 2023. Published by CV. Strata Persada Academia. This is an Open Access article distributed under the terms of the Creative Common Attribution 4.0 International (CC BY 4.0) 119 \| Strata Law Review, 2023, Vol. 1, No. 2, 118–127 Doi: 10.59631/slr.v1i2.94 METHOD The methodology used in this research is qualitative research. In this context, qualitative research methods will allow researchers to explore the social, cultural and legal dynamics involved in the enforcement of Murabahah. 6 The author uses secondary data as a source of data collection, namely through literature studies such as analysis of books, journal articles and analysis of documents such as Murabahah contracts and previous court settlements will be done textually. 7 The data will be analyzed using the theoretical approach of sociology and anthropology of Islamic economic law to identify the social, cultural, and economic factors that influence these conditions. Data analysis techniques in this research include tracing or exploring the understanding of the viewpoints of the parties involved in conservation, and presenting the findings in the form of in- depth narratives to provide richer insights into the context of Islamic economic law in concrete cases. Introduction 5 Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious… Fadli Daud Abdullah et al \| 120 Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious… Fadli Daud Abdullah et al \| 120 120 In this context, we are about to explore how social, cultural and economic factors play a role in the interpretation and implementation of Islamic economic principles in the legal system. Murabahah disputes provide an interesting entry point to delve deeper into how Islamic economic law is reflected in court decisions and the extent to which social and cultural factors influence the final outcome of such agreements. By diving into this issue, we can better understand how sharia economic law thinking interacts with social and cultural realities in Cirebon, as well as how these legal interpretations influence court decisions in concrete cases. This analysis will provide a more comprehensive insight into the dynamics of sharia economic law in the context of real practice and its consequences in the settlement of cases in the Religious Courts. Through this multidisciplinary approach, we hope to provide a more in-depth and contextual understanding of the role of sharia economic law in this case, which in turn can make an important contribution to the development of sharia economic law and legal thinking in the future. Rukajat, Ajat. Pendekatan Penelitian Kualitatif Qualitative Research Approach, (Deepublish, 20 Introduction In the era of globalization and modern legal complexity, multidisciplinary analysis is becoming increasingly important in understanding legal phenomena. One area of interest is the convergence between Sociology and Anthropology with Sharia Economic Law. In this context, the analysis of Murabahah settlements in the Religious Courts is a major concern. This raises profound questions about how social and cultural factors influence the interpretation and application of Islamic economic principles in the legal system.1 The Sociology and Anthropology of Islamic Economic Law provides a deep insight into the way people accommodate sharia values in their economic transactions. Through a sociological perspective, we can explore how social norms and cultural values influence the practice of Islamic finance. On the other hand, the anthropological approach opens a window into the structure of social and cultural life that serves as a foundation for economic decision-making that follows sharia principles.2 Murabahah is a financing in Islamic financial institutions, which often causes devastation between banks and customers. Settlement resolution can be through litigation in court. In court, the parties involved present arguments and evidence. The judge then issues a decision based on the applicable law. However, settlements often arise because one party breaches the contract without acceptable reasons, resulting in disagreements.3 Article 49 of Law No. 3 of 2006, which remains unchanged in Law No. 50 of 2009, grants the Religious Courts authority in several aspects. This includes adjudicating marriage, such as divorce, annulment of marriage, and maintaining joint property. The authority also includes resolving inheritance settlements in Islam, waqf issues, polygamy permits, religious education, as well as religious matters such as worship and fatwas. These powers remain in place without significant change. Case Number 1/Pdt.G.S/2020/Pa.Cn which is the object of this analysis is a battle related to Murabahah transactions in the Cirebon Religious Court. 4 In this context, multidisciplinary analysis from the perspective of Sociology and Anthropology of Sharia Economic Law is important. The Sociology perspective allows us to understand how social and cultural norms influence the practice of Islamic finance, while the Anthropology perspective opens a window into the structure of social and cultural life that underlies economic decision-making that follows sharia principles. 6 Anggito, Albi, and Johan Setiawan. Metodologi Penelitian Kualitatif, (CV Jejak Publisher, 2018), 7. 7 Rukajat, Ajat. Pendekatan Penelitian Kualitatif Qualitative Research Approach, (Deepublish, 2018), 21. 6 Anggito, Albi, and Johan Setiawan. Metodologi Penelitian Kualitatif, (CV Jejak Publisher, 2018), 7. , Albi, and Johan Setiawan. Metodologi Penelitian Kualitatif, (CV Jejak Publisher, 2018), 7. Ajat. Pendekatan Penelitian Kualitatif Qualitative Research Approach, (Deepublish, 2018), 21. Decision of the Cirebon City Religious Court Case Number 1/Pdt.G.S/2020/Pa.Cn regarding Murabaha Dispute Decision of the Cirebon City Religious Court Case Number 1/Pdt.G.S/2020/Pa.Cn regarding Murabaha Dispute Decision of the Cirebon City Religious Court Case Number 1/Pdt.G.S/2020/Pa.Cn regarding Murabaha Dispute A court decision is the final stage in the trial process to resolve a particular case. The provisions regarding the court are regulated in Article 57 paragraph (2) of Law Number 48 of 1989, which designs the structure of the head part of the decision, including the name of the court that handed down the decision, the identity of the parties involved in the case, describing the problem, witness testimony, consideration of the judge and the 121 \| Strata Law Review, 2023, Vol. 1, No. 2, 118–127 Doi: 10.59631/slr.v1i2.94 law, the ruling of the decision, the explanation part of the decision, and the part of the decision endorsement in detail.8 law, the ruling of the decision, the explanation part of the decision, and the part of the decision endorsement in detail.8 Case Number 1/Pdt.G.S/2020/PA.CN follows the same structure and content of the decision, including the identity of the parties, the subject matter, and case chronology. This case involved a lawsuit for default on a murabaha financing product filed by PT Bank Mandiri Cirebon Branch through its attorney Tatang Muliawan as the plaintiff, against Minsya Malik as the defendant. This case began when the defendant applied for a financing facility to the plaintiff in January 2012 for the purchase of a house, and the plaintiff approved it.9 After the financing contract was executed, the respondent was obliged to pay the installments, but he failed to do so in accordance with the agreement. The plaintiff had given several warning letters to the defendant, but there was no adequate response. Therefore, the plaintiff decided to bring this matter to the Cirebon Religious Court, which had the desire to resolve it. The plaintiff requested for the court to declare that the defendant had fulfilled the promise in the financing, to order the defendant to pay an obligation of Rp 189,561,570.90, and to confiscate the property used as collateral, namely a plot of land in Villa Casablanca Housing. In addition, the plaintiff also requested that the defendant pay the court costs incurred.10 During the conference process, the defendant was never present, despite being officially summoned. The plaintiffs have provided written evidence to support their claim, including letters, deeds of financing, and other evidence. Decision of the Cirebon City Religious Court Case Number 1/Pdt.G.S/2020/Pa.Cn regarding Murabaha Dispute In addition, the plaintiffs also called two witnesses who provided evidence regarding this case. In the final decision, the court decided to partially grant the plaintiff's claim, stating that the defendant had breached the promise in the murabaha financing contract. The court ordered the defendant to pay the entire obligation of Rp. 189,561,570.90, including principal, margin/profit sharing, and penalty. The court also stated that a security seizure of a piece of land should be made. The costs of the case were borne by the defendant. Thus, the court decision concluded this settlement by establishing the defendant's obligation to pay to the plaintiff in accordance with the terms described in the financing contract. 10 Triana et al., “Wanprestasi Akad Murabahah Nasabah terhadap Bank Syariah,” 8. 9 Triana et al., “Wanprestasi Akad Murabahah Nasabah terhadap Bank Syariah,” 6. 8 Pasal 57 Ayat (2) Undang-Undang Nomor 48 Tahun 1989. Considerations of the Cirebon City Religious Court in Deciding Case Decisions Consideration of the Panel of Judges in Answering the Plaintiff's Request in the Sharia Economic Dispute regarding Default in the Murabahah Agreement. The Panel of Judges considered the request submitted by the Plaintiff in discussing sharia economics related to default in the Murabahah contract. In essence, this resolution occurred in the breach of promise made by the Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious… Fadli Daud Abdullah et al \| 122 122 Defendant in the context of the Al-Murabahah financing contract Number 19 which occurred on February 13, 2012 before Notary Amilia, S.H. in Cirebon City. As a result of this breach of promise, the Plaintiff suffered a material loss of Rp 189,561,570.90.11 Defendant in the context of the Al-Murabahah financing contract Number 19 which occurred on February 13, 2012 before Notary Amilia, S.H. in Cirebon City. As a result of this breach of promise, the Plaintiff suffered a material loss of Rp 189,561,570.90.11 The Judges noted that the Defendant never attended the conference despite being formally invited, so there was no response or defense from the Defendant. Therefore, the Judge assumed that the Defendant indirectly admitted and confirmed all of the claims and demands made by the Plaintiff. The Defendant's confession, which in this case included a mummy confession, became strong evidence based on Article 174 of the Law of Civil Procedure (HIR).12 In addition, the Panel of Judges also considered the validity of the Al-Murabahah financing contract that was the basis for the rescue. They stated that the contract fulfills these conditions and the pillars of the contract based on various legal considerations, including Article 22 of the Compilation of Sharia Economic Law which regulates the pillars of the contract.13 This contract is an agreement between PT Bank Syariah Mandiri Cirebon Branch as the Plaintiff and Minsya Malik as the Defendant. The agreement letter has been signed by both parties, indicating their understanding and acceptance of all contents as well as obligations and rights arising from the contract.14 In addition, based on Articles 44 and 46 of the Compilation of Sharia Economic Law, a valid contract is valid in accordance with sharia principles for the parties that enter into it. This confirms that the Al-Murabahah financing contract No. Considerations of the Cirebon City Religious Court in Deciding Case Decisions 19 dated February 13, 2012 made by the Plaintiff and the Defendant must be considered valid.15 Finally, the Panel of Judges also considered the claim for compensation submitted by the Plaintiff. Based on legal considerations in accordance with Article 36 of the Compilation of Sharia Economic Law and Article 1238 of the Civil Code, the Judge concluded that the Defendant had broken its promise (default) and this had caused material losses to the Plaintiff. Therefore, the Judge decided to punish the Defendant by paying material losses of Rp. 189,561,570.90 in accordance with the Plaintiff's demands. In the overall analysis and discussion, the Panel of Judges held that the Defendant had reneged on its promise in the Al-Murabahah financing contract and that the contract was valid under sharia law. In addition, the claim for damages filed by the Plaintiff was also accepted. Thus, the Judge's decision was in favor of the Plaintiff in this settlement, and the Defendant was ordered to pay material damages in the amount determined. p y 14 Triana et al., “Wanprestasi Akad Murabahah Nasabah Terhadap Bank Syariah,” 6. p 15 Pasal 44 Dan 46 Kompilasi Hukum Ekonomi Syariah. ( ) 13 Pasal 22 Kompilasi Hukum Ekonomi Syariah. 11 Triana et al., “Wanprestasi Akad Murabahah Nasabah terhadap Bank Syariah,” 6. 11 Triana et al., “Wanprestasi Akad Murabahah Nasabah terhadap Bank Syariah,” 6. 12 Pasal 174 Hukum Acara Perdata (HIR). 1 Triana et al., “Wanprestasi Akad Murabahah Nasabah terhadap Bank Syariah,” 6. 2 Pasal 174 Hukum Acara Perdata (HIR) p 12 Pasal 174 Hukum Acara Perdata (HIR). , p p y , 12 Pasal 174 Hukum Acara Perdata (HIR). ( ) 3 Pasal 22 Kompilasi Hukum Ekonomi Syariah. p y 14 Triana et al., “Wanprestasi Akad Murabahah Nasabah T p 12 Pasal 174 Hukum Acara Perdata (HIR). Analysis of Sociology and Anthropology of Sharia Economic Law on Murabahah Dispute Decisions in the Cirebon City Religious Court Sociology is the scientific study of human society, including interactions, social structures, institutions, and social dynamics. It focuses on understanding human social patterns and behaviors in a variety of contexts, such as urban, rural, communities, families, and 123 \| Strata Law Review, 2023, Vol. 1, No. 2, 118–127 Doi: 10.59631/slr.v1i2.94 organizations.16 Sociology uses scientific methods such as surveys, interviews, statistical analysis, and experiments to collect data that describe and explain social phenomena. The goal is to identify social trends, conflicts, changes, as well as factors that affect society and individuals within society.17 organizations.16 Sociology uses scientific methods such as surveys, interviews, statistical analysis, and experiments to collect data that describe and explain social phenomena. The goal is to identify social trends, conflicts, changes, as well as factors that affect society and individuals within society.17 Anthropology is a branch of social science that emerged from Europeans' interest in the physical, customary and cultural differences in their societies. Anthropology mainly focuses on people living in one geographical area, while sociology focuses more on patterns of social interaction and community life. In essence, anthropology and sociology are similar, but their focus is different in understanding and researching people and societies.18 ociology and anthropology are two social science disciplines that focus on understanding society, culture, and human behavior, albeit with different approaches. Sociology is the scientific study of human society, observing social interactions, structures and dynamics. Anthropology, meanwhile, focuses on human cultures around the world, exploring the differences and similarities in how people live and interact with the environment.19 Both use different research methods, such as surveys and ethnography, and have different goals in explaining culture and society. Despite their different focuses, they work together to answer basic questions about people and society.20 The Sociology and Anthropology of Islamic Economic Law provides a deep understanding of how societies integrate sharia values in their economic transactions.21 From a sociological perspective, we can investigate the impact of social norms and cultural values on the implementation of Islamic financial practices. Meanwhile, the anthropological approach opens up a view of the social and cultural structures that form the basis for economic decision-making that adheres to sharia principles. 22 In the sociological context of sharia economic law, the decision of the Cirebon Religious Court Case Number 1/Pdt.G.S/2020/PA.CN on murabaha exemption reflects how sharia law and values influence and push into the judicial system. 16 Janu Murdiyatmoko. Sosiologi: Memahami Dan Mengkaji Masyarakat, (PT Grafindo Media Pratama, 2007), 6. 17 Sunarto, Kamanto. Pengantar Sosiologi, (Universitas Indonesia Publishing, 2005), 3-12. 18 Tajul Arifin, “Antropologi Hukum Islam,” 1. 19 Damien Short. Sociological and Anthropological Approaches, (Human Rights: Politics and Practice, 2009), 93. 20 Delamont, Sara, and Paul Atkinson. "The Two Traditions in Educational Ethnography: Sociology and Anthropology Compared." British Journal of Sociology of Education 1. 2 (1980): 141. 21 Henrika Kuklick, "Assessing Research in the History of Sociology and Anthropology." Journal of the History of the Behavioral Sciences 35. 3 (1999): 230. 22 Winario, “Masalah Hukum Islam Perspektif Sosiologi Antropologi Hukum,” 269. y f ( ) 2 Winario, “Masalah Hukum Islam Perspektif Sosiologi Antropologi Hukum,” 269. 6 Janu Murdiyatmoko. Sosiologi: Memahami Dan Mengkaji Masyarakat, (PT Grafindo Media 6 p gy p J f gy f ( ) Henrika Kuklick, "Assessing Research in the History of Sociology and Anthropology." J tory of the Behavioral Sciences 35. 3 (1999): 230. 17 Sunarto, Kamanto. Pengantar Sosiologi, (Universitas Indonesia Publishing, 2005), 3-12. Analysis of Sociology and Anthropology of Sharia Economic Law on Murabahah Dispute Decisions in the Cirebon City Religious Court In this context, several social and legal aspects emerge: The decision shows an effort to integrate sharia economic principles into the judicial system. The provisions stipulated in Article 57 paragraph (2) of Law No. 48 of 1989 serve as guidelines for judges in drafting decisions that are in accordance with sharia principles. Article 57 paragraph (2) provides a foundation for judges to consider the values, norms and principles of sharia economics when they have to make decisions in cases relating to the economy. This includes Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious… Fadli Daud Abdullah et al \| 124 124 Fadli Daud Abdullah et al \| matters such as the prohibition of usury, the principle of fairness in business transactions, and other aspects that are in accordance with the principles of sharia economics. Thus, Article 57 paragraph (2) becomes an important tool in bridging between positive law and sharia economic principles. This creates a legal framework that is more in line with sharia economic values in the context of economic law in Indonesia. In a sociological analysis, we can understand how the application of this article affects the actions and decisions of judges and its impact on the development of sharia economics in society. This reflects how religious and ethical aspects play a role in the judicial process.23 The ruling underscores the need to protect the rights of parties who are aggrieved by defaults in murabaha contracts. The court ensured that the aggrieved party was given justice, which is a fundamental principle in Islamic economic law that emphasizes social justice.24 This case involves PT Bank Syariah Mandiri Cirebon Branch, an Islamic financial institution. It reflects how Islamic financial institutions play a role in facilitating economic transactions that comply with sharia principles. These institutions play an important role in encouraging usury- free financial practices.25 This case involves PT Bank Syariah Mandiri Cirebon Branch, an Islamic financial institution. It reflects how Islamic financial institutions play a role in facilitating economic transactions that comply with sharia principles. These institutions play an important role in encouraging usury- free financial practices.25 The absence of the defendant in the judicial process reflects the challenges in resolving Islamic economic rescues. The presence of the disputing party is an important part of the judicial process, and absence can affect the legal proceedings. ( ) 28 Muhammad Ilham and M Taufiq, “Penyelesaian Sengketa dalam Perspektif Antropologi Hukum,” Jurnal Teraju 3, no. 01 (Maret 14, 2021): 19. 27 Uswatun Khasanah, “Perkembangan dan Penerimaan Masyarakat terhadap Perbankan Syariah di Indonesia,” AL-MUTSLA 2, no. 2 (Desember 30, 2020): 150. 23 Erie Hariyanto, “Integrasi Mediasi Sebagai Model Penyelesaian Sengketa Perbankan Syari’ah Masa Depan,” Jurnal Proceedings of Annual Conference for Muslim Scholars 1, no. 110 (2017): 1. ( j p ) ( ) 26 Ahmad Mujahidin, Prosedur Penyelesaian Sengketa Ekonomi Syariah di Indonesia, (Ghalia Indonesia: Bogor, 2010), 61. 23 Erie Hariyanto, “Integrasi Mediasi Sebagai Model Penyelesaian Sengketa Perbankan Syari’ah Masa Depan,” Jurnal Proceedings of Annual Conference for Muslim Scholars 1, no. 110 (2017): 1. 24 Puteri Asyifa Octavia Apandy, Melawati, and Panji Adam, “Pentingnya Hukum Perlindungan Konsumen dalam Jual Beli,” Jurnal Manajemen & Bisnis Jayakarta 3, no. 1 (Juli 25, 2021): 14. 25 Ayief Fathurrahman, “Prospek Pengembangan Ilmu Ekonomi Islam di Indonesia dalam Prespektif Filsafat Ilmu (Sebuah Kajian Epistemik),” Jurnal La Riba 4, no. 2 (Desember 31, 2010): 314. 26 Ahmad Mujahidin, Prosedur Penyelesaian Sengketa Ekonomi Syariah di Indonesia, (Ghalia Indonesia: Bogor, 2010), 61. 27 Uswatun Khasanah, “Perkembangan dan Penerimaan Masyarakat terhadap Perbankan Syariah di Indonesia,” AL-MUTSLA 2, no. 2 (Desember 30, 2020): 150. 28 Muhammad Ilham and M Taufiq, “Penyelesaian Sengketa dalam Perspektif Antropologi Hukum,” Jurnal Teraju 3, no. 01 (Maret 14, 2021): 19. g f f f ( ) Asyifa Octavia Apandy, Melawati, and Panji Adam, “Pentingnya Hukum Perlind n dalam Jual Beli,” Jurnal Manajemen & Bisnis Jayakarta 3, no. 1 (Juli 25, 2021): 14. 23 Erie Hariyanto, “Integrasi Mediasi Sebagai Model Penyelesaian Sengketa Perbankan Syari’ah Masa Depan,” Jurnal Proceedings of Annual Conference for Muslim Scholars 1, no. 110 (2017): 1. 24 Puteri Asyifa Octavia Apandy, Melawati, and Panji Adam, “Pentingnya Hukum Perlindungan 5 Ayief Fathurrahman, “Prospek Pengembangan Ilmu Ekonomi Islam di Indonesia da Filsafat Ilmu (Sebuah Kajian Epistemik),” Jurnal La Riba 4, no. 2 (Desember 31, 2010): 314. 23 Erie Hariyanto, “Integrasi Mediasi Sebagai Model Penyelesaian Sengketa Perbankan Syari’ah Masa Depan,” Jurnal Proceedings of Annual Conference for Muslim Scholars 1, no. 110 (2017): 1. 24 Puteri Asyifa Octavia Apandy, Melawati, and Panji Adam, “Pentingnya Hukum Perlindungan Konsumen dalam Jual Beli,” Jurnal Manajemen & Bisnis Jayakarta 3, no. 1 (Juli 25, 2021): 14. 25 Ayief Fathurrahman, “Prospek Pengembangan Ilmu Ekonomi Islam di Indonesia dalam Prespektif Filsafat Ilmu (Sebuah Kajian Epistemik),” Jurnal La Riba 4, no. 2 (Desember 31, 2010): 314. 26 Ahmad Mujahidin, Prosedur Penyelesaian Sengketa Ekonomi Syariah di Indonesia, (Ghalia Indonesia: Bogor 2010) 61 ef Fathurrahman, Prospek Pengembangan Ilmu Ekonomi Islam di Indonesia dalam P t Ilmu (Sebuah Kajian Epistemik),” Jurnal La Riba 4, no. 2 (Desember 31, 2010): 314. 24 Puteri Asyifa Octavia Apandy, Melawati, and Panji Adam, Pentingnya Hukum Perlindungan Konsumen dalam Jual Beli,” Jurnal Manajemen & Bisnis Jayakarta 3, no. 1 (Juli 25, 2021): 14. 25 Ayief Fathurrahman, “Prospek Pengembangan Ilmu Ekonomi Islam di Indonesia dalam Prespektif Filsafat Ilmu (Sebuah Kajian Epistemik),” Jurnal La Riba 4, no. 2 (Desember 31, 2010): 314. Analysis of Sociology and Anthropology of Sharia Economic Law on Murabahah Dispute Decisions in the Cirebon City Religious Court This represents the importance of the cooperation of the parties involved in finalizing the settlement.26 These rulings reflect how the public can understand and accept the use of sharia economic law in resolving economic settlements. Courts play an important role in educating the public about the importance of complying with sharia principles in economic transactions.27 In the decision of the Cirebon Religious Court Case Number 1/Pdt.G.S/2020/PA.CN regarding the enforcement of murabaha, there are significant anthropological aspects of sharia economic law. This decision reflects the complex interaction between law, economics, and social values in a society that adheres to sharia principles.28 In the view of legal anthropology, conflict has two meanings, namely negative and positive. The negative meaning is that conflict can damage social relations and community cohesion. Meanwhile, the positive meaning is that conflict can strengthen social ties and restore balance 125 \| Strata Law Review, 2023, Vol. 1, No. 2, 118–127 Doi: 10.59631/slr.v1i2.94 between individuals or groups. 29 To achieve the positive meaning, the parties involved in the conflict need to manage it maturely and peacefully, with or without a third party. between individuals or groups. 29 To achieve the positive meaning, the parties involved in the conflict need to manage it maturely and peacefully, with or without a third party. Legal anthropology shows that law is not only limited to written rules or customs, but also involves cultural elements in the resolution of a case. Although courts are a common way to seek justice, mediation is also an important alternative to resolve conflicts.30 Analyzed from the anthropological perspective of Islamic economic law, this decision reflects how Islamic economics, particularly in the context of murabaha contracts, can have a significant social and economic impact on individuals and communities. The default in this transaction resulted in a material loss of Rp. 189,561,570.90 for the plaintiff, which illustrates how serious the economic impact of breaches in the context of Islamic economics can be.31 When viewed from the aspect of sharia norms and ethics, this decision highlights the influence of sharia norms and ethics in regulating economic transactions. The court confirmed that the murabaha financing contract met the requirements and principles of sharia. This reflects the importance of ethical values and adherence to religious principles in doing business and transactions in an Islamic society. p g ( p ) 0 Ilham and Taufiq, “Penyelesaian Sengketa dalam Perspektif Antropologi Hukum,” 21. Abdullah Taib, Asas-Asas Antropologi (Kuala Lumpur: Dewan Bahasa dan Pustaka, 1985), 1. q, y g p p g , 1 Sahlan, The Other Laws Di Era Otonomi Daerah, (Studi Antropologi Hukum), 153. 33 Rina Trisnawati, “Pengukuran Tanggung Jawab Sosial Perusahaan Perbankan Syariah di Indonesia,” Jurnal Akuntansi dan Auditing Indonesia 16, no. 2 (2012): 103. J g ( ) 34 Dewi Sukma Kristianti, “Integrasi Prinsip Syariah dalam Fungsi Intermediasi Lembaga Keuangan Syariah,” Undang: Jurnal Hukum 3, no. 2 (Desember 1, 2020): 318. 32 Achmad Fikri Oslami, “Kedudukan Pengadilan Agama dan Basyarnas dalam Meny Ekonomi Syariah,” AT-TASYRI’: Jurnal Ilmiah Prodi Muamalah, (Juni 26, 2022): 30. ( p g ) 32 Achmad Fikri Oslami, “Kedudukan Pengadilan Agama dan Basyarnas dalam Menyelesaikan Sengketa Ekonomi Syariah,” AT-TASYRI’: Jurnal Ilmiah Prodi Muamalah, (Juni 26, 2022): 30. CONCLUSION The conclusions drawn from the case analysis are as follows: First, the Cirebon City Religious Court ruled that the defendant had breached the Murabahah financing contract and ordered them to pay the outstanding obligations, totaling Rp. 189,561,570.90, including the principal, profit- sharing margin, and penalties. The court also ordered the confiscation of the collateral property, and the defendant was responsible for the case costs. This decision resolved the case by enforcing the defendant's obligation to fulfill the terms of the financing contract. Secondly, the panel of judges, in their decision, carefully considered the plaintiff's claims regarding the Murabahah Agreement and found the defendant in default. They confirmed the validity of the contract under Sharia economic law and accepted the plaintiff's claim for damages. Consequently, the court ruled in favor of the plaintiff, ordering the defendant to pay the specified amount in material damages. g Overall, the Cirebon Religious Court's decision in resolving the Murabahah dispute highlights the significant influence of sociological and anthropological aspects in Islamic economic law within a society adhering to Sharia principles. It emphasizes the intricate interplay between law, cultural values, and social dynamics in Sharia economic case resolutions. Furthermore, it underscores the integration of Islamic financial institutions into the economy and underscores the importance of social responsibility in Islamic economic transactions. This conclusion underscores the complexity and relevance of sociology and anthropology in Sharia economic law within Indonesia's economic legal context. ACKNOWLEDGMENTS We sincerely express our deepest gratitude for the extraordinary guidance and support from Prof. Dr. Tajul Arifin, M.A. and Dr. H. Abdal, M.Si in our research article in the field of Anthropology of Sharia Economic Law. The presence and guidance of both of them has helped us move further in understanding and developing this research. We also want to express our deepest gratitude to our parents who always prayed for us and provided moral support throughout our journey. Sincere prayers from parents are our invaluable source of strength in facing every obstacle and struggle in this research. Thank you again, Prof. Dr. Tajul Arifin, M.A., Dr. H. Abdal, M.Si, and our parents, for this extraordinary help and support. Hopefully the results of this research can be a useful contribution to science and society. Analysis of Sociology and Anthropology of Sharia Economic Law on Murabahah Dispute Decisions in the Cirebon City Religious Court In terms of social responsibility in economic transactions, this decision shows the importance of social responsibility in Islamic economic transactions.32 Although the defendant was not present in the judicial process, the court ensured that the plaintiff's rights were protected and the claim for compensation was in accordance with sharia law. This reflects social responsibility in ensuring fairness in economic transactions.33 In the Integration of Islamic Financial Institutions, this case involves PT Bank Syariah Mandiri Cirebon Branch, which is an Islamic financial institution. This reflects the integration of Islamic financial institutions in the economy of a society that adheres to sharia principles. These institutions play a role in providing financing assistance in accordance with sharia principles.34 The absence of the defendant in the judicial process reflects the challenges in resolving Islamic economic settlements. The court has to work on the assumption that the defendant recognizes the plaintiff's claim, which highlights the importance of collaboration of the parties involved in resolving Islamic economic settlements. Overall, the decision of the Cirebon Religious Court in the murabahah settlement reflects how anthropological aspects of economic law affect social and economic dynamics in a society that adheres to sharia principles. By ensuring the fulfillment of ethical values and religious principles in economic transactions, it participates in maintaining justice and integrity in the context of Islamic economics. Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious… Fadli Daud Abdullah et al \| 126 Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious… Fadli Daud Abdullah et al \| 126 Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious… Fadli Daud Abdullah et al \| 126 Analysis of Sociology and Anthropology of Sharia Economic Law On Murabahah Dispute Settlement at The Cirebon Religious… Fadli Daud Abdullah et al \| 126 126 ) lbi, and Johan Setiawan. Metodologi Penelitian Kualitatif, (CV Jejak Publisher, 2018). Apandy, Puteri Asyifa Octavia, Melawati, and Panji Adam. “Pentingnya Hukum Perlindungan Konsumen dalam Jual Beli.” Jurnal Manajemen & Bisnis Jayakarta 3, no. 1 (Juli 25, 2021): 12–18. Abdullah, Taib. Asas-Asas Antropologi, (Kuala Lumpur: Dewan Bahasa dan Pustaka, 1985). 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https://openalex.org/W2108633424	https://hal.science/hal-01058127/document	English	null	Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome	Oxidative medicine and cellular longevity	2,014	cc-by	8,187	Research Article Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome Cinzia Signorini,1 Silvia Leoncini,1,2 Claudio De Felice,3 Alessandra Pecorelli,1,2 Ilaria Meloni,4 Francesca Ariani,4 Francesca Mari,4 Sonia Amabile,4 Eugenio Paccagnini,5 Mariangela Gentile,5 Giuseppe Belmonte,6 Gloria Zollo,1,2 Giuseppe Valacchi,7 Thierry Durand,8 Jean-Marie Galano,8 Lucia Ciccoli,1 Alessandra Renieri,4,9 and Joussef Hayek2 1 Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy y f y 5 Department of Life Sciences, University of Siena, 53100 Siena, Italy 6Department of Medicine Surgery and Neuroscience, University of Siena, 53100 Siena, Italy 6Department of Medicine Surgery and Neuroscience, University of Siena, 53100 Siena, Italy 7 Department of Life Science and Biotechnologies, University of Ferrara, 44121 Ferrara, Italy 8 7 Department of Life Science and Biotechnologies, University of Ferrara, 44121 Ferrara, Italy 8 8 Institut des Biomol´ecules Max Mousseron (IBMM), UMR 5247-CNRS-UM I-UM II-ENSCM, BP 14491 34093, Montpellier Cedex 5, France p Genetica Medica, Azienda Ospedaliera Universitaria Senese, 53100 Siena, Italy p 9 Genetica Medica, Azienda Ospedaliera Universitaria Senese, 53100 Siena, Italy Correspondence should be addressed to Cinzia Signorini; cinzia.signorini@unisi.it and Claudio De Felice; geniente@gmail.com Correspondence should be addressed to Cinzia Signorini; cinzia.signorini@unisi.it and Claudio De Felice; gen Correspondence should be addressed to Cinzia Signorini; cinzia.signorini@unisi.it and Claudio De Felice; geniente@gmail.com Received 10 March 2014; Revised 9 May 2014; Accepted 12 May 2014; Published 29 May 2014 Academic Editor: Ozcan Erel Academic Editor: Ozcan Erel Copyright © 2014 Cinzia Signorini 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. Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (−43.6%) and GSH/GSSG ratio (−3.05 folds). Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2014, Article ID 195935, 10 pages http://dx.doi.org/10.1155/2014/195935 Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2014, Article ID 195935, 10 pages http://dx.doi.org/10.1155/2014/195935 Research Article Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients. 1. Introduction determines the clinical severity of the disease is a typical feature of RTT [4], key clinical aspects include autistic traits, epileptic seizures, breathing abnormalities, gait ataxia, stereotypies, and loss of finalistic hands use [3]. Mutations in the gene encoding the Methyl-CpG-binding protein 2 (MECP2) account for approximately 90% of cases with typical RTT and are almost exclusively de novo [4]. Nine most Rett syndrome (RTT) predominantly affects females with an incidence of 1 in 10,000–15,000 female births [1, 2]. In its typical form, affected patients exhibit various neuropsychi- atric features after 6–18 months [3] of apparently normal neu- rodevelopment. Although phenotypical heterogeneity which Oxidative Medicine and Cellular Longevity 2 frequent mutations comprise more than 3/4 of all the reported pathogenic ones [5]; in addition, MECP2 gene muta- tions are usually categorized as missense or truncating, including nonsense, frameshift, and large deletions, accord- ing to the type of sequence change. isolated and cultured with standard protocols [28]. Cells were grown in DMEM (Biochrom), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 𝜇g/mL streptomycin, and 2 mM L-glutamine. Cells were incubated at 37∘C with 5% CO2 until 80–90% confluence and routinely passed by trypsin-EDTA (Irvine Scientific). Oxidative stress (OS) indicates a combination of events resulting in a damage to biological molecules due to an imbalance between cellular antioxidant defences and free radicals production. OS appears to be involved in a large number of human diseases, including cancer [6–8] neurode- generative diseases [9, 10] and inflammatory bowel disease [11]. Furthermore, cumulating evidence suggests the presence of a redox imbalance in autism spectrum disorders (ASDs), a condition with a high social impact due to the explosive increase in its prevalence over the last four decades [12–18]. Our group and other laboratories have reported enhanced OS markers levels in plasma and erythrocytes from patients with RTT, thus suggesting the presence of a systemic OS in the disease. However, to date, it is unclear not only why, but also when, and where this OS derangement may occur [19–26]. i Cells for analysis were seeded onto 100 mm tissue culture plates (for NPBI and IsoP evaluations) and 12-well plates (for 4-HNE PAs and GSH/GSSG ratio) containing the indi- cated medium formulations. Fibroblasts at low passage were employed for the analysis.l At confluence, the cells were scraped, transferred in a tube, and washed twice with ice-cold PBS pH 7.4. 1. Introduction Cells were then centrifuged at 600 g for 10 min and the pellet resuspended in a final volume of 2 mL PBS. 2.3. Nonprotein Bound Iron (NPBI). Nonprotein bound iron was determined as desferoxamine- (DFO-) chelatable free iron (DFO-iron complex, ferrioxamine). DFO 25 𝜇M was added to 1 mL of cell suspension to obtain a final concen- tration of 25 𝜇M DFO and the cells were ruptured by the addition of 1 mL water, freeze-thawing, and sonication. The samples were then ultrafiltered in centrifuge filter devices (VIVASPIN 4, Sartorius Stedim Biotech GmbH, Goettingen, Germany) with a 30 kDa molecular weight cutoff and the ultrafiltrate was stored at −20∘C until analysis. The DFO excess was removed by silica (Silicagel 25–40 𝜇m; Merck, Darmstadt, Germany) column chromatography (Varian Inc., CA, USA). The DFO-iron complex was determined by HPLC and the detection wavelength was 229 nm [29]. g y In particular to date, little is known regarding the oxidant- antioxidant status in RTT cellular systems and its possible relationship with the morphological phenotype. Here, we investigated the levels of different OS markers (F2-IsoPs, F4- NeuroPs, NPBI, and 4-HNE PAs) and glutathione in primary skin fibroblasts from patients with typical RTT, as well as possible changes in the cellular/intracellular structure and/or qualitative changes in the synthesized collagen. 2. Materials and Methods 2.1. Subjects. A total of 16 female patients with typical RTT (mean age: 13.06 ± 6.5), as well as 8 healthy female controls of comparable age (mean age: 13.2 ± 6.8), participated in the study. Skin biopsies from the control group were obtained from a skin biobank by selecting subjects without diagnosed skin or collagen diseases (responsibility: Joussef Hayek). 2.4. Total (Sum of Free Plus Esterified) F2-Isoprostanes and F4- Neuroprostanes Determinations. Butylated hydroxytoluene was added to 900 𝜇L of cell suspension as an antioxidant to obtain a final concentration of 90 𝜇M and the samples were frozen at −70∘C until analysis. All isoprostane and neuroprostane determinations were carried out by gas chro- matography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) analysis after solid phase extraction and derivatization steps. F2-Isoprostanes and F4- neuroprostanes levels were normalized for the cell protein content. g p y y All patients were consecutively admitted to the Child Neuropsychiatry Unit of the University Hospital of Siena (Head: Joussef Hayek). For all patients the MECP2 mutation was demonstrated (mutation categories: missense mutation 𝑛= 8; early truncating mutation 𝑛= 6; late truncating mutation 𝑛= 2), and a clinical severity score was calculated using the previously reported scaling system [27] (mean severity score: 15.9 ± 6.47; range: 5.0–28.0). All the examined subjects were on a typical Mediterranean diet.t 2.5. Solid Phase Extraction and Derivatization Procedures. At the time of the determination, the samples were sonicated by ultrasound treatment for 30 seconds and then purified as pre- viously reported [29]. Briefly, aqueous KOH (1 mM, 450 𝜇L) was added to the cellular suspension. After incubation at 45∘C for 45 min, the pH was adjusted to 3 by adding HCl (1 mM, 450 𝜇L). Each sample was spiked with tetradeuterated prostaglandin F2𝛼(PGF2𝛼-d4) (500 pg in 50 𝜇L of ethanol), as an internal standard, and ethyl acetate (10 mL) was added to extract total lipids by vortex-mixing and centrifugation at 1,000 g for 5 min at room temperature. The total lipid extract was applied onto an NH2 cartridge and the isoprostanes in the final eluates were derivatized. The carboxylic group was derivatized as the pentafluorobenzil ester whereas the hydroxyl groups were converted to trimethylsilyl ethers [30]. 2.5. Solid Phase Extraction and Derivatization Procedures. At the time of the determination, the samples were sonicated by ultrasound treatment for 30 seconds and then purified as pre- viously reported [29]. 2. Materials and Methods After fixation and permeabilization for 10 min at room temperature with 0.1% Triton X-100, cells were blocked for 30 min at room temperature with PBS containing 5% BSA. Fibroblasts were then incubated with primary antibodies for collagen I and collagen III (Thermo Fisher Scientific Inc., Rockford, IL, USA) in PBS with 1% BSA at 4∘C overnight. After washing, cells were incubated with secondary antibodies Alexa Fluor 568 and Alexa Fluor 488 (Life Technologies Corporation, Monza, Italy) for 1 h at room temperature. Nuclei were stained with 1 𝜇g/mL DAPI (Sigma-Aldrich S.r.l., Milan, Italy) for 1 min. Coverslips were mounted onto glass slides using antifade mounting medium 1,4-diazabicyclooctane in glycerine (DABCO). Observations and photographs were made with a Leitz Aristoplan light microscope (Leica, Milan, Italy) equipped with fluorescence apparatus. Incubation in primary antibodies was omitted in negative controls.hl 2.8. Intracellular Redox Status. Usually, cellular glutathione (GSH) exists mainly in the reduced form whereas the oxi- dized disulfide form (GSSG) is present in small amounts. The GSH/GSSG ratio is often taken as an indicator of cellular redox status. Intracellular GSH and GSSG levels were determined by an enzymatic recycling procedure according to Tietze [32] and Baker et al. [33]. The SH group of the molecule reacts with 5,5󸀠-dithiobis(2-nitrobenzoic acid) (DTNB), producing a yellow-coloured 5-thio-2-nitrobenzoic acid (TNB), and the disulfide is reduced by NADPH in the presence of GSH reductase. GSSG was determined after derivatization step of GSH by reaction with 2-vinylpyridine. The rate of TNB formation was monitored at 420 nm. GSH and GSSG levels were normalized for protein content.lti At confluence, after medium removal, cultured fibroblasts were washed twice with PBS pH 7.4 and treated with 5% 5- sulfosalicylic acid (w/v) solution for 30 min at 4∘C. The acidic extracts were stored at −70∘C until the assay. The protein extracts were obtained by cellular lysis with NaOH 0.1 M. g The relative intensity of fluorescence was measured in regions of interest (ROI) by using the Software LEICA AF6000 (Leica Microsystems-Germany). 2.9. 4-HNE Protein Adducts. 4-HNE PAs are markers of protein oxidation due to aldehyde binding from lipid perox- idation sources [34], determined by Western blot technique. Cell proteins (30 𝜇g protein, as determined by using Bio-Rad protein assay; BioRad, Hercules, CA, USA) were resolved on 4–20% SDS-PAGE gels (Lonza Group Ltd., Switzerland) and transferred onto a hybond ECL nitrocellulose membrane (GE Healthcare Europe GmbH, Milan, Italy). 2. Materials and Methods Briefly, aqueous KOH (1 mM, 450 𝜇L) was added to the cellular suspension. After incubation at 45∘C for 45 min, the pH was adjusted to 3 by adding HCl (1 mM, 450 𝜇L). Each sample was spiked with tetradeuterated prostaglandin F2𝛼(PGF2𝛼-d4) (500 pg in 50 𝜇L of ethanol), as an internal standard, and ethyl acetate (10 mL) was added to extract total lipids by vortex-mixing and centrifugation at 1,000 g for 5 min at room temperature. The total lipid extract was applied onto an NH2 cartridge and the isoprostanes in the final eluates were derivatized. The carboxylic group was derivatized as the pentafluorobenzil ester whereas the hydroxyl groups were converted to trimethylsilyl ethers [30]. j y A 3 mm skin punch biopsy was performed after obtained written informed consent of either the parents or the legal tutors of the patients (responsibility: Joussef Hayek). Biopsies from control subject were performed in the Dermatology Unit of the University Hospital of Siena. The study was con- ducted with the approval of the Institutional Review Board of the University Hospital Azienda Ospedaliera Universitaria Senese. 2.2. Fibroblasts Isolation and Culture. Following informed consent signature, skin biopsies (about 3-4 mm3) were per- formed using the Punch Biopsy procedure. Fibroblasts were 3 Oxidative Medicine and Cellular Longevity 2.6. F2-Isoprostane GC/NICI-MS/MS. Measured ions were the product ions at m/z 299 and m/z 303 derived from the [M−181]−precursor ions (m/z 569 and m/z 573) pro- duced from 15-F2t-IsoPs and the tetradeuterated derivative of prostaglandin F2𝛼(PGF2𝛼-d4), respectively [29, 30]. for 2 hours at 4∘C, dehydrated in a graded series of alcohol, embedded in Araldite resins, and polymerized in oven for 48 hours at 60∘C. Sixty nm thin sections, obtained with a Reichert ultramicrotome, were routinely stained with uranyl acetate and lead citrate and observed with a TEM Fei Tecnai G2 spirit at 100 Kv. 2.7. F4-NeuroPs GC/NICI-MS/MS. Measured ions were the product ions at m/z 323 and m/z 303 derived from the [M−181]−precursor ions (m/z 593 and m/z 573) produced from oxidized DHA and the tetradeuterated derivative of PGF2𝛼, respectively [29, 31]. 2.11. Immunofluorescence Double Staining. The localization of two main type of collagen synthetized by skin fibroblasts was evaluated by an immunofluorescence assay. Primary human fibroblasts, grown on glass coverslips at a density of 2 × 104 cells/cm2, were fixed in 4% paraformaldehyde for 10 min at 4∘C. 2. Materials and Methods After blocking in 3% nonfat milk (BioRad, Hercules, CA, USA), the membranes were incubated overnight at 4∘C with goat polyclonal anti 4- HNE adduct antibody (cod. AB5605; Millipore Corporation, Billerica, MA, USA). Following washes in TBS Tween and incubation with specific secondary antibody (mouse anti- goat horseradish peroxidase-conjugated, Santa Cruz Biotech- nology Inc., CA, USA) for 1 h at room temperature, the membranes were incubated with ECL reagents (BioRad, Hercules, CA, USA) for 1 min. The bands were visualized by autoradiography. Quantification of the relevant bands was performed by digitally scanning the Amersham Hyperfilm ECL (GE Healthcare Europe GmbH, Milan, Italy) and mea- suring immunoblotting image densities with ImageJ software. 2.12. Statistical Analysis. Results were expressed as medians with interquartile ranges or means ± standard deviation. Differences between groups were evaluated by the non- parametric Mann-Whitney rank sum test, Wilcoxon rank test, or Kruskal-Wallis test analysis of variance (ANOVA), as appropriate. The MedCalc ver. 12.1.4 statistical software package (MedCalc. Software, Mariakerke, Belgium) was used for data analysis. Two-sided 𝑃values <0.05 were considered as significant. 3. Results 3.1. Cell Oxidant and Antioxidant Status. Biochemical signs of lipid and protein oxidative damage, together with the antioxidant cellular defense, were evaluated in skin fibroblasts from RTT and healthy control subjects. Increased lipid oxidative damage was evidenced as indicated by increased levels of total (i.e., sum of free and esterified form) F2-IsoPs (7.5-folds) and F4-NeuroPs (12-folds), both deriving from membrane polyunsaturated fatty acids, that is, arachidonic (AA) and docosahexaenoic (DHA) acid, respectively (Figures 1(a) and 1(b)). Biochemical signs of protein oxidative damage consequent to a lipid peroxidation event were indicated by increased (1.48-folds) 4-HNE PAs levels for which a significant increase was also detected (Figure 1(c)). Oxidative 2.10. Transmission Electron Microscopy (TEM). Cultured fibroblasts were fixed in 2.5% glutaraldehyde in 0.1 M cacody- late buffer pH 7.2 (CB) for 3 hours at 4∘C. After a rinse in CB, the material was postfixed in 1% osmium tetroxide in CB Oxidative Medicine and Cellular Longevity 4 0 200 400 600 800 1000 (pg/mg protein) Healthy controls Typical RTT Total F2-IsoPs ∗ (a) 0 200 400 600 800 1000 (pg/mg protein) Total F4-NeuroPs ∗ Healthy controls Typical RTT (b) 0 5 10 15 20 25 4-HNE PAs (×106 a.u.) ∗∗ Healthy controls Typical RTT (c) 0 0.2 0.4 0.6 0.8 1 NPBI (nmol/mg protein) ∗ Healthy controls Typical RTT (d) Figure 1: Increased levels of total (i.e., sum of free and esterified form) F2-IsoPs, total F4-NeuroPs, 4-HNE PAs, and NPBI in RTT skin fibrob- last as compared to the control cells. ∗𝑃< 0.0001, ∗∗𝑃= 0.0013. Data are expressed as means ± standard deviation. Legend: F2-IsoPs, F2-isoprostanes; F4-NeuroPs, F4-neuroprostanes; 4-HNE PAs, 4-hydroxy-2-nonenal protein adducts; NPBI, nonprotein bound iron. (b) 0 0.2 0.4 0.6 0.8 1 NPBI (nmol/mg protein) ∗ Healthy controls Typical RTT (d) (d) (c) Figure 1: Increased levels of total (i.e., sum of free and esterified form) F2-IsoPs, total F4-NeuroPs, 4-HNE PAs, and NPBI in RTT skin fibrob- last as compared to the control cells. ∗𝑃< 0.0001, ∗∗𝑃= 0.0013. Data are expressed as means ± standard deviation. Legend: F2-IsoPs, F2-isoprostanes; F4-NeuroPs, F4-neuroprostanes; 4-HNE PAs, 4-hydroxy-2-nonenal protein adducts; NPBI, nonprotein bound iron. 0 0.4 0.8 1.2 1.6 Healthy controls Typical RTT Reduced GSH (nmol/mg protein) ∗ (a) 0 0.4 0.8 1.2 1.6 GSSG ∗∗ (nmol/mg protein) Healthy controls Typical RTT (b) Figure 2: Significant reduction in cellular GSH and significant increase of GSSG in RTT skin fibroblast as compared to control cells. 3. Results ∗𝑃< 0.0001, ∗∗𝑃= 0.0033. Data are expressed as means ± standard deviation. Legend: GSH reduced glutathione; GSSG, oxidized glutathione. 0 0.4 0.8 1.2 1.6 GSSG ∗∗ (nmol/mg protein) Healthy controls Typical RTT (b) (b) (a) Figure 2: Significant reduction in cellular GSH and significant increase of GSSG in RTT skin fibroblast as compared to control cells. ∗𝑃< 0.0001, ∗∗𝑃= 0.0033. Data are expressed as means ± standard deviation. Legend: GSH reduced glutathione; GSSG, oxidized glutathione. and total cellular F4-NeuroPs (Rho = 0.84, 𝑃= 0.001) and NPBI versus total cellular F2-IsoPs (Rho = 0.69, 𝑃= 0.019). No significant relationships between each of the investigated molecules (i.e., F2-IsoPs, F4-NeuroPs, 4-HNE Pas, GSH and GSSG) and the MECP2 mutation categories were observed (𝑃 value range: 0.461–0.981). damage was concomitant to an imbalance in the principal antioxidant cytoplasmic agent in so far as a significant reduction (−43.6%) in cellular GSH, a significant increase (1.44-folds) of GSSG (Figures 2(a) and 2(b)), and a significant reduction (−3.05 folds) of GSH/GSSG ratio were reported. Lipid peroxidation events in RTT skin fibroblasts were found to be related to the levels of NPBI, a prooxidant agent. NPBI was significantly increased (2.3-folds) (Figure 1(d)), with significant positive correlations observed between NPBI 3.2. Cell Morphology Study. To evaluate the effect, if present, of the oxidant/antioxidant imbalance evidenced in RTT 5 Oxidative Medicine and Cellular Longevity M RER G V 1𝜇m (a) RER V MLB 1𝜇m (b) Figure 3: Transmission electron microscopy of control (a) and RTT (b) fibroblasts cultures. Skin fibroblasts, either from control subjects or RTT patients, show a flattened morphology with extensive tapering cytoplasmic processes. An euchromatic and oval-shaped nucleus was present in central position of the cells, with clumps of heterochromatin next to the nuclear envelope. The cytoplasm contains many vesicles with variable electron density, a prominent Golgi complex, and mitochondria. Rough endoplasmic reticulum (RER) cisternae in RTT fibroblasts appear more dilated than in control. Some large multilamellar bodies (MLB) are frequently detectable in the cytoplasm of the RTT fibroblast cells. (G) Golgi complex, (M) mitochondrion, and (V) vesicle. Bar = 1 𝜇m. RER V MLB 1𝜇m (b) M RER G V 1𝜇m (a) (b) (a) Figure 3: Transmission electron microscopy of control (a) and RTT (b) fibroblasts cultures. Skin fibroblasts, either from control subjects or RTT patients, show a flattened morphology with extensive tapering cytoplasmic processes. 3. Results An euchromatic and oval-shaped nucleus was present in central position of the cells, with clumps of heterochromatin next to the nuclear envelope. The cytoplasm contains many vesicles with variable electron density, a prominent Golgi complex, and mitochondria. Rough endoplasmic reticulum (RER) cisternae in RTT fibroblasts appear more dilated than in control. Some large multilamellar bodies (MLB) are frequently detectable in the cytoplasm of the RTT fibroblast cells. (G) Golgi complex, (M) mitochondrion, and (V) vesicle. Bar = 1 𝜇m. Healthy controls Typical RTT Col III Col III Col I Col I Figure 4: Double immunofluorescence staining shows the localization of type I collagen (central column, red color) and type III collagen (left column, green color). Images are merged in the right panel and the yellow color indicates overlap of the staining. The colocalization of types I and III collagen is reduced in RTT skin fibroblasts. Legend: Col I, type I collagen; Col III, type III collagen. Col I Col I Figure 4: Double immunofluorescence staining shows the localization of type I collagen (central column, red color) and type III collagen (left column, green color). Images are merged in the right panel and the yellow color indicates overlap of the staining. The colocalization of types I and III collagen is reduced in RTT skin fibroblasts. Legend: Col I, type I collagen; Col III, type III collagen. granules were also detectable (Figure 4). Likewise, in these same cells, fluorescence relative intensity for type I collagen was significantly reduced (𝑃= 0.00997) (Figure 5). fibroblasts, the physiological cellular condition was evaluated by observation of two key typical features: the morphology and the collagen distribution.i At transmission electron microscope significant ultra- structural differences between control and RTT fibroblasts were observed. In particular, a marked dilation of the rough endoplasmic reticulum cisternae was detectable in the skin RTT fibroblasts, along with evidence of cytoplasmic multil- amellar bodies (Figures 3(a) and 3(b)). Immunofluorescence double staining showed a reduced degree of colocalization of type III and type I collagen in RTT skin fibroblasts when compared to control cells. Staining for type I collagen was found to be more evident in RTT cells, where perinuclear Oxidative Medicine and Cellular Longevity Although enhanced plasmatic OS markers levels suggest a systemic OS status, to date no indications are present in order to infer what tissues/cellular systems other than blood could be potentially damaged by the OS imbalance in RTT. A preliminary suggestion that an oxidative process should occur at the cellular level was con- tained in our recent study where oxidative posttranslational modifications on SRB1 receptor in primary fibroblast cultures were observed [36]. Fibroblasts have been used in the RTT scientific research to evaluate gene mutation and epigenetic process [37], differentiation to induced pluripotent stem (iPS) [38], cellular response subsequent to MECP2 mutation [39], reduction of stathmin-like 2 [40], and the effects of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT [41]. Fibroblasts have been extensively employed to investigate OS in different pathophysiological processes such as genetic neurodegenerative diseases [42], Parkinson’s disease [43], aging [44], and response to etiological agents [45, 46]. Moreover, lipid peroxidation events are known to be relevant to the fibroblast function, as relationship between mechanotransduction and lipid metabolites has been previ- ously investigated in keloids [47].h Our reported results of decreased GSH levels and GSH/GSSG ratio indicate a reduced antioxidant defence in RTT cells and suggest that oxidative events are likely to be chronic, thus determining a consumption of glutathione in its reduced form. An alternative explanation could be that the MECP2 mutation-harboring cells may have coexisting defects in the synthesis and/or recycling of glutathione, thus leading to an uncontrolled free radicals action. Of course, further study is needed in order to clarify this point.i Overall, our data indicate that a significant oxidation of DHA, the precursor acid for F4-NeuroPs, and AA, precursor of F2-IsoPs, likely triggered by NPBI, occurs in skin fibrob- lasts of patients with MECP2 mutation and clinical RTT and confirm the biological relevance of such key mediators of lipid peroxidation. Skin dermis is known to be consisting of 80% collagen type I, with its remaining fraction being made mostly of collagen type III [54]. In this context, the fibroblast shows a pivotal role for collagen production, [55]. The biochemical markers so far employed for measuring OS are not to be considered as equal in terms of the con- veyed information. Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity 6 0 100 200 300 400 500 600 700 Relative intensity N.S. Col III Col I ∗ Healthy controls Typical RTT Figure 5: Relative intensity of fluorescence for types I and type III collagen in RTT and control skin fibroblasts. Software LEICA AF6000 (Leica Microsystems-Germany). Data are expressed as median ± semiinterquartile range ∗𝑃= 0.0062; N.S.: no significant difference (𝑃= 0.4361). Legend: Col I, type I collagen; Col III, type III collagen. this fatty acid represents a normal constituent of all cell membranes. In particular, human fibroblasts are able to synthetize DHA [51] and incorporate it in their membrane phospholipids [52]. Furthermore, variations of the DHA content in the membrane of fibroblasts have been implicated in neuropsychiatric disorders, with lowered levels evidenced in patients with schizophrenia and bipolar disorder [53]. Our results indicate an increased oxidation of DHA in RTT fibrob- lasts, thus underlying an increased susceptibility of these cells to oxidative damage. This process, in turn, could contribute to changes in the fatty acid composition of membranes with consequent cellular damage. 4HNE, which is formed from arachidonic acid or other unsaturated fatty acids following free radical attack, can bind, by Michael addition, to proteins, particularly, to cysteine, his- tidine, or lysine residues. Thanks to its ability to form adducts to the proteins, 4HNE is not only considered as a reliable marker of OS, but also has a biological impact by changing protein function [34]. Our findings of increased 4HNE-PAs can be considered as a long-term consequence of enhanced lipid peroxidation, further contributing to cellular damage. Figure 5: Relative intensity of fluorescence for types I and type III collagen in RTT and control skin fibroblasts. Software LEICA AF6000 (Leica Microsystems-Germany). Data are expressed as median ± semiinterquartile range ∗𝑃= 0.0062; N.S.: no significant difference (𝑃= 0.4361). Legend: Col I, type I collagen; Col III, type III collagen. Glutathione, the main cellular antioxidant defence, pre- venting the damage to key cellular components induced by reactive oxygen species, exists in both reduced (GSH) and oxidized (GSSG) states. In its reduced state, the thiol group of cysteine is able to donate a reducing equivalent (H+ + e−) to other unstable molecules, such as ROS, whereas in donating an electron, glutathione itself becomes reactive but is ready to react with another reactive glutathione to form GSSG. erythrocytes samples [26, 35]. 4. Discussion Our findings demonstrate, for the first time, the presence of an extensive redox imbalance in primary skin fibroblasts cultures obtained from patients with RTT by adding new evidence to the concept of an OS imbalance as key pheno- typical features of MeCP2 deficiency in RTT. Earlier clues for an abnormal OS balance in RTT patients harbouring MECP2 gene mutations are to date limited to plasma and Abbreviations 4-HNE: 4-Hydroxy-2-nonenal 4-HNE PAs: 4-Hydroxy-2-nonenal protein adducts AA: Arachidonic acid DHA: Docosahexaenoic acid F2-IsoPs: F2-isoprostanes F4-NeuroPs: F4-neuroprostanes GSH: Reduced glutathione GSSG: Oxidized glutathione IsoPs: Isoprostanes MECP2: Methyl-CpG-binding protein 2—human gene Authors’ Contribution The conception of the paper was performed by Cinzia Sig- norini, Claudio De Felice, Silvia Leoncini, Lucia Ciccoli, Alessandra Renieri, and Joussef Hayek. The experimental design was performed by Cinzia Signorini and Claudio De Felice. MECP2 mutation determinations were performed by Ilaria Meloni, Francesca Ariani, Francesca Mari, Sonia Amabile, and Alessandra Renieri. Clinical assessement was performed by Joussef Hayek, Claudio De Felice, and Alessan- dra Renieri. Skin biopsies were performed by Joussef Hayek. Samples were prepared by Cinzia Signorini, Silvia Leoncini, Alessandra Pecorelli, Gloria Zollo, Giuseppe Belmonte, Mar- iangela Gentile, Eugenio Paccagnini, Sonia Amabile, and Ilaria Meloni. NPBI assays were performed by Silvia Leoncini. Isoprostanes and neuroprostanes assays were performed by Cinzia Signorini, Thierry Durand, and Jean-Marie Galano. 4-HNE-PAs assays were carried out by Alessandra Pecorelli, and Giuseppe Valacchi. Gluthatione assays were carried out by Silvia Leoncini and Gloria Zollo. Immunocytochem- istry was performed by Giuseppe Belmonte and Alessandra Pecorelli. TEM was done by Eugenio Paccagnini and Mari- angela Gentile. Data analysis was done by Claudio De Felice, Cinzia Signorini, and Silvia Leoncini. All the authors equally contributed in data interpretation, paper drafting, and paper approval. Given that OS is known to be a major determinant of aging [73, 74], it is conceivable that aging phenomena in RTT, including skin fibroblasts morphological changes, may be caused through OS mechanisms. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Disclosure Cofirsts: Cinzia Signorini, Silvia Leoncini, Claudio De Felice, and Alessandra Pecorelli. Colasts: Lucia Ciccoli, Alessandra Renieri, and Joussef Hayek. p gy p p g A condition of oxidative stress has been previously reported in aging skin [67]. Skin aging is known to be mainly due to fragmentation/loss of type I collagen fibrils, conferring strength, and resiliency in association with metalloproteinase activation, involved in type I collagen degradation [68]. Although, no evidence of wound healing alterations or derma laxity has not been reported in RTT, an accelerated ageing process is known to occur in the affected patients. Our results suggest the occurrence of a possible reduction in type I collagen, a feature of aging skin, while they show a clear redox imbalance in RTT skin fibroblasts. Therefore, it is conceivable that a premature skin aging may occur in RTT patients, a hypothesis which is in line with prior evidence of premature senescence phenomena in the disease [69–72]. Oxidative Medicine and Cellular Longevity In particular, F2-IsoPs, the gold standard molecules for the OS in vivo evaluation [35, 48], are the end-products of arachidonic acid (AA) oxidation, a polyun- saturated fatty acid abundant in both brain grey and white matter. On the other hand, F4-NeuroPs are the oxidative end-products of docosahexanoic acid (DHA), abundant in neuronal membranes [49]. NPBI is a prooxidant factor, asso- ciated with hypoxia, hemoglobin oxidation, and subsequent heme iron release [50]. Besides being a major component of the extracellular matrix in a variety of internal organs and skin in adults, type III collagen is critical for fibrillogenesis in the development of apparatus such as skin and cardiovascular system [56, 57]. Overall in such tissues, type III collagen is colocalized, within the same fibril, with the most abundant member of the family, type I collagen, and regulates the diameter of type I collagen fibrils, which have to meet the physiological requirements of different tissues at different developmental stages [54, 58–62].h Our data demonstrate that both AA and DHA undergo significant oxidation in RTT fibroblasts. Although DHA is known to be particularly abundant in neuronal membranes, ff The reported ultrastructural changes are to be considered as a new finding to be added to the mitochondrial changes 7 Oxidative Medicine and Cellular Longevity MeCP2: Methyl-CpG-binding protein 2—human protein NPBI: Nonprotein bound iron OS: Oxidative stress PUFAs: Polyunsaturated fatty acids ROS: Reactive Oxygen Species RTT: Rett syndrome TEM: Transmission electron microscopy. previously reported in RTT skin fibroblasts [63]. Since no abnormalities in wound repair and/or healing have been reported in RTT patients, these ultrastructural features are to be considered as subclinical characteristics of the disease. Marked dilation of the rough endoplasmic reticulum cister- nae and cytoplasmic multilamellar bodies were observed for the first time in this study. Dilated rough endoplasmic retic- ulum cisternae can be considered as a nonspecific adaptive response due to either increased secretory activity or as the result of the cellular response to noxae of different nature [64, 65]. On the other hand, the presence of multilamellar bodies could be interpreted as an expression of cellular damage. In particular, those structures suggest the occurrence of autophagy phenomena of unclear pathogenesis [66]. 5. Conclusion Our study demonstrates for the first time the occurrence of lipid peroxidation in RTT fibroblasts, together with a reduced antioxidant cellular defense with a major impact on cell morphology. We speculate about a possible functional involvement of these changes in the skin fibroblasts of RTT patients which must be taken into account when evaluating this cellular model of the disease. 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https://openalex.org/W4226141622	https://zenodo.org/records/6410659/files/CAS-EurASc%20WG1_2022-04-04.pdf	English	null	Ocean-based climate action	HAL (Le Centre pour la Communication Scientifique Directe)	2,022	cc-by	5,704	Lead authors: Jean-Pierre Gattuso (CNRS-Sorbonne University-Iddri, France), Nianzhi Jiao (Xiamen University, China), Fahu Chen (Institute of Tibet Plateau), Jean Jouzel (Laboratoire des Sciences du Climat et de l’Environnement, France), Corinne Le Quéré (University of East Anglia, UK), Yonglong Lu (Xiamen University, China), Paul Tréguer (University of Western Brittany, France), Karina von Schuckmann (Mercator Ocean, France), Zhong Lin Wang (Beijing Institute of Nano Energy and Systems, China), Jing Zhang (East China Normal University/Shanghai Jiao Tong University, China) Contributing authors: Alexandre K. Magnan (Iddri, France), Jihua Liu (Shandong University, China), Faming Wang (Chinese Academy of Sciences, China), Phillip Williamson (University of East Anglia, UK), Jiangning Zeng (Second Institute of Oceanography, China) Ocean-based CLIMATE ACTION Ocean-based CLIMATE ACTION FRONTIER FORUM ON PROGRESS IN OCEAN SCIENCE AND TECHNOLOGY Oc FRONTIER FORUM ON PROGRESS IN OCEAN SCIENCE AND TECHNOLOGY Chinese Academy of Sciences (CAS) – European Academy of Sciences (EurASc) Progress in Ocean Science and Technology Ocean-Based Climate Action Citation: Gattuso J.-P., Jiao N., Chen F., Jouzel J., Le Quéré C., Lu Y., Tréguer P., von Schuckmann K., Wang Z. L. & Zang J., 2022. Ocean-based climate action. 12 p. Chinese Academy of Sciences and European Academy of Sciences. DOI: 10.5281/zenodo.6410659 Gattuso J.-P., Jiao N., Chen F., Jouzel J., Le Quéré C., Lu Y., Tréguer P., von Schuckmann K., Wang Z. L. & Zang J., 2022. Ocean-based climate action. 12 p. Chinese Academy of Sciences and European Academy of Sciences. DOI: 10.5281/zenodo.6410659 Abstract As a consequence of anthropogenic perturbations the global ocean is warming, acidifying, losing oxygen and sea ice, and sea level is rising. While drastic reduction of the emission of greenhouse gases is urgently needed, which includes ocean energy substitution for fossil energy, we show that the ocean offers numerous opportunities to reduce the causes and consequences of climate change, globally and locally. A wide range of ocean-based measures to enhance societal climate adaptation are currently implemented worldwide to deal either with coastal risks or changes in ocean resources. Ocean-related measures should not be considered as a substitute for climate mitigation on land or non ocean-based adaptation measures, which must be strongly pursued for the benefit of the atmosphere, the ocean, and socio-ecological systems worldwide. Ocean-Based Climate Action Ocean-Based Climate Action Foreword The China–Europe Frontier Forum on “Progress in Ocean Science and Technology” (POST) was held in Shanghai and on line on 20-21 October 2020 in the framework of the imminent launch of the UN decade of Ocean Science for Sustainable Development (2021-2030). The POST Frontier Forum was attended by about 300 participants. Thirteen invited speakers made presentations during three sessions on: the Earth System and the future ocean in a changing climate; progress in ocean sciences and technology; and ocean sustainable development. The POST Frontier Forum was intended to enhance collaborations between China and Europe, and identified the following frontier topics for China-Europe action, which includes contributions on: The POST Frontier Forum was intended to enhance collaborations between China and Europe, and identified the following frontier topics for China-Europe action, which includes contributions on: - Ocean based solutions for climate change mitigation and adaptation that offer a policy-relevant framework for decision and action; - The global coastal ocean to make it sustainable for the well-being of human societies; - The "Digital Twin of the Ocean" as component of the “Digital Earth” initiatives, which would contribute to enhance the understanding of the role of the ocean in the changing climate. The present report deals with “Ocean-based climate action”. Co-lead by Jean-Pierre Gattuso (EurASc) and Nianzhi Jiao (CAS), a working group gathered the efforts of 16 lead and contributing authors (9 from Europe and 9 from China). It aims to be a landmark on the preparation on the POST Frontier Forum n°2 to be held in Europe and on-line. Paul Tréguer University of Brest, France Paul Tréguer University of Brest, France Jing Zhang East China Normal University, Shanghai East China Normal University, Shanghai 2 2 1. Introduction The global ocean is warming, acidifying and losing oxygen and sea ice, and sea level is rising. As a result, keystone species and ecosystems such as warm-water coral reefs, seagrass meadows and kelp forests face high to very high risks even under low carbon dioxide (CO2) emissions (Bindoff et al., 2019). Moreover, unless comprehensive and intense adaptation efforts are undertaken, low-lying coastal settlements will face moderate to high sea-level rise risks by the end of the century, even under full and timely implementation of the Paris Agreement, with nearly all the world!s regions are projected to experience an increase in coastal flooding throughout the 21st century (IPCC, 2021). This calls for a dramatic scaling up of efforts towards ambitious mitigation and adaptation. The ocean offers opportunities to reduce the causes and consequences of climate change, globally and locally, as shown by The Ocean Solutions Initiative (Gattuso et al., 2018) and other recent reports (Jiao et al., 2011, Hoegh-Guldberg et al., 2019; Because the Ocean 2019; von Schuckmann et al., 2020). However, countries have poorly used ocean-based measures for tackling climate change and its impacts, in their Nationally Determined Contributions (NDCs; Gallo et al. 2017) under the Paris Agreement. Opportunities for countries to adopt more ocean-inclusive mitigation and adaptation strategies, include the first UNFCCC Global Stocktake in 2023, the UN 2030 Sustainable Development Goals, and the UN Ocean Decade for Sustainable Development (Ryabinin et al., 2019) In this report we assess 19 ocean-based measures to support climate policies and the revision of NDCs in the areas of mitigation and adaptation (Fig. 1). The categories considered are closely based on Gattuso et al. (2018) and Magnan et al. (2018). The US National Academies of Sciences, Engineering, and Medicine (2019, 2021a,b) produced three reports which are at least partly relevant to the ocean. In this report we assess 19 ocean-based measures to support climate policies and the revision of NDCs in the areas of mitigation and adaptation (Fig. 1). The categories considered are closely based on Gattuso et al. (2018) and Magnan et al. (2018). The US National Academies of Sciences, Engineering, and Medicine (2019, 2021a,b) produced three reports which are at least partly relevant to the ocean. 3 3 Figure 1. Overview of the main ocean-based measures for climate action. Modified from Gattuso et al. (2018) and Abram et al. (2019). 1. Introduction Marine renewable energy Pollution reduction Restoration and conservation of vegetation Enhancing open-ocean productivity Enhancing weathering and alkalinization Carbon capture and storage Marine bioenergy with carbon capture and storage Enhancing societal adaptation Addressing the causes of climate change Cloud brightening Pollution reduction Conservation Assisted evolution Restoration and enhancement of habitats Supporting biological and ecological adaptation (including ecosystem-based management) Managing solar radiation Relocate and diversify economics activities Relocate people Improving risk reduction policies Community-based adaptation Infrastructure-based adaptation Early warning, precautionary approach, emergency response system Surface albedo enhancement Taking action Conservation Restoration and enhancement of habitats Assisted evolution Pollution reduction Supporting biological and ecological adaptation (including ecosystem-based managemen Addressing the causes of climate change Enhancing societal adaptation Managing solar radiation Cloud brightening Surface albedo enhancement Figure 1. Overview of the main ocean-based measures for climate action. Modified from Gattuso et al. (2018) and Abram et al. (2019). 2.1. Addressing the causes of climate change Renewable energy. Ocean energy substitution for fossil energy. The focus is on the extraction of physical (and potentially chemical) energy from or over the marine environment that is relatively rapidly replenished from natural sources, including from offshore wind. Hydrogen generation from seawater or by algae is not covered here; despite recent technical advances, such approaches are still very much at early stages of development. Restoration and conservation of vegetation. (hereafter "Vegetation”) Restoration and conservation of coastal vegetation to enhance CO2 uptake and avoid further emissions. Vegetation refers to the implementation of the solution at the global scale (e.g. restoring all the human-induced degraded salt marshes, mangroves and seagrass habitats of the planet). Vegetation (l) refers to the ongoing implementation of the solution at the local scale (< ~100 km2). Seaweed cultivation can also contribute to reduced emissions if the biomass is either stored in the deep sea, or used to manufacture lasting products, or used in bioenergy with carbon capture and storage (BECCS). Enhancing Open Ocean Productivity. (hereafter “Fertilization”) is the enhancement of ocean productivity by adding nutrients. The focus here is on i) the open ocean; ii) primary production by phytoplankton (and hence carbon uptake); and iii) the use of iron as the added (micro)nutrient. Alkalinization is the addition of natural or man-made alkalinity, including intentional dissolution of calcium carbonate or other minerals such as olivine to enhance CO2 removal and/or carbon storage, this may be particularly beneficial for areas with hypoxia and acidification. Hybrid methods correspond to measures involving both land and ocean components. These methods include: i) Marine-fueled bioenergy with carbon capture and storage (marine BECCS); ii) marine- or land-fueled biomass energy + accelerated weathering of limestone; iii) marine or land 4 renewable energy based negative emissions electricity/H2 with ocean alkalinity production; iv) soil or ocean storage of marine organic carbon and biochar; v) burial of land crop waste in ocean sediment; and vi) subsurface ocean or under sea ﬂoor storage of land-based direct air capture (DAC) or CO2 generated by BECCS. Other methods initially considered but not included in this analysis are: i) abiotic seawater extraction and use/storage of CO2; and ii) Increasing marine consumption of and/or reducing marine emissions of other GHGs such as CH4, N2O, etc. 2.2. Supporting biological and ecological adaptation Pollution reduction corresponds to the reduction of marine and land-based pollution, including non-CO2 drivers of ocean acidiﬁcation (e.g. nitrogen, phosphorus and organic carbon from agricultural, industrial, urban and domestic sources causing eutrophication). Restoring hydrology is about the maintenance and restoration of hydrological regime and delivery of water and sediment from watersheds to the coastal marine environment. Eliminating over-exploitation of living resources (including vegetation and ﬁsh stocks) and over- extraction of nonliving resources (e.g., sand, minerals) through management measures. Protection of marine habitats and ecosystems through spatial measures including marine protected areas and notake reserves. Assisted evolution is the human intervention to accelerate the rate of naturally occurring evolutionary processes. The purpose of assisted evolution is to change certain characteristics of an organism; for example, the resistance of corals and bivalves to stress such as elevated temperature and lower pH. Synthetic biology, which involves genome editing using natural or synthetic genes, is not considered because, to our knowledge, its feasibility has not been evaluated on marine species other than in the aquaculture industry. Relocation and reef restoration. Relocation refers to introduction of species, ecosystems and habitats where they were not historically present, but current and future climatic conditions are expected to allow them to exist. Restoration refers to the enhancement of degraded habitats and ecosystems, and the creation of new habitats. Only coral and oyster reefs are considered here, as vegetated ecosystems (mangroves, salt marshes and seagrass habitats) are already considered in the "Restoration and conservation of vegetation” measure above. 2.4. Enhancing societal adaptation A wide range of ocean-based measures to enhance societal climate adaptation are currently implemented worldwide to deal either with coastal risks (including shoreline erosion and temporary flooding; Oppenheimer et al. 2019) or changes in ocean resources (e.g. in fish stocks at the coast and in the open ocean; Bindoff et al. 2019). Various categorizations exist to package such a diversity —for example with respect to coastal risks (protect, advance with ground elevation, accommodate, and retreat; Nicholls 2018, Oppenheimer et al. 2019)— and to provide decision- makers and practitioners with simplified guidance to develop adaptation strategies at various scales. Here we describe six measures: (a) Community-based adaptation; (b) Improving risk reduction policies; (c) Infrastructure-based adaptation; (d) Relocate people; (e) Relocate and diversify economic activities; and (f) Early warning, precautionary approach, emergency response system. One of the main conclusions from scientific literature and experience gained through practice is that the relevance of any of these (categories of) measures in terms of their potential to reduce climate- related risk, highly depends on the specificities of the context and conditions under which they are implemented (e.g., institutional leadership, social acceptability, affordability, legal frameworks, etc.; Duvat et al. 2020, Magnan and Duvat 2020, Magnan et al. 2020). As a result, policy clustering is complicated for societal adaptation measures as characteristics (effectiveness, feasibility, co- benefits, disbenefits, governability, etc.) necessarily vary from one context to another. One measure could provide a solution in one case but a maladaptation in another (i.e. when vulnerability to climate change is insidiously exacerbated instead of being reduced), for example over time or if it implies negative collateral effects on neighboring or economically/socially connected systems. Some general lessons can however be highlighted distinguishing between: measures that do not raise major concern whatever the context of implementation; measures for which climate relevance cannot be decided a priori; and measures for which we still lack experience. A first set of measures, comprising (a) and (b) described above, have low disbenefits (i.e., associated adverse impacts and other undesirable consequences, including opportunity costs), have a more or less direct but proven influence on climate risk reduction, and do not face strong barriers in terms of their governability. Community-based adaptation addresses a major concern raised in the scientific literature: the need to engage local people and communities in both the design and implementation of adaptation-related responses. This is important for three reasons. 2.3. Solar radiation management Solar radiation management (or modification) refers to the intentional modification of the Earth!s shortwave radiative budget with the aim of reducing warming. Two approaches have been described in connection with the ocean. Marine cloud brightening involves the spraying of seawater into the lower atmosphere to enhance the production, longevity and brightness of stratocumulus clouds. This approach is also called marine sky brightening because sea spray climate engineering is sometimes as efficient in clear-sky conditions as in cloudy-sky conditions. Albedo enhancement. Increase surface ocean albedo by producing long-lived ocean foam or by artificially increasing sea-ice cover in polar regions. Only the former is considered here. 5 5 The term "low regret!$#is used to show that there are costs as well as benefits for these actions. However, on balance 1 the benefits are considered greater, and therefore many of these measures could also be considered as "no regret”. 6 2.4. Enhancing societal adaptation First, because these people usually play a central role in implementation (e.g., when raising risk-awareness); second, because they are the ultimate beneficiaries of any adaptation measure; and third, because social acceptability is a critical criteria to decide the feasibility of any adaptation-related measure. Improving risk reduction policies (e.g. limit new buildings in risk-prone areas at the coast) or shifting toward less fish-dependent consumption patterns and markets can be considered relatively low regret in that they contribute to reduce exposure to climate events and/or pressure to climate- 1 sensitive natural resources. It is however important to note that despite these categories apparently being low regret, they are not cost-free and require well-informed planning and effective coordination over a wide range of stakeholders, scales and timeframes. For a second set of measures, comprising (c) to (f), whether or not they contribute to adaptation or maladaptation, will intrinsically depend on the geographical, policy and societal context of their implementation. While seawalls and other Infrastructure-based adaptation can protect against sea level rise in densely populated coasts, they can be counterproductive to natural environments (e.g. sand-dune systems, mangroves and coral reefs) as they tend to undermine ecosystems!# natural The term "low regret!$#is used to show that there are costs as well as benefits for these actions. However, on balance 1 the benefits are considered greater, and therefore many of these measures could also be considered as "no regret”. 6 6 adjustments to ocean changes, inland migration, and buffering function against storm surges. In the former case, Infrastructure-based adaptation describes a decisive, unavoidable climate solution, while in the latter it carries a high risk of maladaptation. The benefits of Relocating people are also context-dependent: if planned and at a local-scale (e.g. within an island), such relocation can help save lives, and it is therefore decisive (Magnan and Duvat 2020); but if unplanned or forced, relocation can generate multiple and ramifying detrimental effects on both the displaced people and host communities, and therefore must be considered risky and maladaptive (Haasnoot et al. 2021). The same caution applies to Relocating and diversifying economic activities, which can either secure local jobs and economies, or generate the opposite effect. Early warning, precautionary approach, emergency response system is another illustration of adaptation measures that can play a decisive role as a vehicle for maladaptation over time. It is clear that early warning systems (e.g. 2.4. Enhancing societal adaptation tsunamis superimposed on sea-level rise) are a critical tool to alert decision-makers and populations in case of the occurrence of potentially damaging extreme events such as tropical cyclones or ocean heat waves. In the same way, developing cyclone shelters for example is a way to provide secure places to people in the case of high impacts, particularly useful in low-lying countries. These measures could however have counterproductive effects if not accompanied with a series of measures including awareness campaigns and sustainable funding. It has been shown for example in Bangladesh that when too frequent, precautionary early warnings about cyclones that finally do not hurt the area can lead to lowering the trust people have in alerts, and therefore a decrease in local communities!#preparedness to extreme events. Similarly, the development of cyclone shelters in areas affected by intense cyclones but of low occurrence (e.g., Tuamotu atolls in French Figure 2. Policy clusters of the main ocean-based measures for climate action. Modiﬁed from Gattuso et al. (2019). 3. Policy clusters of ocean-based action Ocean-based measures to enhance both global mitigation and local coastal adaptation can be clustered into overarching policy-relevant clusters (Fig. 2). Two clustering exercises were recently performed based on key features according to their state of implementation, effectiveness to reduce climate-related ocean drivers globally, e%ectiveness to reduce impacts/risks locally, and potential co-beneﬁts and disbeneﬁts. Gattuso et al. (2019) grouped 18 ocean-related measures into four policy-relevant categories: Decisive, Low Regret, Unproven and Risky. Another clustering (Gattuso et al., 2021) grouped a subset of 12 measures into three clusters (Decisive, Low Regret, Concept Stage). We use here the more complete groupings of Gattuso et al. (2019). Decisive measures address the causes of climate change: Marine renewable energy and Carbon Capture and Storage (CCS). Marine renewable energy has the theoretical potential to meet all global electricity requirements, although requiring further infrastructure development. Whilst some local adverse impacts are inevitable, these can be minimized. Importantly, CCS is a Decisive measure only if implemented in a way which avoids significant leakage. When ocean-based CCS is deployed for enhanced oil recovery, that application negates its climate mitigation benefits. Four societal adaptation measures can also be considered Decisive (Infrastructure-based adaptation, Relocating and diversifying economic activities, Relocating people, and early warning and emergency systems), but can also be Risky depending on the context within which they are implemented, as discussed below. Low Regret measures provide both climatic and non-climatic benefits, with few disbenefits (associated adverse impacts and other undesirable consequences, including opportunity costs). For example, Conservation measures can protect carbon-rich coastal ecosystems from direct human disturbance and loss, and play an important role in limiting local climate impacts. Similarly, Restoring and enhancing coastal vegetation supports ecological adaptation whilst providing storm protection, contributing to food security, and enhancing biodiversity. It can also increase carbon uptake, at levels that may be locally and/or nationally significant. Nevertheless, because of the limited total area for restoring such blue carbon ecosystems, this action can only make a very small contribution to climate mitigation at the global scale (Bindoff et al., 2019). Pollution reduction in coastal waters removes contaminants and excess nutrients that impair ecosystem function, thereby supporting ecosystem-based adaptation (Jiao et al., 2011). Reduced pollution from shipping can also, to a limited degree, address the causes of climate change. The societal measures Community- based adaptation and Improving risk reduction policies are also considered as Low Regret, providing self-evident benefits. 2.4. Enhancing societal adaptation Decisive Policy clusters Already implemented in the real-world High effectiveness to reduce climate-related ocean drivers globally (for mitigation actions) Range of low to high effectiveness to reduce impacts/risks locally Relatively limited uncertain- ties, and few disbenefits Currently at concept stage Potentially low to moderate effectiveness to reduce climate-related ocean drivers globally Potentially low to moderate effectiveness to reduce impacts/risks locally Potentially low-to- moderate disbenefits Unproven Risky Currently at concept stage Potentially high effectiveness to reduce climate-related ocean drivers globally Potentially low to high effectiveness to reduce impacts/risks locally Potentially high disbenefits Already implemented in the real-world Low effectiveness to reduce climate-related ocean drivers globally Moderate-to-high effectiveness to reduce impacts/risks locally High non-climatic co- benefits and no-to-very- limited disbenefits Low Regret Ocean-based measures = Mitigation (reducing sources of GHG) Address the causes of climate change Address the impacts of climate change = Mitigation (increasing sinks of GHG) = Solar Radiation management = Ecological/Societal Adaptation Bioenergy with carbon capture and storage Enhancing open-ocean productivity Surface albedo enhancement Relocating & diversifying economic activities Relocating people Restoring and increasing coastal vegetation Restoring and enhancing habitats Reducing atmospheric pollution Pollution reduction Marine renewable energy Infrastructure-based adaptation Improving risk-reduction policies Enhancing weathering and alkalinization Early warning and emergency systems Cloud brightening Assisted evolution Carbon capture and storage Conservation Community-based adaptation Policy clusters Policy clusters Decisive Already implemented in the real-world High effectiveness to reduce climate-related ocean drivers globally (for mitigation actions) Range of low to high effectiveness to reduce impacts/risks locally Relatively limited uncertain- ties, and few disbenefits Low effectiveness to reduce climate-related ocean drivers globally Moderate-to-high effectiveness to reduce impacts/risks locally Hi h li ti Currently at concept stage Potentially low to moderate effectiveness to reduce climate-related ocean drivers globally Potentially low to moderate effectiveness to reduce impacts/risks locally Potentially low-to- moderate disbenefits Currently at concept stage Potentially low to moderate effectiveness to reduce climate-related ocean drivers globally Potentially low to moderate effectiveness to reduce impacts/risks locally Potentially low-to- moderate disbenefits Figure 2. Policy clusters of the main ocean-based measures for climate action. Modiﬁed from Gattuso et al. (2019). 7 Polynesia, Pacific) can be perceived by local populations as useless investments conflicting with more urgent needs (e.g. support to improve roads and houses, or to educational and economic activities), and therefore been disregarded as a risk prevention option. 2.4. Enhancing societal adaptation Making shelters a place for daily life activities, even if not focussed on reducing climate risk (e.g., social and traditional activities), is often critical to sustain the relevance of this measure as an adaptation solution. 3. Policy clusters of ocean-based action However, these actions are not cost-free, requiring well-informed planning and effective coordination over a wide range of spatial and temporal scales. It is critical to note that, in many contexts, Low Regret measures will only be effective under the lowest levels of warming (Bindoff et al., 2019). Unproven measures are illustrated by Marine bioenergy with carbon capture and storage, and some forms of Assisted evolution. The former would use macroalgae (seaweed) or cultivated microalgae as the biomass source for bioenergy. Such measures have potential but their practicality and cost-effectiveness for climatic benefits have yet to be demonstrated. Assisted evolution 8 8 envisages alterations to species and genetics; the feasibility of such actions is uncertain, and they are also considered Risky. Risky measures are mostly defined based on their potentially high disbenefits: Enhancing open- ocean productivity through ocean fertilization; Enhancing weathering and alkalinization, by adding CO2-absorbing materials to the ocean; and the solar radiation management techniques of Marine cloud brightening and Surface albedo enhancement, neither of which would ameliorate ocean acidification. Whilst all these actions have a very large theoretical potential to address climate change globally, only the first one has been tested in the field, with limited success. Risky measures may also have unintended adverse consequences and some are short-lived, implying a long-term commitment. Much more attention needs to be given to their international governance and public acceptability before they can be considered for implementation as climate policy responses. Some societal adaptation measures cannot be classified in a single policy cluster because their effectiveness critically depends on the environmental and societal contexts of their implementation (Fig. 2). While seawalls and other infrastructure-based adaptation can protect against sea level rise in densely populated coasts, they can be counterproductive to natural environments (e.g. sand-dune systems, mangroves and coral reefs) as they tend to undermine ecosystems!#natural adjustments to ocean changes, inland migration, and future ability to provide coastal protection. In the former case, Infrastructure-based adaptation is Decisive while in the latter it is Risky. The benefits of Relocating people are also context-dependent: if planned and at a local-scale (e.g. within an island), such relocation can help save lives, and it is therefore Decisive; but if unplanned or forced, relocation can generate multiple and ramifying detrimental effects on both the displaced people and host communities, and it is therefore Risky. 3. Policy clusters of ocean-based action The same caution applies to Relocating and diversifying economic activities, which can either secure local jobs and economies, or generate the opposite effect; and to Early warning and emergency systems (see above). 4. Ongoing and future activities Several programmes, projects and other activities are currently on-going or planned. First, some of the measures described above are rapidly being implemented. Significant progress is being made on deployment of capacity of marine renewable energy, which was deemed decisive in previous assessments. For example, China built more offshore wind capacity in 2021 than the rest of the world in the last 5 years . Its 26 GW now accounts for half of the world's 54 GW total. 2 Relevant research projects planned or on-going include Ocean Carbon Negative Emissions (ONCE; Jiao at al., 2020) and harvesting energy from waves (Wang, 2017) in China, Ocean-based Negative Emission Technologies (OceanNETs; www.oceannets.eu) and Marine carbon sinks in decarbonisation pathways (CDRmare; https://cdrmare.de/en/) in Europe. It should also be mentioned that the United Nations has proclaimed a Decade of Ocean Science for Sustainable Development (2021-2030) to support efforts to reverse the cycle of decline in ocean health and gather ocean stakeholders worldwide behind a common framework that will ensure ocean science can fully support countries in creating improved conditions for sustainable development of the Ocean (Ryabinin et al., 2019). 9 5. Conclusion It is now clear that the ocean is a key driver of Earth!s climate system and a critical element in the response to rising greenhouse gas. It moderates global warming by absorbing more than 90% of the excess heat and about a quarter of anthropogenic carbon emissions. Ocean-based actions could provide major benefits to society but it is critical that their implementation is sustainable, and that they do not add further stress to the already severe consequences of ocean warming, acidification, deoxygenation and sea level rise. Ocean-related measures should not be considered as a substitute for climate mitigation on land or non ocean-based adaptation measures, which must also be strongly pursued for the benefit of the atmosphere, the ocean and socio-ecological systems worldwide. Ocean-based climate action should prioritise Decisive and Low Regret measures, improve knowledge on the Unproven measures, and very cautiously weigh the Risky ones. 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https://openalex.org/W2055148375	https://dash.harvard.edu/bitstream/1/26640484/1/nejm_11.15.12.pdf	English	null	Reducing Administrative Costs and Improving the Health Care System	New England journal of medicine/The New England journal of medicine	2,012	cc-by	3,438	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 Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:26640484 Published Version doi:10.1056/nejmp1209711 Published Version doi:10.1056/nejmp1209711 Citation Cutler, David, Elizabeth Wikler, and Peter Basch. 2012. “Reducing Administrative Costs and Improving the Health Care System.” N Engl J Med 367 (20) (November 15): 1875–1878. doi:10.1056/nejmp1209711. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility PERSPECTIVE Preparing for Medicare Bundled Payments marks of true success or failure. require treatment. CMS recognizes the importance of random varia- tion and the need for actuarially stable populations in its Medicare Shared Savings Program, in which organizations cannot participate unless they serve at least 5000 beneficiaries. The bundled-pay- ment initiative sets no such lim- its, and medium-sized hospitals typically have 100 to 200 cases for their highest-volume types of Medicare episodes. At these lower volumes, hospitals may have large year-to-year shifts in average spending for particular episode types owing to random variation in illness severity. risk corridors that allow hospi- tals to share both gains and loss- es as they acclimate to the pro- gram. CMS has begun to discuss changes to the proposed finan- cial model with applicants. If hospitals are confident that the program will financially reward successful clinical performance, many more will be willing to pursue the opportunities for care improvement that this program seeks to encourage. The Bundled Payments for Care Improvement Initiative has great potential to engage hospitals in clinical redesign and care coordi- nation that could improve both care and efficiency. Hospitals al- ready have incentives through Medicare’s DRG payment system to make inpatient care more effi- cient, but few have invested in managing care after discharge. The bundled-payment initiative provides an opportunity for hos- pitals to gain experience with coordinating care across a con- tinuum of services for discrete clinical conditions. Nonetheless, program managers must be vigi- lant to ensure that the financial incentives don’t cause stinting on care or avoidance of high-risk pa- tients.4 Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. From the Heller School for Social Policy and Management, Brandeis University, Waltham, MA. For example, our simulations showed that 25% of hospitals with 100 to 125 annual admis- sions for congestive heart failure would incur financial losses of at least 11% simply because their patients in the program’s first year differ from those in the pe- riod used in setting their target prices.3 Another 25% of these hospitals would achieve finan- cial gains of 6.1% or more. Share Your Story Such random variation in illness sever- ity could overwhelm the effect of performance improvements in determining a hospital’s short- term financial outcomes — making early wins and losses artifacts of variation rather than 1. Welch WP. Bundled Medicare payment for acute and postacute care. Health Aff (Millwood) 1998;17:69-81. 2. RTI International. Post-acute care epi- sodes: expanded analytic file data chart book: report to the Assistant Secretary of Planning and Evaluation, June 2011. The success of the initiative will depend on whether it protects participating hospitals against losses resulting from both ran- dom and systematic variation in illness severity. Certain design features will make it much more attractive to hospitals, including risk adjustment, stop-loss pro- tection for high-cost cases, an ability to exclude cases with high- cost primary diagnoses from epi- sode definitions, and so-called g J 3. Tompkins C, Ritter G, Mechanic R, Perloff J, Chapman J. Analysis of financial risk and risk mitigation options in the Medicare Bun- dled Payment for Care Improvement pro- gram. Waltham, MA: Brandeis University, June 28, 2012 (http://sihp.brandeis.edu/ihs/ publications/Bundled%20Payments%20 4. Mechanic RE, Altman SA. Payment reform options: episode payment is a good place to start. Health Aff (Millwood) 2009;28:w262- w271. DOI: 10.1056/NEJMp1210823 Copyright © 2012 Massachusetts Medical Society. From the Heller School for Social Policy and Management, Brandeis University, Waltham, MA. Reducing Administrative Costs and Improving the Health Care System David Cutler, Ph.D., Elizabeth Wikler, B.A., and Peter Basch, M.D. Health Care Efficiency Index 2010 National Progress Report, the UnitedHealth Center for Health Reform and Modernization, the McKinsey Report: Overhauling the U.S. Healthcare Payment System 2007, and Milliman: Electronic Transaction Savings Opportunities for Physician Practices, 2006. † This solution includes electronic adoption and automation of prior authorization. Potential Savings for Health Care Providers from Reforms to Simplify Administrative Activities.* * The estimate for adoption of electronic transactions represents potential savings per year for the electronic processing of claim submissions, eligibility inquiries and requests, claims status requests, payment, and remittance transactions. It assumes uptake of 85%. The amount of savings per physician is based on estimates of the total number of physicians in the United States in 2010, published by the Bureau of Labor Statistics. ACA denotes Affordable Care Act, HITECH Health Information Technology for Eco nomic and Clinical Health Act, and HHS Health and Human Services. Data are from the U.S. Health Care Efficiency Index 2010 National Progress Report, the UnitedHealth Center for Health Reform and Modernization, the McKinsey Report: Overhauling the U.S. Healthcare Payment System 2007, and Milliman: Electronic Transaction Savings Opportunities for Physician Practices, 2006. † This solution includes electronic adoption and automation of prior authorization. with the history of health care, shows how. attempts have been made to use federal policy to lead this sort of reform, they have so far come up short. The Health Insurance Por- tability and Accountability Act (HIPAA) of 1996 introduced man- datory standards for the elec- tronic processing of common ad- ministrative transactions. Before HIPAA, more than 400 different formats were used for electronic health care claims; after HIPAA, there was just one. But HIPAA was not detailed enough, was not implemented rapidly enough, and allowed payers to request addi- tional data from providers. The Department of Health and Hu- man Services (DHHS) was slow to put out HIPAA regulations, and those it issued were suffi- Institute of Medicine (IOM), the United States spends $361 billion annually on health care adminis- tration2 — more than twice our total spending on heart disease and three times our spending on cancer. Also according to the IOM, fully half of these expenditures are unnecessary. Standardization is a central factor in reducing administrative costs. The Federal Reserve low- ered the administrative costs of banking by standardizing the way that computer systems from dif- ferent banks communicated with one another. Reducing Administrative Costs and Improving the Health Care System David Cutler, Ph.D., Elizabeth Wikler, B.A., and Peter Basch, M.D. David Cutler, Ph.D., Elizabeth Wikler, B.A., and Peter Basch, M.D. istrative tasks is one of the most frustrating aspects of modern medicine. T he average U.S. physician spends 43 minutes a day in- teracting with health plans about payment, dealing with formularies, and obtaining authorizations for procedures.1 In addition, physi- cians’ offices must hire coders, who spend their days translating clinical records into billing forms and submitting and monitoring reimbursements. The amount of time and money spent on admin- T Indeed, for the system as a whole, administrative tasks are ex- tremely costly. According to the n engl j med 367;20 nejm.org november 15, 2012 1875 1875 PERSPECTIVE Reducing Administrative Costs Potential Savings for Health Care Providers from Reforms to Simplify Administrative Activities.* Solution Total Annual Savings for Providers ($) Steps Needed to Achieve Savings Adoption of electronic transactions 11.0 billion Implement the ACA, regulate the integration of billing and administrative systems, encourage provider adoption Integrated administrative and clinical health systems 2.1 billion Implement HITECH and expand certification criteria for elec- tronic health records to include administrative provisions National provider enrollment and credentialing program 0.9 billion Implement the ACA and promote regulations to standardize and unify provider enrollment and credentialing sys- tems across public and private entities Standardized reporting requirements 0.1 billion Promote the HHS National Strategy for Quality Improve ment and support regulatory and legislative efforts to standardize federal, state, and private-market reporting requirements Stabilized enrollment in public programs 1.8–2.9 billion Implement the ACA, promote federal and state legislation to further streamline enrollment (including continuous eligibility policies for adults), and endorse regulations to coordinate benefits and networks across programs Widespread automation† 2–6 billion Implement the ACA, work to integrate with HITECH provi- sions, and support public and private innovation Total savings accrued to providers 17.9–23.0 billion Savings per physician 29,000 * The estimate for adoption of electronic transactions represents potential savings per year for the electronic processing of claim submissions, eligibility inquiries and requests, claims status requests, payment, and remittance transactions. It assumes uptake of 85%. The amount of savings per physician is based on estimates of the total number of physicians in the United States in 2010, published by the Bureau of Labor Statistics. ACA denotes Affordable Care Act, HITECH Health Information Technology for Eco nomic and Clinical Health Act, and HHS Health and Human Services. Data are from the U.S. n engl j med 367;20 nejm.org november 15, 2012 * The estimate for adoption of electronic transactions represents potential savings per year for the electronic processing of claim submissions, eligibility inquiries and requests, claims status requests, payment, and remittance transactions. It assumes uptake of 85%. The amount of savings per physician is based on estimates of the total number of physicians in the United States in 2010, published by the Bureau of Labor Statistics. ACA denotes Affordable Care Act, HITECH Health Information Technology for Eco nomic and Clinical Health Act, and HHS Health and Human Services. Data are from the U.S. Health Care Efficiency Index 2010 National Progress Report, the UnitedHealth Center for Health Reform and Modernization, the McKinsey Report: Overhauling the U.S. Healthcare Payment System 2007, and Milliman: Electronic Transaction Savings Opportunities for Physician Practices, 2006. † This solution includes electronic adoption and automation of prior authorization. Reducing Administrative Costs and Improving the Health Care System David Cutler, Ph.D., Elizabeth Wikler, B.A., and Peter Basch, M.D. The ACA requires the DHHS to set more detailed rules for processing administrative interactions. To im- prove compliance and strengthen enforcement, it imposes finan- cial penalties on health plans that don’t adopt these standardized procedures. Currently, providers are not required to adopt elec- tronic transactions, but the hope is that the promise of increased efficiency will promote adoption. If ACA implementation goes as intended and widespread utiliza- tion and automation are achieved, providers could save about $11 bil- lion per year.3 Finally, coverage policies will need to be addressed. Currently, many Medicaid patients “churn” The possibilities for reducing administrative complexity are immense. The reforms we describe could save as much as $20 billion annually for providers (roughly $29,000 per physician), or $40 billion annually for all stakeholders. may be costly to transmit. For example, a provider may bill for magnetic resonance imaging, but a payer’s rules may stipulate that it will approve the charge only if the provider demonstrates that the procedure was medically nec- essary — for example, by sub- mitting documentation of a work- ing diagnosis with relevant test results and prescriptions. Such information is contained only in the medical record. To address this problem, the ACA will make the transmission of claims at- tachments more uniform; how- ever, a more advanced approach would automatically integrate ap- propriate information from bill- ing systems with the correspond- ing clinical information from electronic health records (EHRs). from one policy to another fre- quently. This movement adds ad- ditional expense to practice, since staff in providers’ offices must determine which insurer is cover- ing the patient each month and how much to collect from the in- surer and the patient. If the fed- eral government allowed and en- couraged states to have an annual open-enrollment period, as pri- vate insurers do, coupled with 12-month continuous eligibility policies for nonelderly adults (giving them continuous access to Medicaid coverage even if fam- ily income fluctuates throughout the year), it could save providers nearly $3 billion annually. There are areas that the ACA omitted that could also benefit from standardization. Credential- ing and other systems that are used to establish contracts be- tween providers and health plans are riddled with redundancy, with many organizations collecting vir- tually identical information from providers. Reducing Administrative Costs and Improving the Health Care System David Cutler, Ph.D., Elizabeth Wikler, B.A., and Peter Basch, M.D. Similarly, Walmart made suppliers conform to its computer standards if they want- ed to sell to the retail giant — which led to enormous standard- ization of retail information systems. What can be done to reduce these costs? Though some argue that a single-payer system would eliminate many administrative ex- penses, that solution seems un- likely to be embraced in the United States. Nevertheless, administra- tive expenses are one area of health care in which large sav- ings might be realized particu- larly rapidly (see table). The expe- rience of other industries, along In health care, only one orga- nization has the sort of influence that Walmart and the Federal Re- serve have in their domains: the federal government. But though 1876 n engl j med 367;20 nejm.org november 15, 2012 PERSPECTIVE Reducing Administrative Costs ciently vague that payers and providers never agreed on a truly common standard. Granted, there was only one form, but payers’ additional data requests, which varied both among payers and among health plans offered by a given payer, defeated the stan- dardization effort. The result for providers was a continued mess, which discouraged the investment necessary to achieve electronic interchange. cess, but a coordinated, nation- wide credentialing system that is employed across the public and private sectors could save nearly $1 billion per year for providers. Further savings could be achieved if processes for establishing payer– provider contracts were also stan- dardized and conducted electroni- cally.5 The DHHS could mandate both changes under Section 10109 of the ACA, but so far it hasn’t. There is a mechanism for doing that: the Health Information Tech- nology for Economic and Clinical Health (HITECH) Act, which pro- vides financial incentives for the acquisition of EHRs and specifies standards for the electronic trans- mission of clinical data; the sec- retary of health and human ser- vices could expand these criteria to include standards for electron- ic transmission of administrative data (such as billing information). Doing so could save providers as much as $2 billion annually.5 Standardization will not solve all the problems, however. Payers may be required to accept stan- dard billing forms electronically, but the supplemental informa- tion required to approve a claim Building on recent state-level and private-sector successes, the Affordable Care Act (ACA) took a second bite at the apple, and so far things are going better. Reducing Administrative Costs The cost of administrative com- plexity: administrative intricacies add no value to health care — but the costs keep stacking up. MGMA Connex 2004;4:36-41. 5. Health care cost containment — how technology can cut red tape and simplify health care administration: working paper 2. Minneapolis: UnitedHealth Center for Health Reform and Modernization, 2009. The lack of this kind of lead- ership was a key obstacle to reduc- ing administrative costs through HIPAA. Since the ACA’s passage, the need is greater than ever. Doctors have thousands of rea- sons to hope that reforms are on the way. DOI: 10.1056/NEJMp1209711 Copyright © 2012 Massachusetts Medical Society. DOI: 10.1056/NEJMp1209711 Copyright © 2012 Massachusetts Medical Society. DOI: 10.1056/NEJMp1209711 Copyright © 2012 Massachusetts Medical Society. Reducing Administrative Costs Reducing Administrative Costs form and push through changes that benefit the system as a whole. Only the federal government has the clout in health care to effect these changes. Thus, it may be necessary to establish a senior- level office in the DHHS focused solely on implementation and in- novation in the realm of admin- istrative simplification. Such an office would lead regulatory ef- forts in standardization and inte- gration of billing with clinical records, advocate for federal and state legislation to reduce churn- ing in public programs, and co- ordinate new regulations affect- ing administrative transactions. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. could save as much as $20 billion annually for providers (roughly $29,000 per physician), or $40 bil- lion annually for all stakeholders. And $2 billion of these savings would accrue to the federal gov- ernment — a relatively small but valuable contribution to reducing the deficit. For the individual physician, these savings could translate into more time and re- sources for direct patient care — and therefore into improved pro- fessional satisfaction. From the Department of Economics (D.C.) and the Ph.D. Program in Health Policy (E.W.), Harvard University, Cambridge, MA; and MedStar Health, Washington, DC (P.B.). 1. Casalino LP, Nicholson S, Gans DN, et al. What does it cost physician practices to in- teract with health insurance plans? Health Aff (Millwood) 2009;28:w533-w543. ( ) 2. Yong PL, Saunders RS, Olsen L, eds. The healthcare imperative: lowering costs and improving outcomes — workshop series summary. Washington, DC: National Acad- emies Press, 2010. Unlike other proposals for re- ducing costs, such as restricting access to care, reducing adminis- trative complexity could garner broad bipartisan support. The major stumbling block is that the constituency for administra- tive simplification is broad but diffuse. Achieving these reforms will require bold steps by policy- makers to rise above the commo- tion surrounding health care re- 3. US healthcare efficiency index: national progress report on healthcare efficiency. Nashville: Emodeon Business Services, 2010. 4. Pope C. The cost of administrative com- plexity: administrative intricacies add no value to health care — but the costs keep stacking up. MGMA Connex 2004;4:36-41. 3. US healthcare efficiency index: national progress report on healthcare efficiency. Nashville: Emodeon Business Services, 2010. 4. Pope C. Reducing Administrative Costs and Improving the Health Care System David Cutler, Ph.D., Elizabeth Wikler, B.A., and Peter Basch, M.D. The typical physician spends more than 3 hours annu- ally submitting nearly 18 differ- ent credentialing forms, with staff spending an additional 20 hours.4 Some private-sector initiatives are working to streamline this pro- The possibilities for reducing administrative complexity are im- mense. The reforms we describe 1877 n engl j med 367;20 nejm.org november 15, 2012 PERSPECTIVE The Reciprocity of Recognition — What Medicine Exposes about Self and Other Rita Charon, M.D., Ph.D. left ear. I remembered in a flash how hard the social worker and I had worked to arrange an ENT evaluation despite Mrs. N.’s lack of insurance and citizenship. I remembered the couple barging into my office some weeks later, terrified, crying, breathless, con- vinced that she was going to die. They had been told she needed an MRI to rule out a brain tumor, but they’d left the ENT clinic be- lieving she had a brain tumor. Happily, I could reassure them otherwise. Now, hearing her say “79,” I realized that she was hearing and on her way to speak- M rs. N. announced trium- phantly that she had be- come a U.S. citizen. Her husband — on whose English she depend- ed, though she’d arrived from Al- bania 5 years earlier — said, “She took the test, and then she said the words with her hand like this.” He put his hand over his heart. She grinned and blushed, seeming younger than her 50 years. When she answered one of my questions with a quick “79,” I startled. Wait a minute, I thought, she doesn’t speak English. ing fluent English. I wasn’t sur- prised to find her blood pressure normal, her dizzy spells and head- aches gone. M rs. N. announced trium- phantly that she had be- come a U.S. citizen. Her husband — on whose English she depend- ed, though she’d arrived from Al- bania 5 years earlier — said, “She took the test, and then she said the words with her hand like this.” He put his hand over his heart. She grinned and blushed, seeming younger than her 50 years. When she answered one of my questions with a quick “79,” I startled. Wait a minute, I thought, she doesn’t speak English. Thanks to Mrs. N., I also saw something within myself that I treasure and cultivate and can sometimes claim: the leaning- forward internist, the sleeves- rolled-up doctor. With the ascen- dance of practice-based learning and its emphasis on self- assessment and lifelong learning, medicine has come to value the capacity of practice to provide a mirror not only for occasional self- reflection but for constant self- It was then that she shyly pointed to the hearing aid in her 1878 n engl j med 367;20 nejm.org november 15, 2012
https://openalex.org/W4214823849	https://vtechworks.lib.vt.edu/bitstream/10919/113008/2/Could%20a%20Focus%20on%20the%20Why%20of%20Taxonomy%20Help%20Taxonomy%20Better%20Respond%20to%20the%20Needs%20of%20Science%20and%20Society.pdf	English	null	Could a Focus on the “Why” of Taxonomy Help Taxonomy Better Respond to the Needs of Science and Society?	null	2,022	cc-by	2,967	Could a Focus on the “Why” of Taxonomy Help Taxonomy Better Respond to the Needs of Science and Society? Leighton Pritchard 1, C. Titus Brown 2, Bailey Harrington 1, Lenwood S. Heath 3, N. Tessa Pierce-Ward 2 and Boris A. Vinatzer 4* 1 Strathclyde Institute for Pharmacy and Biomedical Sciences (SIPBS), University of Strathclyde, Glasgow, United Kingdom, 2 Department of Population Health and Reproduction, University of California, Davis, Davis, CA, United States, 3 Department of Computer Science, Virginia Tech, Blacksburg, VA, United States, 4 School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, United States Genomics has put prokaryotic rank-based taxonomy on a solid phylogenetic foundation. However, most taxonomic ranks were set long before the advent of DNA sequencing and genomics. In this concept paper, we thus ask the following question: should prokaryotic classification schemes besides the current phylum-to-species ranks be explored, developed, and incorporated into scientific discourse? Could such alternative schemes provide better solutions to the basic need of science and society for which taxonomy was developed, namely, precise and meaningful identification? A neutral genome-similarity based framework is then described that could allow alternative classification schemes to be explored, compared, and translated into each other without having to choose only one as the gold standard. Classification schemes could thus continue to evolve and be selected according to their benefits and based on how well they fulfill the need for prokaryotic identification. Edited by: Iain Sutcliffe, Northumbria University, United Kingdom Reviewed by: Barny Whitman, University of Georgia, United States Stephanus Nicolaas Venter, University of Pretoria, South Africa Correspondence: Boris A. Vinatzer vinatzer@vt.edu Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology Received: 01 March 2022 Accepted: 29 April 2022 Published: 19 May 2022 Specialty section: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology PERSPECTIVE published: 19 May 2022 doi: 10.3389/fmicb.2022.887310 PERSPECTIVE published: 19 May 2022 doi: 10.3389/fmicb.2022.887310 PERSPECTIVE published: 19 May 2022 doi: 10.3389/fmicb.2022.887310 Received: 01 March 2022 Accepted: 29 April 2022 Published: 19 May 2022 Received: 01 March 2022 Accepted: 29 April 2022 Published: 19 May 2022 THE WHY OF TAXONOMY In an insightful article in 2021 in the International Society of Microbial Ecology Journal, Hugenholtz and colleagues provided a comprehensive review of the history of prokaryotic taxonomy and highlighted current and future challenges (Hugenholtz et al., 2021). The article contributed rich context for the ongoing debate over taxonomy and nomenclature, in particular, in regard to the uncultured majority of prokaryotes (Rinke et al., 2013), and built a strong argument for genome-based taxonomy. However, we believe that improving taxonomy using genomics should not stop us from more fundamentally rethinking both its structure and applications and answering the question of why we practice taxonomy in the first place.h Keywords: taxonomy, genomics, identification, prokaryotes, phylogeny, taxonomic ranks, species Citation: Pritchard L, Brown CT, Harrington B, Heath LS, Pierce-Ward NT and Vinatzer BA (2022) Could a Focus on the “Why” of Taxonomy Help Taxonomy Better Respond to the Needs of Science and Society? Front. Microbiol. 13:887310. doi: 10.3389/fmicb.2022.887310 i The why of taxonomy becomes clear when we consider all three elements of taxonomy: classification, nomenclature, and identification (Cowan, 1965). Importantly, only when taxonomy permits identification of an unknown as a member of a named group, a taxon, with characteristics that are distinct and relevant (and, therefore, predicts that the unknown has these same distinct May 2022 \| Volume 13 \| Article 887310 Frontiers in Microbiology \| www.frontiersin.org 1 Pritchard et al. Why of Taxonomy Some of the assumptions relating to genome-based taxonomy can also be questioned if we consider the why of taxonomy. For example, an assumption that phylogenetic clades based on the alignment of core genes or proteins should be the only basis for the circumscription of named groups prioritizes vertical transfer of genomic information. However, in many societally-important circumstances, such as antimicrobial resistance or the presence of pathogenicity islands, the phenotype of interest may be governed by horizontally-transferred genes (Soucy et al., 2015). In these circumstances, a classification that prioritizes the phenotype and considers lateral transfer of genomic information might be more useful. Such a classification might be better facilitated by a system of individual genome accessions with flexible labelling (like tags in a Google Mail inbox) than a hierarchical naming scheme. Hugenholtz et al. (2021) argue that hierarchical taxonomic ranks and binomial species are a necessity because of biologists’ reluctance to take up new systems, such as the rank-free PhyloCode (Cantino and de Queiroz, 2020). However, reluctance to change is not an argument against the utility of change and, in any case, might very well apply more to taxonomists than to biologists overall. and relevant characteristics), can it answer to scientific and societal needs. In this context, classification becomes a prerequisite for meaningful identification by creating clear and distinct boundaries of practical relevance between groups of microbes. The need for nomenclature also follows from identification as unambiguous naming is critical for effective communication, and possibly societal action (e.g., selection of the appropriate clinical treatment), following identification. i Here are some examples of what we intend by meaningful identification of microbes. Citation: In basic science, identification may simply consist in finding the position of an organism in a phylogenetic tree to reveal its evolutionary relationship to other organisms. Beyond single organisms, reliable identification of community members may delineate community structure to understand system-level responses central to the environmental roles of microbes. From a societal and policy-making perspective, meaningful identification of an unknown as a member of a taxon may predict a threat, such as the potential to cause disease, and may trigger regulatory action under national or international laws, such as import/export restrictions or implementation of quarantine or isolation. On the other hand, identification as a member of a group with beneficial characteristics, may lead to intellectual property protection. Therefore, from the perspective of identification, it becomes clear that taxonomy answers important needs in both basic science and society. Nonetheless, there are challenges inherent to taxonomy that cannot be avoided. Circumscription of existing groups requires revision as new knowledge becomes available, and such reclassification necessarily requires translation from one named taxon to another, for example, when reading literature about the same organism before and after reclassification occurred. Also, there is more than one reasonable hierarchical classification system. For this reason, and because we do not have perfect knowledge and understanding of the hierarchical process of evolution, it is inevitable that no single human-created model will capture all useful categorizations of organisms—regardless of the claim that “biologists now agree that taxonomy should be based on evolutionary relationships as the most natural way of arranging organisms” (Hugenholtz et al., 2021). QUESTIONING THE CURRENT PRACTICE OF TAXONOMY When we focus on identification, aspects of current taxonomy that some taxonomists may consider to be unchangeable tenets and needs of taxonomy can be seen to serve only the how of the current practice of taxonomy, but not the why of taxonomy itself. In our view, several elements of classical taxonomy can be reassessed on these grounds, such as: the reliance on historic and subjective taxonomic ranks when a different number and distribution of ranks might be more useful; making a distinction between “taxonomy” and “strain typing” when the boundary between the two is arguably subjective; considering species and subspecies to be the smallest recognizable taxonomic units when the possession of laterally-transferred functional regions, such as pathogenicity islands, may be a more appropriate reason for demarcating a group of isolates; requiring name-bearing type strains to describe species when the vast majority of prokaryotes are likely fastidious or otherwise practically unculturable; the attachment to Latin binomials which draw attention to a limited set of properties; and the insistence on a single scheme of stable and unique hierarchical names to describe a collection of items that could be partitioned usefully in many other ways. In the current era of databases that can provide persistent stable IDs to individuals, we believe it is time for microbiology to adopt the advantages that technology brings to data organization, while preserving the best features of classical taxonomy. Frontiers in Microbiology \| www.frontiersin.org WHERE TO GO FROM HERE? If we accept that our current taxonomic system is conditioned by history and just one of many reasonable choices, perhaps we could dare to try some unconventional solutions to answer the why of taxonomy within the landscape of genomic data. For example, what if we acknowledged that the distinction between named species and subspecies on one hand and informal within-species clades, clonal complexes, and outbreak strains on the other hand is subjective and these taxa could all be handled using a single set of rules? Would it help us understand biology better if we were to give species complexes, within-species clades, and other monophyletic groups, the same importance as current taxonomic ranks, as proposed in the PhyloCode (Cantino and de Queiroz, 2020)? What if we could use a neutral framework of genome identifiers to explore and compare new ways to infer May 2022 \| Volume 13 \| Article 887310 Frontiers in Microbiology \| www.frontiersin.org 2 Why of Taxonomy Pritchard et al. a coordinate in genome space can be automatically assigned and used as an identifier even before the genome is classified as a member of an already described, or still to be named, taxonomic group. If this genome is of an emerging pathogen, the identifier can be used for clear communication about an ongoing disease outbreak from the moment the genome has been sequenced without having to wait for a validly published name. An example from virology is that one cannot search for the earliest reports of SARS-CoV-2 by searching for “SARS-CoV-2” since scientists referred to it as “2019-nCoV” until the Coronaviridae study group of the International Committee on Taxonomy of Viruses decided on its name (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020). A LIN-based identifier for the first SARS-CoV-2 genome, as we propose, may have enabled ready tracing of SARS-CoV-2 genomes, without the confusion imposed by nomenclature changes. evolutionary relationships between organisms, beyond core gene phylogenies? What value could be gained from approaches that combine genome similarity with similarity in gene content reflecting adaptation to different ecological niches? Is it possible to construct complementary phylogenetic or even non-phylogenetic classification systems, similar to library cataloging systems, such as the subject-based Dewey system? WHERE TO GO FROM HERE? Specialized schemes could be based on the content of functional classes of genes (corresponding to subjects in the Dewey system), such as pathogenicity/virulence genes that are important in a biosecurity context, where the risk is governed by gene content more than evolutionary relationship. p To implement, compare, and perhaps unify these alternative classification schemes, we would need a “Rosetta Stone” to translate between them. We are thus building a “genomic coordinate system” as part of the genomeRxiv platform using the Life Identification Number (LIN) approach (Vinatzer et al., 2017; Tian et al., 2020) analogous to a map grid reference, which hierarchically subdivides and labels the entire prokaryotic genome space into uniquely-labelled volumes (or voxels) of sequence-similar genomes that, at their finest resolution, contain a single genome uniquely identified with its “coordinate” (its LIN). We believe that this is a practical, quantitative, automatable, stable, and robust solution to the problem of translating among classification schemes, for example, between validly published prokaryotic named species and genome-based species clusters (Sanford et al., 2021). More in general, classifications of prokaryote genomes made by one scheme, such as descent from a common ancestor, or “bona fide species definitions,” can then be expressed as combinations of uniquely-labelled voxels and compared to similar combinations obtained by alternative classification schemes, such as presence or absence of specific genes. For example, besides assigning plant growth promoting bacteria to named species based on common ancestry, they could also be assigned to a taxon called “plant growth promoters.” When additional bacteria are found to promote plant growth, they could be added to this function- based taxon. The LINs of the genomes reclassified as “plant growth promoters” however would not change, maintaining stability. In conclusion, we propose to treat genome sequence data neutrally to build a genotypic framework. We do not propose to privilege a specific set of genes, or a specific evolutionary model or reconstruction method as an immutable truth, or “gold standard,” against which all other schemes would be measured. Instead, we propose a whole-genome framework on which alternative choices of phylogenetic and phenotypic classification schemes can be compared. Like a coordinate system on a map, this framework provides an address for each genome and links to any classification information within any taxonomic system. WHERE TO GO FROM HERE? We expect this framework to provide a landscape on which classification systems that best respond to the needs of science and society can continue to be developed based on the latest biological, biotechnological, and ecological discoveries. 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. AUTHOR CONTRIBUTIONS The LIN-based coordinate scheme is hierarchical, but purely descriptive. It neither requires nor imposes an evolutionary model. It is neutral on the questions of nomenclature and classification. It requires no consensus on a single scheme but instead enables meaningful translation among alternative schemes. For example, the validly published species names, informal phylotypes, and population clusters within the Ralstonia solanacearum species complex were all circumscribed using LINs allowing the identification of any newly sequenced genome as a member of each of these taxa, simultaneously (Sharma et al., 2022). Our proposal is also by nature able to accommodate the many, as yet unknown and unclassified, prokaryotes whose sheer number currently poses a significant problem for nomenclature and classification. So long as a genome sequence is available, LP, CB, BH, LH, NP-W, and BV: concept development and writing. All authors contributed to the article and approved the submitted version. Frontiers in Microbiology \| www.frontiersin.org REFERENCES members of crowdsourced taxa. Nucleic Acids Res. 48, W529–W537. doi: 10.1093/nar/gkaa190 members of crowdsourced taxa. Nucleic Acids Res. 48, W529–W537. doi: 10.1093/nar/gkaa190 Vinatzer, B. A., Tian, L., and Heath, L. S. (2017). A proposal for a portal to make earth’s microbial diversity easily accessible and searchable. Antonie Van Leeuwenhoek 110, 1271–1279. doi: 10.1007/s10482-017-0849-z Cantino, P. D., and de Queiroz, K. (2020). International Code of Phylogenetic Nomenclature (PhyloCode): Version 6. 1st Edn. Boca Raton, Florida, USA: CRC Press. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses (2020). The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 5, 536–544. doi: 10.1038/s41564-020-0695-z Conflict of Interest: Life Identification Number and LIN are registered trademarks of this Genomic Life Inc. LH and BV report in accordance with Virginia Tech policies and procedures and their ethical obligation as researchers, that they have a financial interest in this Genomic Life Inc. that may be affected by the publication of this manuscript. They have disclosed those interests fully to Virginia Tech, and they have in place an approved plan for managing any potential conflicts arising from this relationship. Cowan, S. T. (1965). Principles and practice of bacterial taxonomy. J. Gen. Microbiol. 39, 143–153. doi: 10.1099/00221287-39-1-143 Hugenholtz, P., Chuvochina, M., Oren, A., Parks, D. H., and Soo, R. M. (2021). Prokaryotic taxonomy and nomenclature in the age of big sequence data. ISME J. 15, 1879–1892. doi: 10.1038/s41396-021-00941-x The remaining 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. Rinke, C., Schwientek, P., Sczyrba, A., Ivanova, N. N., Anderson, I. J., Cheng, J.-F., et al. (2013). Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437. doi: 10.1038/ nature12352 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Sanford, R. A., Lloyd, K. G., Konstantinidis, K. T., and Löffler, F. E. (2021). Microbial taxonomy run amok. Trends Microbiol. 29, 394–404. doi: 10.1016/j. tim.2020.12.010 Sharma, P., Johnson, M. A., Mazloom, R., Allen, C., Heath, L. S., Lowe-Power, T. FUNDING Development of the genomeRxiv platform is supported by the United States National Science Foundation (DBI-2018522) and the United Kingdom Biotechnology and Biological Sciences Research Council (BB/V010417/1). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. May 2022 \| Volume 13 \| Article 887310 Frontiers in Microbiology \| www.frontiersin.org 3 Why of Taxonomy Pritchard et al. Frontiers in Microbiology \| www.frontiersin.org REFERENCES M., et al. (2022). Meta-analysis of the Ralstonia solanacearum species complex (RSSC) based on comparative evolutionary genomics and reverse ecology. Microb. Genom. 8. doi: 10.1099/mgen.0.000791 Copyright © 2022 Pritchard, Brown, Harrington, Heath, Pierce-Ward and Vinatzer. 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) 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. Soucy, S. M., Huang, J., and Gogarten, J. P. (2015). Horizontal gene transfer: building the web of life. Nat. Rev. Genet. 16, 472–482. doi: 10.1038/ nrg3962 Tian, L., Huang, C., Mazloom, R., Heath, L. S., and Vinatzer, B. A. (2020). LINbase: a web server for genome-based identification of prokaryotes as May 2022 \| Volume 13 \| Article 887310 Frontiers in Microbiology \| www.frontiersin.org
https://openalex.org/W2002465847	https://zenodo.org/records/5420814/files/source.pdf	English	null	Assessment of condition in pejerrey Odontesthes bonariensis (Atheriniformes: Atherinopsidae) populations: which index works best?	Neotropical ichthyology/Neotropical Ichthyology	2,009	cc-by	6,136	Assessment of condition in pejerrey Odontesthes bonariensis (Atheriniformes: Atherinopsidae) populations: which index works best? Claudio R. M. Baigún1, Darío C. Colautti1 and Fabian Grosman2 The pejerrey Odontesthes bonariensis is the most important target species in temperate freshwater fisheries of Argentina, and assessment of condition has been a regular practice and common diagnostic tool. Most pejerrey fishery studies have used Fulton’s (K) index, without testing whether underlying assumptions or requirements were met. We analyzed and contrasted the applicability of K, Kn and Wr indices to assess condition status in several pejerrey populations inhabiting Pampean lakes. Our results showed that whereas Wr and Kn displayed significant condition changes across length at some study lakes, Kn portrayed a small range of variation. We also noted that pejerrey maximum length and size structure strongly varied among populations probably due to the characteristics of trophic niche changes through lifespan, depending on lake limnological characteristics and zooplankton availability. We conclude that the K index should be disregarded in those cases where populations show allometric growth and size ranges strongly vary. In turn, the Kn index appears to be only appropriate for regular within population assessment, being difficult to apply when comparisons between populations are needed and when they exhibit different weight-length relationship slopes. Finally, the Wr index should be strongly preferred if the objective is to perform comparisons between pejerrey populations, particularly when population structure is not well known, stocking has been used for population recovery, lakes are strongly supported by limnological changes, data are limited to only one or few samplings and metaphoetesis is suspected in pejerrey populations. O peixe-rei Odontesthes bonariensis é a espécie mais importante na pesca de água doce da região temperada da Argentina, e a avaliação de sua condição corpórea tem sido uma prática normal e uma ferramenta de diagnóstico bastante utilizada. Grande parte dos estudos sobre a pesca do peixe-rei utiliza o índice de Fulton (K), sem testar se seus pressupostos são atendidos. Nós analisamos e comparamos a aplicabilidade dos índices K, Kn e Wr para a avaliação do fator de condição em várias populações de peixe-rei em lagos dos Pampas. Nossos resultados mostram que, enquanto o Wr e o Kn dependeram significativamente do comprimento corporal em alguns lagos, o Kn apresentou uma pequena amplitude de variação. Foi também notado que o comprimento máximo e a estrutura de tamanho variaram fortemente entre populações, provavelmente devido às mudanças ontogenéticas no nicho trófico, dependendo das características limnológicas e da disponibilidade de zooplâncton. 1IIB-INTECH, Camino de Circunvalación Laguna, Km 6, 7130 Chascomús, Argentina. claudiobaigun@intech.gov.ar 2Instituto Multidisciplinario sobre Ecosistemas y Desarrollo Sustentable, Facultad Ciencias Veterinarias, Universidad Nacional del Centro, 7300 Azul, Argentina. Neotropical Ichthyology, 7(3):439-446, 2009 Copyright © 2009 Sociedade Brasileira de Ictiologia Neotropical Ichthyology, 7(3):439-446, 2009 p y gy ( ) Copyright © 2009 Sociedade Brasileira de Ictiologia Introduction The increasing importance of pejerrey recreational fisheries and the development of some commercial fisheries in large shallow Pampean lakes have challenged managers and biologists to provide comprehensive management guidelines and cues for assessing pejerrey population status. This is a critical issue since Pampean lakes exhibit highly dynamic changes in their trophic status related to natural hydrological modifications associated with rain cycles (López et al., 2001). Such modifications may affect pejerrey populations by changing recruitment, mortality, growth patterns, food availability and, ultimately, body condition. Since rapid assessment methods for pejerrey population have included condition estimates as a regular and effective diagnostic method, even outside Pampean lakes and also for culture purposes, it is therefore essential to review and define how well different indices portray population condition under different ecological scenarios and population characteristics. The aim of this study was to analyze and discuss the application of the most common condition indices used for pejerrey populations in Pampean lakes, testing their application for regular fish population assessment. We also analyze here how lake ecological characteristics may influence condition assessments and how interpretation may be affected by intrinsic index characteristics. The increasing importance of pejerrey recreational fisheries and the development of some commercial fisheries in large shallow Pampean lakes have challenged managers and biologists to provide comprehensive management guidelines and cues for assessing pejerrey population status. This is a critical issue since Pampean lakes exhibit highly dynamic changes in their trophic status related to natural hydrological modifications associated with rain cycles (López et al., 2001). Such modifications may affect pejerrey populations by changing recruitment, mortality, growth patterns, food availability and, ultimately, body condition. Since rapid assessment methods for pejerrey population have included condition estimates as a regular and effective diagnostic method, even outside Pampean lakes and also for culture purposes, it is therefore essential to review and define how well different indices portray population condition under different ecological scenarios and population characteristics. A common practice in freshwater fisheries biology is to scrutinize fish condition as an indicator of well being and for comparing populations or stocks. Condition indices portrayed by morphometric measurements represent a basis for developing an explanatory hypothesis about biological responses or different ecological scenarios for populations (Liao et al., 1995). Several indices have been proposed in the literature (Bolger & Connolly, 1989) and reviewed by Froese (2006). Material and Methods During January-February 2004, we sampled pejerrey populations from eight Pampean lakes located in Buenos Aires Province (Fig. 1). At each lake, three sites (inshore and open waters) were sampled using two sets of standardized gill-net with mesh sizes of 30, 38, 42, 50, 60, 66, 76 and 80 mm) measured between opposite knots, each with a length of 25 m, and a seine beach net, with the aim of obtaining representative samples of fish populations and fish community. Capture per unit effort was estimated from gill nets considering one fishing night (12 h) as a standard effort unit and mesh size (Baigún, 1989). g p y ( ) Most pejerrey studies have focused on the use of Fulton’s (K) index (Ricker, 1975) and the cephalic index (Ringuelet, 1964). The K index has been used, for example, by Freyre et al., (1969) in Chascomús, Salada Grande, El Carpincho, Monte, Alsina and Cochicó lakes and by Grosman & Sergeña (1996) in the Encadenadas de Tornquist lakes. Several other examples are found in technical reports prepared by the Undersecretary of Fishing Activities of Buenos Aires province (http:// www.maa.gba.gov.ar/pesca/index.php), which performed regular surveys in different lakes for several years. Since some of these surveys were carried out on a punctual basis, even comparing condition among populations from different lakes, it seems necessary to clarify the constraints of using condition indices based on the characteristics of samples and populations. Other alternatives such as relative weight (Wr) (Wege & Anderson, 1983) have been neglected or ignored, despite the fact that such index has become popular in assessing North American fish populations (Blackwell et al., 2000). For pejerrey, Baigún & Anderson (1994) provided for the first time the required standard relationship for the species, whereas Colautti et al. (2006) developed a standard regression for only Pampean lake populations. The weight (W), standard length (SL) and total length (TL) of the fishes were measured with 1 g or 1 mm precision. Thirty pejerrey stomachs were collected per lake and preserved in 10% formalin for determining food items. Assessment of condition in pejerrey Odontesthes bonariensis (Atheriniformes: Atherinopsidae) populations: which index works best? Concluímos que o índice K deve ser desconsiderado nos casos em que as populações apresentam variações alométricas marcantes no crescimento e na amplitude de tamanhos. O índice Kn parece ser apropriado apenas para avaliações de uma única população, não sendo apropriado para comparações entre populações quando elas exibem diferentes inclinações na relação peso- comprimento. Finalmente, o índice Wr é recomendado se o objetivo for fazer comparações entre populações de peixe-rei, particularmente quando a estrutura populacional não é bem conhecida, a estocagem tenha sido usada para a recuperação populacional, os lagos apresentam grandes variações limnológicas, os dados se limitam a uma ou poucas amostras e suspeita- se da ocorrência de mudanças ontogenéticas na dieta. Key words: Fish plumpness, Pampean lakes, Fishery management. 439 440 Assessment of condition in Odontesthes bonariensis: which index works best? Introduction Physiological indices such as hepatosomatic, mesenteric and visceral indices represent a direct measure of fish nutritional status, whereas indirect indices such as condition factor, relative condition factor and relative weight provide external measurements of overall health (Brown & Murphy, 2004). Such indices have been widely used as indicators of the nutritional state of fish (Gutreuter & Childress, 1990), assuming that condition changes are a consequence of physiological or ecological alterations (Sutton et al., 2000). Some constraints for the application of condition indices are obvious, but several limitations are more subtle and are therefore usually ignored. The aim of this study was to analyze and discuss the application of the most common condition indices used for pejerrey populations in Pampean lakes, testing their application for regular fish population assessment. We also analyze here how lake ecological characteristics may influence condition assessments and how interpretation may be affected by intrinsic index characteristics. Condition assessment has been a regular practice and has been applied as a diagnostic tool for pejerrey (Odontesthes bonariensis) population surveys (Freyre, 1976). The pejerrey is the most valuable target species in most of the warm temperate lakes and reservoirs of Argentina. This species originally inhabits lakes and lagoons of Pampean region (Argentina) and Rio Grande do Sul, Brazil (Dyer, 2006), but has also been successfully stocked in other basins of Brazil, Argentina and even other South American countries due to its fishing values. The high quality of pejerrey has also encouraged its culture in places far from its native distribution range, such as Japan and Italy (Somoza et al., 2008). p j y Wr = (W/Ws).100 The Ws value derived from a standardized equation represents the weight of a fish exhibiting better than average for a given species. According to Anderson & Gutreuter (1983), a Ws value of 100 ± 5 should be considered indicative of an optimum condition within a physiological and ecological framework. Baigún & Anderson (1994) defined a standard regression based on populations inhabiting temperate lakes and reservoirs of Argentina. However, to estimate Ws values, we used the equation presented by Colautti et al. (2006) established for only Pampean pejerrey populations using the regression-line-percentile technique (Murphy et al., 1990). This equation that predicts weight from standard length is: log Ws= 5.2669 + 3.1625 log SL Fig. 1. Geographical location of sampled lakes in the Pampean region of Argentina. by measuring conductivity, dissolved oxygen and lake area (Table 1). Zooplankton composition at each lake was also characterized by estimating the abundance of cladocerans and copepods (macrozooplankton) and determining the qualitative food items in pejerrey stomachs. Samples were taken in at least two locations per lake by filtering twenty liters each in order to determine organisms/l using the methods described in Paggi & Paggi (1995). To determine if index values were related to fish length, we tested the significance of the slope regression between all pooled data. We also estimated mean values and their respective coefficient of variation by length interval and pooled lengths for each lake. Length intervals were obtained from Baigún & Anderson (1994) who defined a stock, preferred, quality, memorable and trophic length range. Total length determined by these authors was transformed to standard length. However, we added a substock group which was defined as the number of fish between 12 cm and 21 cm standard length. To determine the trends in variation of each index as a function of length, we plotted the individual index data and displayed also the average index values for each length category by lake. Finally, to assess size distribution and its potential effect on condition, we also estimated a proportional reproductive stock index (PRS). The PRS is defined as the ratio between the number of pejerrey larger than 21 cm (Ls) and the number of pejerrey equal to or larger than 12 cm. The PRS is conceptually equivalent to the proportional stock density index (Anderson, 1980) and is a suitable indicator of size distribution within a population. Material and Methods In order to test pejerrey condition for each population and to compare the results, we applied different indices as summarized below: a) Fulton’s condition index (K): a) Fulton’s condition index (K): Fulton’s index (Ricker, 1975) is the ratio of the observed weight (W) at a specific length with respect to a fish whose length (L) increases following a cubic (isometric) relationship (Ricker, 1975). The formula for this index is: ( , ) K = (W/L3).C where C is a constant, usually 105 when metric units are used. b) Relative condition factor (Kn) Le Cren (1951): Kn = W/We where C is a constant, usually 105 when metric units are used. ( ) Kn = W/We where (We) is the length specific mean weight predicted by the population weight-length relationship, i.e., We = a Lb, where a and b are the regression coefficients. Unlike the Fulton 441 C. R. M. Baigún, D. C. Colautti & F. Grosman Fig. 1. Geographical location of sampled lakes in the Pampean region of Argentina. index, the Le Cren index considers that weight could increase in an allometric way if b differs from 3. The Kn index represents the true population variation around the mean condition value independent of fish size, allowing comparisons of different fish from the same sample, independent of length. c) Relative weight (Wr): This index, originally developed by Wege & Anderson (1978), represents a refinement of Kn and is defined as the ratio of the weight (W) of a fish to the length specific standard weight (Ws) predicted by a weight-length regression for the species, which must be obtained using the regression-line- percentile (RLP) developed by Murphy et al. (1991). Unlike the simple regression required by the Le Cren index, RLP uses data from different populations fitting a curve to the 75th-percentile weights and must meet several requirements (Murphy et al., 1990; Brown & Murphy, 1996). Such constraints were considered by Colautti et al. (2006), who developed a standard regression for pejerrey in Pampean lakes. In accordance with these authors, we used 12 cm as the minimum reliable size for pejerrey Wr estimation. p j y Wr = (W/Ws).100 Results The study lakes differed in general limnological characteristics displaying the mosaic of environmental conditions usually exhibited by Pampean lakes. Also, general fish abundance and pejerrey composition varied according to lake (Table 1). In turn, Table 2 exhibits the parameters a (intercept), b (regression coefficient), r (correlation coefficient), and N (number of specimens analyzed) of the length-weight relationships, whereas statistical parameters of the indices used are shown in Table 3. Mean K index showed values greater than 1 in almost all lakes, whereas mean Wr exhibited values smaller than 100 except in only one lake. A Wr between 95 and 100 has been considered indicative of good condition in the literature (Wege & Anderson, 1978). In turn Kn values ranged around 1 and, as expected, exhibited the lowest variation. Also, general limnological characteristics were obtained Assessment of condition in Odontesthes bonariensis: which index works best? 442 Fig. 2. Relationship between standard length and (a) K index, (b) Kn index and (c) Wr index for pejerrey populations in Pampean lakes. In a first attempt to determine to what extent condition indices vary across length, we analyzed the relationship between the different indices and pejerrey length for the pooled populations (Fig. 2). Only the K-length regression showed a significant correlation and slope (r = 0.32; p < 0.0001). However, since K has been the most used index but at the same time has demonstrated instability due to ignoring allometric growth, we explored in more detail to what extent condition values were affected across length in individual lakes (Fig. 3). In Colon and Igartua lakes, the general condition of fish was good, where Wr and Kn followed a similar trend but differed from K. In Puan and Los Chilenos lakes where both K and Wr increased with length, the K slope was larger and population values showed a broader range compared to Wr. For both indices, there was a large contrast between substock and memorable fish size condition, changing for instance the K index from 0.9 to 1.84 and Wr index from 74 to 125 in Los Chilenos Lake. In turn, Kn did not show a significant variation across length. In both lakes, pejerrey populations were dominant and those individuals larger than 200 mm showed a piscivorous diet, with condition values being above the average condition and the highest among sampled populations. Results In La Salada Lake, Wr and K exhibited the lowest mean values, which can be related to very low zooplankton abundance and lack of piscivory. Finally, in Segovia and Tranier lakes, all indices showed same variation in profiles. The K and Wr indices assumed high mean values suggesting a good condition across length. Nonetheless, Kn remained around one, and followed closely the Wr variation due to the b parameter of the weight-length equations being very similar to the b of the pejerrey standard weight equation. In both lakes, pejerrey appeared as the highly dominant species with zooplankton abundance also being high. Fig. 2. Relationship between standard length and (a) K index, (b) Kn index and (c) Wr index for pejerrey populations in Pampean lakes. Discussion Lake N a b r2 Tranier 196 -5.176 3.136 0.992 Puan 160 -5.971 3.440 0.994 Los Chilenos 162 -6.371 3.604 0.988 La Segovia 117 -5.245 3.147 0.976 La Brava 190 -4.972 3.006 0.986 Igartua 391 -5.066 3.071 0.978 Colon 176 -5.107 3.080 0.988 La Salada 137 -4.788 2.890 0.993 Such constraints are probably a main limitation for pejerrey population assessment on a regional scale, since lakes strongly differ in terms of ecological characteristics, thus influencing fish bionomic features (growth, length, weight). Moreover, its application would not be recommended where only punctual or occasional surveys are performed. As proposed by Froese (2006), the Kn index should be used only within samples, and it could be useful and applicable where species condition is monitored for long time periods in the same population. However, it could be indicative of spawning period and/or decrease in foraging activity (Gomiero & Braga, 2005). Since in Pampean lakes, managers are often interested in allocating both recreational and commercial activity, Kn index appears to be of little value to support decisions based on length or growth comparisons among lakes. Table 3. Statistical parameters of K, Kn and Wr indices in sampled lakes. SD: standard deviation. Index Colon Igartua Puan Los Chilenos La Brava La Salada La Segovia Tranier Mean Wr 94.17 97.82 89.04 81.34 80.60 70.64 97.07 107.13 Max Wr 117.45 146.86 123.33 125.40 104.92 82.61 120.39 126.17 Min Wr 69.28 59.88 71.01 48.79 59.69 59.84 66.81 86.98 SD Wr 8.33 7.81 11.84 17.29 7.09 5.601 11.85 8.15 Mean K 1.18 1.24 1.16 1.04 1.10 0.99 1.27 1.37 Max K 1.54 1.85 1.80 1.84 1.43 1.00 1.67 1.65 Min K 0.84 0.77 0.86 0.53 0.87 0.82 0.89 1.01 SD K 0.13 0.11 0.23 0.30 0.10 0.053 0.17 0.13 Mean Kn 1.00 1.00 1.00 1.01 1.00 1.00 1.01 1.00 Max Kn 1.23 1.51 1.26 1.36 1.29 1.10 1.25 1.18 Min Kn 0.73 0.62 0.79 0.67 0.78 0.90 0.69 0.82 SD Kn 0.09 0.08 0.09 0.14 0.08 0.053 0.12 0.08 In turn, Wr application has not been a common practice Fig. 3. Relationship between K, Kn and Wr values and length intervals for study lakes. average conditions. However, most of these studies obtained unrealistic condition values since slope of weight-length relationship was rarely isometric (b = 3). For example, slopes from weight-length relationships computed from 74 pejerrey populations in different lakes or time periods presented by Colautti et al. Discussion In this paper, we tested the ability of different indices to assess pejerrey condition in Pampean lakes. We compared the application of such indices during the summer period when pejerrey populations do not exhibit reproductive patterns, thus removing the effects of gonad development on fish weight. Condition indices have been historically used by biologists as surrogate indicators of changes in physiological characteristics related to bioecological factors. In Pampean lakes by far, the K index has been used as the main index for assessing pejerrey populations relating condition with trophic conditions, and spawning periods. Moreover, some previous studies presented K values associated with their confidence limits across length as suggested by Freyre (1976) to determine departures from Table 1. Limnological characteristics and pejerrey population features in study lakes. Z: zooplankton; F: fish; I: insects. PRS: proportional reproductive stock. CPUE: catch per unit effort. Table 1. Limnological characteristics and pejerrey population features in study lakes. Z: zooplankton; F: fish; I: insects. PRS: proportional reproductive stock. CPUE: catch per unit effort. Lake Colon Igartua La Brava Segovia Puan Los Chilenos Tranier La Salada Area (ha) 36 51 420 89 793 450 57 5500 Conductivity (µS/cm) 5970 1980 963.3 2210 3380 1500 624 6896 Zooplankton (individuals x10-3/100L) 1.38 150 176.44 217.73 42.68 17.6 150.63 12.5 Species richness 9 5 6 3 2 7 2 6 CPUE 24.30 28 43.13 14.52 12.63 4.22 52.60 37 Pejerrey population characteristics PRS 31.5 48 97 37 50 27 58.4 47 % pejerrey 19.7 99.5 51.93 95 97.7 87.5 99.5 66.5 Food items <200mm Z I Z Z I Z I Z Z Z Z Food items >200mm F Z Z Z F F F Z Z C. R. M. Baigún, D. C. Colautti & F. Grosman 443 Table 2. Estimated parameters of the weight-length- relationships for the studied lakes. N: number of sampled fish; a: intercept; b: regression coefficient; r: correlation coefficient. populations should have the same slope (b) in the weight- length relationship (Bolger & Connolly, 1989). However, the b coefficient changes with season (Lima et al., 2002) and populations, thus precluding proper analysis. Our results showed that whereas Wr and K displayed significant condition changes across length in some study lakes, Kn showed a small variation range (0.9-1.1). This feature indicates that Kn exhibits low sensitivity to portray condition differences, particularly for large-size species. Discussion In the case of pejerrey, Wr was related to length structure indices (Baigún & Anderson, 1994) and CPUE (Colautti et al., 2006), showing its potential as a reliable tool for measuring fisheries response to management regulations. An additional advantage of Wr but not found in other condition indices is that management objectives can be recommended based on target values (Murphy et al., 1991). Yet, Wr would not be free of constraints, and some authors cautioned about its length dependence (Murphy et al., 1990; Willis et al., 1991; Cone, 1989, 1990; Hansen & Nate, 2005). However, Neuman & Murphy (1991) for assessing and comparing pejerrey populations, although a standardized weight-length relationship was available (Baigún & Anderson, 1994). Recently, Colautti et al. (2006) proposed a new relationship based only on Pampean populations, taking into account main requirements for properly establishing such equation (Murhy et al., 1990; Brown & Murphy, 1996). Relative weight can be related to biological features such as growth, prey availability, population density, stocking density, P/B ratio, fishing regulations, hydrological variations, water quality, etc (see Blackwell et al., 2000 for review). In the case of pejerrey, Wr was related to length structure indices (Baigún & Anderson, 1994) and CPUE (Colautti et al., 2006), showing its potential as a reliable tool for measuring fisheries response to management regulations. An additional advantage of Wr but not found in other condition indices is that management objectives can be recommended based on target values (Murphy et al., 1991). Yet, Wr would not be free of constraints, and some authors cautioned about its length dependence (Murphy et al., 1990; Willis et al., 1991; Cone, 1989, 1990; Hansen & Nate, 2005). However, Neuman & Murphy (1991) We conclude that although condition indices will still be used as diagnostic and assessment tools for pejerrey populations due to their simplicity, their application should be considered with caution taking into account their advantages and inherent limitations. Fulton’s index should be disregarded in those cases where populations show allometric growth and where size range strongly varies, as seems to be the case for most of the pejerrey populations reviewed. The Kn index appears to be only appropriate for regular within population assessment and therefore of more limited utility, where it is difficult to apply when comparisons between populations are needed and when they exhibit different weight-length relationship slopes. Discussion 6. Variability of weight-length regression slopes within lakes sampled at different years (adapted from Colautti et al., 2006) Fig. 4. Distribution of weight-length regression slopes of 74 sampled populations (adapted from Colautti et al., 2006). Fig. 6. Variability of weight-length regression slopes within lakes sampled at different years (adapted from Colautti et al., 2006). Fig. 4. Distribution of weight-length regression slopes of 74 sampled populations (adapted from Colautti et al., 2006). Fig. 4. Distribution of weight-length regression slopes of 74 sampled populations (adapted from Colautti et al., 2006). Fig. 6. Variability of weight-length regression slopes within lakes sampled at different years (adapted from Colautti et al., 2006). Fig. 5. Distribution of maximum standard length from 74 sampled populations (adapted from Colautti et al., 2006). considered that Wr variation as a function of length can be attributed to different bioecological characteristics. In our study, we adopted length interval criteria according to Baigún & Anderson (1994) guidelines, by assuming that length intervals should have some fisheries meanings (Murphy et al., 1990). We propose that zooplankton quantity and quality are a major factor influencing pejerrey body condition (Colautti et al., 2003). Unlike other Pampean fish species, pejerrey can modify its trophic niche, where adult growth depending on zooplankton structure and fish prey abundance changes from strictly zooplanktivorous to piscivorous through its lifespan (Ringuelet et al., 1980; Escalante, 2001). In Puan and Los Chilenos, for example, pejerrey larger than 200 mm showed a piscivorous diet and the largest condition as reflected by both K and Wr indices but not by Kn. In such cases, it was not unexpected that Wr may be related to length as trophic niche changes according to growth. This effect should be reflected by condition changes, and thus we encourage estimating condition indices by length intervals when possible. Fig. 5. Distribution of maximum standard length from 74 sampled populations (adapted from Colautti et al., 2006). for assessing and comparing pejerrey populations, although a standardized weight-length relationship was available (Baigún & Anderson, 1994). Recently, Colautti et al. (2006) proposed a new relationship based only on Pampean populations, taking into account main requirements for properly establishing such equation (Murhy et al., 1990; Brown & Murphy, 1996). Relative weight can be related to biological features such as growth, prey availability, population density, stocking density, P/B ratio, fishing regulations, hydrological variations, water quality, etc (see Blackwell et al., 2000 for review). Discussion (2006) showed that most slope values were not isometric (b > 3) and above 3 (Fig. 4). In fact, isometric slope only represented 16% of study cases, demonstrating that a slope of b = 3 is a common assumption that was mostly not fulfilled, ranging in our study lakes from 2.89 to 3.60. A major limitation for K index use is its inability to compare condition between populations unless similar length intervals are used. Length dependence by this index is easy to demonstrate by replacing W by its respective length relationship (aLb) in the index formula. Clearly, if length increment is isometric, K is defined by only the parameter “a” and therefore becomes length independent; otherwise, length dependence becomes positive or negative according to the b = 3 value. We propose that K is only appropriate for indexing fish of close length and populations where b = 3, which may be quite uncommon in Pampean lakes. Using maximum length as proxy for infinite length (Froese & Binohlan, 2000), it is apparent that growth strongly varies among populations from 27 to 53 cm (Fig. 5), probably depending on zooplankton quality and limnological differences between lakes. Moreover, even within the same population, the weight-length relationship slope showed a noticeable variation over years (Fig. 6). Fig. 3. Relationship between K, Kn and Wr values and length intervals for study lakes. Le Cren’s Kn index relaxes length dependence, but this index appears suitable for species or body waters where Assessment of condition in Odontesthes bonariensis: which index works best? 444 Fig. 6. Variability of weight-length regression slopes within lakes sampled at different years (adapted from Colautti et al., 2006). Fig. 4. Distribution of weight-length regression slopes of 74 sampled populations (adapted from Colautti et al., 2006). considered that Wr variation as a function of length can be attributed to different bioecological characteristics. In our study, we adopted length interval criteria according to Baigún & Anderson (1994) guidelines, by assuming that length intervals should have some fisheries meanings (Murphy et al., 1990). We propose that zooplankton quantity and quality are a major factor influencing pejerrey body condition (Colautti et al., 2003). Unlike other Pampean fish species, pejerrey can modify its trophic niche, where adult growth depending on zooplankton structure and fish prey abundance changes from Fig. 6. Variability of weight-length regression slopes within lakes sampled at different years (adapted from Colautti et al., 2006). Fig. Literature Cited Froese, R. & C. Binohlan. 2000. Empirical relationships to estimate asymptotic length, length at first maturity and length at maximum yield per recruit in fishes, with a simple method to evaluate length frequency data. Journal of Fish Biology, 56: 758-773. Anderson, R. O. 1980. Proportional stock density (PSD) and relative weight (Wr): interpretative indices for fish populations and communities. Pp. 27-33. In: Gloss, S. & B. Shupp (Eds.). Practical fisheries management: more with less in the 1980’s. New York Chapter American Fisheries Society, Bethesda, MD. Gomiero, L. M. & F. M. S. Braga. 2005. The condition factor of fishes from two river basins in São Paulo state, Southeast of Brazil. Acta Scientiarum, 27: 73-78. Anderson, R. O. & S. J. Gutreuter. 1983. Length, weight and associated structural indices. Pp. 283-300. In: Nielsen, L. A. & D. L. Johnson (Eds.). Fisheries Techniques. American Fisheries Society, Bethesda, MD. Grosman, F. & S. Sergueña. 1996. Parámetros biológicos y sociales de una pesquería deportiva de pejerrey (Odontesthes bonariensis). Pp. 133-141. In: Actas VI Jornadas Pampeanas de Ciencias Naturales, Santa Rosa (La Pampa). Baigún, C. R. M. 1989. Evaluación de recursos pesqueros en aguas continentales mediante el uso de redes enmalladoras. Clímax, 7: 1-79. Gutreuter, S. & M. Childress. 1990. Evaluation of condition indices for estimation of growth of Largemouth Bass and White Crappie. North American Journal of Fisheries Management, 10: 434-441. Baigún, C. R. M. & R. O. Anderson. 1994. Structural indices for stock assessment and management recommendations for pejerrey Odonthestes bonariensis in Argentina. North American Journal of Fisheries Management, 13: 600-618. Hansen, M. J. & N. A. Nate. 2005. A method for correcting the relative weight (Wr) index for seasonal patterns in relative condition (Kn) with length as applied to Walleye in Wisconsin. North American Journal of Fisheries Management, 25: 1256- 1262. Blackwell, B., M. L. Brown & D. W. Willis. 2000. Relative weight (Wr) status and current use in fisheries assessment and management. Review in Fisheries Sciences, 88: 1-44. Le Cren, E. D. 1951. The length-weight relationship and seasonal cycle in gonad weight and condition in the perch Perca fluviatilis. Journal of Animal Ecology, 20: 201-219. Bolger, T. & P. L. Connoly. 1989. The selection of suitable indices for the measurements and analysis of fish condition. Journal of Fish Biology, 34: 171-182. Brown, M. L. & M. L. Murphy. 1996. Selection of a minimum sample size for application of the regression-line-percentile technique. Literature Cited North American Journal of Fisheries Management, 16: 427-432. Liao, H., H. C. Pierce & D. Whal. 1995. Relative weight (Wr) as a field assessment tool: relationship with growth, prey biomass and environmental conditions. Transactions of the American Fisheries Society, 124: 387-400. Brown, M. L. & M. L. Murphy. 2004. Seasonal dynamics of direct and indirect condition indices in relation to energy allocation in largemouth bass Micropterus salmoides (Lacepede). Ecology of Freshwater Fish, 13: 23-36. Lima Jr., S. E., I. Braz Cardone & R. Goitein. 2002. Determination of a method for calculation of allometric condition factor of fish. Acta Scientiarum, 24(2): 397-400. López, H. L., C. R. M. Baigún, J. M. Iwaszkiw, R. L. Delfino & O. Padin. 2001. La cuenca del Salado: uso y posibilidades de sus recursos pesqueros. La Plata, Universidad Nacional de la Plata, 60p. Colautti, D. C., M. Remes Lenicov & G. E. Berasain. 2003. Vulnerabilidad del pejerrey Odontesthes bonariensis a la pesca deportiva en función de su condición. Biología Acuática, 20: 49-55. Murphy, B. R., M. L. Brown & T. A. Springer. 1990. Evaluation of the relative weight (Wr) index with new applications to walleye. North American Journal of Fisheries Management, 10: 85-97. Colautti, D. C., M. Remes Lenicov & G. E. Berasain. 2006. A standard weight equation to assess the body condition of pejerrey Odontesthes bonariensis. Biocell, 30: 131-135. Cone, R. S. 1989. The need to reconsider the use of condition indices in fishery science. Transactions of the American Fisheries Society, 119: 510-514. Murphy, B. R., D. W. Willis & T. A. Springer.1991. The relative weight index in fisheries management: status and needs. Fisheries, 16: 30-38. Cone, R. S. 1990. Properties of relative weight and other condition indices. Transactions of the American Fisheries Society, 119: 1052-1057. Neuman, R. M. & B. R. Murphy. 1991. Evaluation of the relative weight (Wr) index for assessment of the white crappie and black crappie populations. North American Journal of Fisheries Management, 11: 543-555. Dyer, B. S. 2006. Systematic revision of the South American silversides (Teleostei, Atheriniformes). Biocell, 30: 69-88. Paggi, J. de & J. Paggi. 1995. Determinación de la abundancia y biomasa zooplanctónica. Pp. 315-323. In: Lopretto, E. E. & G. Tell (Eds.). Ecosistemas de aguas continentales. Metodologías para su estudio. La Plata, Ediciones Sur. Escalante, A. 2001. Alimentación natural del pejerrey. Pp. 67-75. In: Grosman, F. (Ed.). Discussion The Wr index should be strongly preferred in regular surveys if the objective is to perform comparisons between pejerrey populations, particularly when population structure is not well known, C. R. M. Baigún, D. C. Colautti & F. Grosman 445 Freyre, R. L., C. Togo, J. Zetti & S. Mollo. 1969. Estudios ictiológicos sobre poblaciones, correlaciones somáticas y correlaciones biocenóticas en lagunas bonaerenses. Convenio Estudio Rique- za Ictícola. Trabajos Técnicos Cuarta Etapa (1968-1969). Consejo Federal de Inversiones, Ministerio de Asuntos Agrarios, La Plata. stocking has been used for population recovery, lakes are supported strongly by limnological changes, data are limited to only one or few samplings, and metaphoetesis is suspected in pejerrey populations. We believe that the Wr index is better than K since it minimizes the skewness due to length effect and is preferable to Kn because it portrays the condition not only at the population level but also from a species perspective. Froese, R. 2006. Cube law, condition factor and weight-length relationships: history, meta-analysis and recommendations. Journal of Applied Ichthyology, 22: 241-253. Literature Cited Fundamentos biológicos, económicos y sociales para una correcta gestión del recurso pejerrey. Azul, Buenos Aires, Editorial Astyanax, 212p. Freyre, R. L. 1976. Normas para la inspección y determinación del estado actual de ambientes pesqueros pampásicos. Ministerio de Asuntos Agrarios de la Provincia de Buenos Aires. Ricker, W. E. 1975. Computation and interpretation of biological statistics of fish populations. Bulletin of Fisheries Research Board of Canada, 191: 1-382. Assessment of condition in Odontesthes bonariensis: which index works best? 446 Ringuelet, R. A. 1964. Un ejemplo de criterio normativo para la explotación de un recurso íctico de aguas continentales. La pes- ca comercial del pejerrey en la laguna de Chascomús (1958). Agro, 6: 61-78. Ringuelet, R. A., R. Iriart & A. H. Escalante. 1980. Alimentación del pejerrey (Basilichthys bonariensis bonariensis, Atherinidae) en laguna de Chascomús (Buenos Aires, Argentina). Relaciones ecológicas de complementación y eficiencia trófica del plancton. Limnobios, 1: 447-600. Somoza, G. M., L. A. Miranda, G. E. Berasain, D. Colautti, M. Remes Lenicov & C. A. Strüssmann. 2008. Historical aspects, current status, and prospects of pejerrey aquaculture in South America. Aquaculture Research, 39: 784-793. Sutton, N. S. G., T. P. Bult & R. L. Haedrich. 2000. Relationships among fat weight, body weight, water weight, and condition factors in wild Atlantic salmon parr. Transactions of the American Fisheries Society, 129: 527-538. Wege, G. J. & R. O. Anderson. 1978. Relative weight (Wr): a new index of condition for largemouth bass. Pp: 79-91. In: Novinger, G. & J. Dillard. (Eds.). New approaches to the management of small impoundments. American Fisheries Society, North Cen- tral Division, Special Publication 5, Bethesda, Maryland, 132p. Willis, D. W., C. S. Guy & B. R. Murphy. 1991. Development and evaluation of a standard weight (Ws) equation for yellow perch. North American Journal of Fisheries Management, 11: 374-380. Accepted July 2, 2009 Published September 30, 2009 Accepted July 2, 2009 Published September 30, 2009
https://openalex.org/W4361833163	https://figshare.com/articles/journal_contribution/Figure_S3_from_Modeling_Acquired_Resistance_to_the_Second-Generation_Androgen_Receptor_Antagonist_Enzalutamide_in_the_TRAMP_Model_of_Prostate_Cancer/22424281/1/files/39870541.pdf	English	null	Figure S3 from Modeling Acquired Resistance to the Second-Generation Androgen Receptor Antagonist Enzalutamide in the TRAMP Model of Prostate Cancer	null	2,023	cc-by	96	Supplementary Figure S3 Supplementary Figure S3 Supplementary Figure S3 Supplementary Figure S3. Time courses of pH (A) and oxygen (B) for 30 samples in presence of enzalutamide continuous treatment. The plots correspond to the simulation in figure 3B. Supplementary Figure S3. Supplementary Figure S3. Time courses of pH (A) and oxygen (B) for 30 samples in presence of enzalutamide continuous treatment. The plots correspond to the simulation in figure 3B. Time courses of pH (A) and oxygen (B) for 30 samples in presence of enzalutamide continuous treatment. The plots correspond to the simulation in figure 3B.
https://openalex.org/W4224120787	https://recyt.fecyt.es/index.php/retos/article/download/91463/68256, https://repositori.udl.cat/bitstream/10459.1/84900/1/retos_a2022n45p326.pdf	es		Percusión corporal a través de la Esku Dantza. Efectos sobre las emociones en estudiantes de grado en Ciencias de la Actividad Física y el Deporte (Body percussion through Esku Dantza. Effects on emotions in students of Physical Activity and Sport Scienc	Retos digital/Retos	2,022	cc-by	7,884	2022, Retos, 45, 326-336 © Copyright: Federación Española de Asociaciones de Docentes de Educación Física (FEADEF) ISSN: Edición impresa: 1579-1726. Edición Web: 1988-2041 (https://recyt.fecyt.es/index.php/retos/index) Percusión corporal a través de la Esku Dantza. Efectos sobre las emociones en estudiantes de grado en Ciencias de la Actividad Física y el Deporte Body percussion through Esku Dantza. Effects on emotions in students of Physical Activity and Sport Sciences Silvia Garcías de Ves, Alfredo Joven Pérez, Eloísa Lorente-Catalán Universidad de Lérida (España) Resumen. La Percusión Corporal es un contenido que se está utilizando en Educación Física (EF) para desarrollar competencias vinculadas a la Expresión Corporal.Tras observar que existen evidencias científicas de sus posibles beneficios socioemocionales sobre las personas que la practican, este estudio pretende analizar si una práctica de percusión corporal, concretamente la Esku Dantza, una danza colectiva que utiliza patrones rítmicos con palmadas para relacionarse con sus compañeros/as, puede producir cambios en el estado emocional de los/las estudiantes de formación inicial del profesorado de EF. Se administró el cuestionario POMS a 82 estudiantes universitarios, 28 mujeres (34.1%) y 54 hombres (65.9%) al llegar y finalizar la sesión. La propuesta siguió un proceso pedagógico progresivo que dio lugar a aumentar los valores de tensión y vigorosidad así como disminuir los valores depresión, rabia y fatiga. Al finalizar la intervención, los/las estudiantes expresaron palabras como sentirse enérgicos/as, felices y motivados/as. Estos resultados podrían reafirmar los posibles beneficios de estas prácticas, promoviendo la actividad física a través de prácticas artísticas, no competitivas; así como generar impacto emocional que, sumado a un aprendizaje significativo, les haga interiorizar esta práctica y genere la motivación necesaria para implementarla en sus futuros ámbitos profesionales (educativos, deportivos, terapéuticos, etc) o incluso pueda utilizarse como estrategia para incorporar la danza entre el género masculino. Palabras clave. Educación física; formación inicial del profesorado; expresión corporal; estados de ánimo; aprendizaje cooperativo. Abstract. Body Percussion is a content that is being used in Physical Education (PE) to develop skills related to Body Expression. After observing that there is scientific evidence of its possible socioemotional benefits on people who practice it, this study aims to analyze whether a practice of body percussion, specifically Esku Dantza, a collective dance that uses rhythmic patterns with clapping to interact with their peers, it can lead to changes in the emotional state of students in initial PE teacher training.. The POMS questionnaire was administered to 82 university students, 28 women (34.1%) and 54 men (65.9%) upon arrival to class (Pre) and upon completion (Post).The proposal followed a progressive pedagogical process that resulted in increasing the values of tension and vigor as well as decreasing the values of depression, anger and fatigue. At the end of the intervention, the students expressed words such as feeling energetic, happy and motivated. These results could reaffirm the possible benefits of these practices, promoting physical activity through artistic practices, not competitive; as well as generating emotional impact that, added to a significant learning, makes them internalize this practice and generates the necessary motivation to implement it in their future professional fields (educational, sports, therapeutic, ...) or can even be used as a strategy to incorporate dance between the male gender. Keywords. Physical education; initial teacher training; body expression; states of mood; cooperative learning. Introducción El constante cambio de la sociedad actual sugiere una continua y reflexiva adecuacion del sistema educativo. Las demandas de aprendizaje emergentes requieren formar personas activas con autoconfianza, curiosas, emprendedoras e innovadoras, deseosas de participar en la sociedad a la que pertenecen, de crear valor individual y colectivo, capaces de asumir como propio Fecha recepción: 01-10-21. Fecha de aceptación: 11-03-22 Silvia Garcías de Ves sgarcias@inefc.es - 326 - el valor del equilibrio entre el esfuerzo y la recompensa. Una enseñanza diferente para poder satisfacer a unos alumnos y alumnas que han ido cambiando con la sociedad (LOMCE, 2013). La calidad educativa debe promover el desarrollo de todos los dominios de aprendizaje (físicos, de estilo de vida, afectivos, sociales y cognitivos), y al mismo tiempo debe presentar oportunidades para que el alumnado desarrolle habilidades esenciales, como el liderazgo, la comunicación y el trabajo en equipo (UNESCO, 2015). En este estudio nos centramos en la formación inicial de los futuros profesores y profesoras de EF. La EF es una materia obligatoria en la educación obligatoria Retos, número 45, 2022 (3º trimestre) cuya calidad vendrá determinada, en parte, por el desarrollo de la motricidad a través de metodologías activas, dinámicas y reflexivas, dando mucha importancia al aprendizaje continuo (Cañabate, Tesouro, Puigggali, & Zagalaz, 2019). Algunos autores se enfocan en modelos pedagógicos consolidados como el aprendizaje cooperativo (Palomares-Montero & Chisvert-Tarazona, 2016; Sánchez-Molina, González-Martí, & HernándezMartínez, 2021;Velázquez, Fraile & López-Pastor, 2014). Por otro lado, los contenidos donde la educación emocional, la igualdad y la perspetiva de género se han integrado en las recomendaciones de la LOMLOE (Ley Organica 3/2020, de 29 de diciembre, por la que se modifica la Ley Organica 2/2006, de 3 de mayo, de Educacion). Esta nueva ley manifiesta utilizar la EF y el deporte para favorecer el desarrollo personal y social, afianzar los habitos de actividades fisico-deportivas para favorecer el bienestar fisico y mental, asi como medio de desarrollo personal y social. Cuevas (2019) menciona la necesidad de crear propuestas didácticas para las clases de EF que faciliten experiencias positivas del alumnado. Para ello se plantea cinco elementos con los cuales se puede jugar para estructurar el proceso de enseñanza – aprendizaje: el espacio, el tiempo, el material, el alumnado y el/la docente, siendo este último el más relevante para conseguir una EF de calidad. El papel activo del alumnado para la construcción del aprendizaje y el tipo de animación que promueve el/la docente condiciona la motivación del alumnado. Cañabate et al. (2019) afirman que uno de los factores más influyentes en el proceso de enseñanza-aprendizaje para garantizar el éxito en la EF es la actitud y la motivación del profesorado. Aunque son escasos los estudios específicos que tengan como objetivo de investigación las actitudes hacia la Expresión Corporal (EC) (Arias, Fernández, & San Emeterio, 2020), alguno de estos estudios muestran cómo los estudiantes ven influenciada su actitud frente a la EC dependiendo de la actitud del docente, existiendo diferencias dependiendo del género (Arias, Fernández, & Valdés, 2021). También defienden la necesidad, por ejemplo en el caso de la danza, de que «el educador reconozca que este contenido permite un trabajo para la educacion del cuerpo y del movimiento, permite el desarrollo de la creatividad, la relacion entre compañeros, el conocimiento de uno mismo, el conocimiento de otras culturas y el desarrollo de la capacidad expresiva del alumnado» (García, Pérez, & Calvo, 2015, p.36). Actualmente, las emociones han cobrado relevancia Retos, número 45, 2022 (3º trimestre) en educación (Duran, Lavega, Planas, Muñoz, & Pubill, 2014; Lavega-Burgués, March-Llanes, & Moya-Higueras, 2018; Torrents, Mateu, Planas, & Dinusova, 2011). Por ello, se cree necesario seguir investigando sobre las emociones que genera la EC (Armada, 2017; Górritz, Garcías, & Mateu, 2020; Rovira, López-Ros, Lagardera, Lavega, & March, 2014; Ruano, 2004) ya que, sabiendo que la EC y la danza son herramientas que permiten conocer y revelar el mundo interno, sensaciones, emociones o ideas del propio alumnado (Gómez-Carmona, Redondo-Garrido, Bastida-Castillo, Mancha-Triguero, & Gamonales-Puerto, 2019; Ugena, 2014), el profesorado de EF sigue implementando la EC en sus clases en menor medida que el resto de bloques de contenido (actividad física saludable, deportes, actividad física y tiempo de ocio). Se sigue destacando la falta de formación específica en EC y los miedos o vergüenzas que sienten los/las docentes para aplicarla en clase, siendo aún el género masculino quien se muestra como el más afectado frente a este tipo de prácticas artísticas (Conesa & Angosto, 2017; Lafuente & Hortigüela, 2021). Por esta razón, se cree necesario un cambio en la raíz, en la formación inicial y contínua del profesorado (Abilleira & Fernández, 2017). Por otro lado, la Percusión Corporal (PC) comienza a estar presente en las clases de EF como contenido que permite desarrollar competencias relacionadas con la EC, el sentido del ritmo y la coordinación, entre otras capacidades y cualidades motrices (Garcías, 2021a, 2021b, 2021c; Garcías, Joven, Lorente-Catalán, & Planas, 2020). Contenido que suele aparecer explícito en las guías docentes de las materia de EC y Danza, en los Grados de Ciencias de la Actividad Física y Deporte (CCAFyD) (Gil, 2016). Además, existen evidencias científicas sobre los beneficios de la utilización de la PC en diferentes niveles del ser como por ejemplo a nivel social donde la PC puede utilizarse como medio para unir y crear lazos a través del trabajo cooperativo e inclusivo (Romero-Naranjo, 2020). La Esku Dantza es una danza tradicional vasca realizada con patrones rítmicos de PC (Di Russo & RomeroNaranjo, 2021; García et al., 2018; Romero-Naranjo, 2013). En las danzas vascas aparecen formaciones que son las figuras geométricas que los dantzaris proyectan con sus cuerpos en el espacio y podría utilizarse de manera interdisciplinar al trabajar competencias de otras áreas e incluso fomentar el dominio de la orientación espacial (Mateu, 2010). También se observan los «juegos», principalmente de manos, que hacen los dantzaris. Por último, se aprecian desplazamientos y giros como - 327 - conjunto de pasos o unidades coreográficas realizadas con el tren inferior, principalmente pies. La danza vasca se considera verdaderamente gimnástica (Araolaza, 2020). Por tanto, dependiendo de la forma de trabajar la EC mediante la Esku Dantza, no sólo se trabajan las cualidades y capacidades motrices anteriormente citadas, sino también las habilidades motrices básicas (desplazamientos, saltos, giros…) y dependiendo de la forma de trabajarlo, el nivel de condición física del alumnado (con su correspondiente trabajo de las capacidades físicas), sus habilidades sociales y sus respectivos beneficios de salud y rendimiento académico y cognitivo ampliamente demostrados. Elaborar situaciones didácticas óptimas requiere dominar el contenido y saber estructurar los objetivos, planificar las secuencias didácticas motrices, utilizar los errores como herramienta de conocimiento, realizar una adecuada progresión de los aprendizajes motrices, ajustar la actividad al nivel y a las posibilidades motrices del alumnado, tener en cuenta la heterogeneidad del grupo-clase, desarrollar la cooperación y la ayuda mutua entre estudiantes, estimular el autoaprendizaje, motivar la práctica de actividad física saludable, fomentar el debate, etc (Contreras, 2019). Aspectos a tener en cuenta para la propuesta pedagógica que se va a implementar para esta investigación. está compuesto de 29 ítems valorados con una escala de Likert de 0 a 4 donde 0 es nada, 1 es un poco, 2 corresponde a moderadamente, 3 es bastante y 4, mucho.También se les pidió expresar en una sola palabra cómo se sentían al terminar la sesión. Procedimiento y análisis de datos Desarrollo de la implementación La sesión específica de PC tuvo una duración de 75 minutos y se dividió en cuatro partes: la administración voluntaria y anónima del cuestionario POMSPre como evaluación inicial de su estado de ánimo; parte A (primera propuesta para entrar en calor con patrones rítmicos a desarrollar en cánon), Parte B (Progresión de la Esku Dantza) y parte final (cierre de la sesión con administración del cuestionario POMSPost). Parte inicial o de bienvenida (POMSPre) El cuestionario POMSPre se administró al llegar a clase. Parte A de calentamiento En círculo. La docente propone un patrón rítmico cantando «Si lo sabes cantar, lo sabes tocar». El resto de practicantes replica sólo con la voz, en loop, hasta integrar (Figura 1). Objetivo Este estudio pretende conocer los cambios que produce la Esku Dantza en el estado de ánimo de un grupo de estudiantes en la formación inicial del profesorado de EF. Metodología Contexto y Participantes Se trata de una muestra intencionada por conveniencia ya que todos/as los/las participantes cursaban la materia de EC y Danza en el Grado en CCAFyD del INEFC de Lleida (curso 2020 – 2021). Como muestra final participaron un total de 82 estudiantes, de los cuales 28 son mujeres (34.1%) y 54 son hombres (65.9%) y tienen un rango de edad de 19 a 35 años. Figura 1. Patrón Rítmico cantado. La docente pregunta cómo se podrían ejecutar los sonidos de Tum y los sonidos de Pa con PC (golpeos sobre el cuerpo). De este modo, ellos/as proponen y ejecutan motrizmente en modo loop hasta integrar. Ejemplo:Tum con un golpe de pie en el suelo (Stomp) y Pa con un golpe de palmada (Figura 2). Figura 2. Patrón Rítmico motriz y con acompañamiento vocal. Instrumento de medida Se utilizó el Cuestionario Profile of Moods States Scale (POMS), una versión reducida adaptada y validada al español de Balaguer et al. (1993) que se administró al inicio y al finalizar la clase. El cuestionario POMS - 328 - Una vez se ha integrado el patrón rítmico motrizmente, se organizan, aleatoriamente, en 4 subgrupos donde hacen exposición 1 a 1. Expone el Subgrupo 1, seguido del Subgrupo 2, seguido del Retos, número 45, 2022 (3º trimestre) Subgrupo 3, seguido del Subgrupo 4 y finalmente TODOS juntos (Figura 3). Figura 6. Organización por subgrupos para el cánon 3. Figura 3. Pasación por Subgrupos del Patrón Rítmico motriz y con acompañamiento vocal. La pasación es un procedimiento coreográfico que podría entenderse como un relevo, es decir, el Subgrupo 1 expone su frase musical, ejecutada motrizmente y acompañada de la voz, y cuando termina, el siguiente subgrupo interviene sin perder el tempo (pulsación). También se propone un cánon, es decir, cada Subgrupo repetirá 4 veces la secuencia y parará. Cada subgrupo comienza 2 pulsaciones más tarde que el grupo anterior. Para facilitar la reproducción motriz del patrón rítmico sin interferencias se hace en pequeños círculos con la finalidad de poder escucharse entre ellos/as mismos/as (Figura 4). Figura 4. Organización por subgrupos para el cánon 1. Cuando entienden qué hacer vuelven a la posición de círculo grupal y se diferencian los Subgrupos para su entrada e intervención en cánon. Observación para el/ la docente que guía la actividad: las dos últimas palmadas son las que te sitúan en el cánon y nos facilita reconocer si algún subgrupo no está situado y retomar la actividad si fuera necesario (Figura 5). patrón rítmico generado con los sonidos corporales que crean los diferentes golpeos propuestos sobre el cuerpo, PC. Parte B o principal: Esku Dantza La docente expone el patrón rítmico cantando y al mismo tiempo ejecuta los movimientos a modo de progresión y de manera individual, de más sencillo a más complejo. A medida que la docente siente que el estudiantado ha integrado el patrón rítmico de la propuesta va cambiando la forma (el lugar espacial) en el que se ejecuta la palmada. Para aquellos/as que no conozcan el patrón rítmico motriz de la Esku Dantza se detalla seguidamente: Se choca la mano derecha con un/a compañero/a, palmada, se choca la mano izquierda con el/ la compañero/a, palmada, palmada por debajo de la pierna derecha alzada, palmada, palmada por debajo de la pierna izquierda alzada, palmada, palmada por detrás del tronco, es decir, parte posterior del cuerpo, palmada y pausa con las manos juntas, como un rezo, contactan con las manos del compañero/a que tenemos delante. Observación: Se trata de una variante que pone el acento en la primera pulsación y por ello comienza con el choque de manos (palmada) con el/la compañero/a ya que la danza original se inicia con el silencio y los dos choques de manos seguidos. En círculo. Se ejecuta el patrón rítmico sin contacto físico, después en parejas, se añade medio giro para cambiar de pareja, se hace un giro completo sobre sí mismo/a manteniendo el/la mismo/a compañero/a o cambiando de pareja (Figura 7). Estos cambios motrices se proponen ejecutar en la cuarta repetición. Figura 5. Organización por subgrupos para el cánon 2. Observación: De manera espontánea propusieron entremezclarse de tal modo que se hicieron subgrupos con representantes de cada uno de los grupos. Esta opción para realizar el cánon 3, es algo más compleja ya que no disponen de la fuerza sonora de sus compañeros/as para seguir con su propuesta rítmica (Figura 6). Todas las propuestas de cánon anteriores pueden ejecutarse sin el acompañamiento vocal y escuchando el Retos, número 45, 2022 (3º trimestre) Figura 7. Representación de la Esku Dantza en parejas y en círculo. En dos hileras. Se explica cómo tradicional y culturalmente, esta danza se bailaba en un lado los hombres y en el otro lado las mujeres. En clase será indiferente pero para conocer su origen y trayectoria se explica la historia-procedencia de esta danza (figura 9). - 329 - Figura 8. Organización de grupo en hileras sin desplazamiento. Figura 9. Organización de grupo en hileras con desplazamiento. Posición inicial. Figura 10. Organización de grupo en hileras con desplazamiento. Posición Final. En hileras con desplazamiento. En la 4ª repetición se desplazan a la derecha. Tener en cuenta que si ambas filas se mueven al mismo tiempo el/la compañero/a siguiente no será el consecutivo sino que habrá que saltar a una persona. Ejemplo: Si el 1 va con 4 (centrados) y ambos se desplazan hacia la derecha, en la siguiente repetición el 1 irá con el 2. También tener en cuenta que los extremos que van a desplazarse a la derecha, si no tienen más compañeros/as, deberán pasar a la fila de delante (Figura 9 y 10). En círculos concéntricos. En la 4ª repetición se mueven a la derecha sólo el círculo de fuera (Ver Figura 11). Una vez se ha integrado se pone la música de la Esku Dantza modificada. La música original de la Esku Dantza se caracteriza por acelerarse, de tal modo que la parte final es tan rápida que dificulta la ejecución. Por este motivo se recomienda parar la música cuando haya un cambio o bien se edite. En este caso fue editada en loop a 63 ppm, a 74 ppm, y a 85 ppm (pulsaciones por minuto). Figura 11. Organización de grupo en círculos concéntricos con desplazamiento (sólo el círculo de fuera). Figura 12. Organización de grupo en círculos concéntricos con desplazamiento (sólo el círculo de dentro). Figura 13. Organización de grupo en círculos concéntricos con desplazamiento ambos círculos a la derecha al mismo tiempo. Variantes: En la 4ª repetición se mueven a la derecha sólo el círculo de dentro (Figura 12); en la 4ª repetición se mueven todos hacia la derecha. Recordar saltar a un/a compañero/a (Figura 13); solo se desplazan los del círculo de fuera pero en cada repetición se mueven hacia la derecha y en la 4ª repetición cambian con el círculo de dentro (Figura 14). Observación: Las preguntas de reflexión se proponen en cada una de las propuestas. Se escuchan las vivencias, las emociones y se reflexiona sobre lo sucedido Parte final o de cierre y reflexión (POMSPost) Para terminar la sesión se pasó nuevamente el cuestionario POMSPost (participación voluntaria y anónima y que no repercute sobre su evaluación). Análisis de datos Los 29 ítems del cuestionario POMS se aglomeran conformando cinco estados emocionales: la Tensión– Ansiedad (Factor T) que se atribuye a sentirse intranquilo/a, inquieto/a, agitado/a, nervioso/a, tenso/a y relajado/a (ítems 1, 8, 11, 18, 21 y 28); la Depresión– Abatimiento (Factor D) como sentirse desamparado/ a, deprimido/a, desdichado/a, triste e infeliz (ítems 3, 6, 13, 23 y 26); la Rabia-Hostilidad (Factor A) como sentirse furioso/a, molesto/a, luchador/a, irritable, amargado/a, enfadado/a, enojado/a y de mal genio (ítems 4, 9, 12, 14, 16, 19, 24 y 29); la Fatiga-Inmovilidad (Factor F) se atribuye a las palabras sin fuerzas, agotado/a, cansado/a, exhausto/a y fatigado/a (ítems 5, 10, 15, 20 y 25); y el Vigor-Actividad (Factor V) que corresponde a sentirse enérgico/a, lleno/a de energía, animado/a, vigoroso/a y activo/a (ítems 2, 7, 17, 22 y 27). La combinación de los resultados anteriores da lugar al estado de ánimo general donde el valor de la Tensión (T) + el valor de la Depresión (D) + el valor de la Rabia (R) + el valor de la Fatiga (F), se resta al valor obtenido en vigor (V) y al resultado, se le suma 100. PGEA= ((T +D + R + F) – V) + 100 Para su análisis se utilizó el programa Excel (16.49), calculado las medias (M) y desviaciones estándar (SD) de cada uno de los factores (F, V, D, T y A). La palabra que resumía la experiencia se categorizó señalando aquellas que se repiteron más veces. Resultados Figura 14. Organización de grupo en círculos concéntricos con desplazamiento el círculo de fuera y en la 4ª repetición cambian con el círculo de dentro. - 330 - En este estudió se analizó el cuestionario POMS de Retos, número 45, 2022 (3º trimestre) manera generalizada y por géneros. De manera generalizada se observa como la fatiga disminuye pasando de una MPre de 8.12 a una MPost de 6.82. Destacar que las respuestas presentan más disparidad al llegar a clase y con la vivencia práctica se reduce la desviación estándar en relación a los ítem que hacen referencia a la tensión, depresión y la rabia. Sin embargo, en los ítems vinculados con la fatiga se mantiene y el de vigor aumenta. (Ver Tabla 1). de un esfuerzo físico y se repiten 17 veces. En esta categorgía aparecen también otras palabras como muerto/a, fatigado/a y reventado/a sumando un total de 21. Finalmente, la categoría otras se atribuye a aquellas que no encajan en las anteriores como por ejemplo, rítmico/a, bailarín/a, flow, concentrado/a y coordinado/a. Seguidamente se muestran los resultados de los factores analizados por separado y según el género. La puntuación Total(Pre) del estado de la Tensión – Ansiedad (Factor T) sumó 425, Tabla 1 laTotal(Post) 446, laTmujPre Comparativa de los resultados obtenidos en este estudio con los estudios de Muñoz et al. (2017) y Mateu et al. (2021). Estudio Actual Estudio 1 Merce et al. (2021) Estudio 2 Merce et al. (2021) Estudio Muñoz et al. (2017) 170, la TmujPost 176, la Pre Post Pre Post Pre Post Psicomotores Cooperativos M(SD) M(SD) M(SD) M(SD) M(SD) M(SD) M(SD) M(SD) ThomPre 255 y la ThomPost Tensión (T) 5.18 (4.12) 5.44 (3.95) 9.25 (4.39) 5.40 (3.46) 9.87 (3.77) 5.42 (3.17) 6.60 (.249) 6.66 (.227) Depresión (D) 1.98 (2.81) 1.12 (2.12) 1.50 (2.28) 1.12 (2.61) .97 (1.61) .78 (1.89) .95 (.100) .89 (.175) 270. Rabia (R) 4.49 (5.07) 3.32 (3.32) 5.04 (3.70) 4.11 (3.90) 4.15 (2.66) 3.82 (3.25) 3.23 (.210) 3.63 (.252) Fatiga (F) 8.12 (4.83) 6.82 (4.88) 2.91 (3.31) 5.06 (4.73) 2.88 (3.15) 4.56 (4.37) 3.46 (.237) 3.43 (.208) Se observa como los Vigor (V) 13.10 (4.08) 13.63 (4.37) 14.40 (4.10) 13.39 (4.91) 14.41 (3.98) 12.60 (5.24) Estado de ánimo (PGEA) 126.70 (13.48) 127.73 (103.47) 104.29 (11.23) 102.29 (11.05) 103.47 (8.34) 101.97 (9.59) 105.92 (.571) 105.70 (.663) valores de T de las muM=Media; SD=Desviación Estandar jeres están por encima que los valores obteniTabla 2 Comparativa de los resultados obtenidos en este estudio con los estudios de Muñoz et al. (2017), según el género. dos por los hombres Estudio Actual Muñoz et al. (2017) Estudio Actual Muñoz et al. (2017) Mujeres Hombres aunque para ambos gruPre Post Comp Sin Comp Pre Post Comp Sin Comp M M M M M M M M pos de población auTensión (T) 6.07 6.28 6.77 6.27 4.72 5.00 7.35 6.60 Depresión (D) 1.89 .96 .59 .58 2.01 1.20 1.53 1.43 mentan al finalizar la Rabia (R) 4.42 3.57 2.48 2.53 4.48 3.18 4.38 3.79 Fatiga (F) 7.53 5.92 3.14 3.67 8.42 7.27 4.40 4.27 sesión. Vigor (V) 7.50 12.14 6.62 10.46 Estado de ánimo (PGEA) 127.42 128.89 103.48 104.73 126.31 127.12 107.73 107.44 La puntuación M=Media; Com=Competición; Sin Comp=Sin competición Total(Pre) del estado de la Depresión-Abatimiento Los valores de tensión y vigorosidad en las mujeres (Factor D) sumó 162, laTotal(Post) 92, la DmujPre 53, la DmujPost son los que aumentan una vez finalizada la clase aunque 27, la DhomPre 109 y la DhomPost 65. esta última en mayor medida, hasta 4 puntos. Mientras Los valores medios de depresión-abatimiento están que los demás disminuyen, siendo la fatiga el más alto muy próximos entre ambos grupos aunque en este fac(Tabla 2). tor los valores obtenidos por los hombres son algo más Al finalizar la sesión expresaron en una palabra cómo se sentían y se han podido agrupar en tres categorías Estados de la Depresión 3,00 (inspiradas en Hawkins, 2014): emociones de vibración 2,02 1,98 1,89 2,00 1,20 1,12 0,96 elevada, emociones de baja vibración y otras. La cate1,00 0,00 goría de emociones de vibración elevada se identifican Total (Pre) Mujeres (Pre) Hombres (Pre) Total (Post) Mujeres (Post) Hombres (Post) con las palabras de sentirse feliz, enérgico/a, motivaFigura 16. Resultados del estado de la Depresión-Abatimiento (Factor D) de manera global y do/a, activo/a que se han repetido 35 veces. Otras exsegún el género analizados antes (Pre) y después de la sesión (Post). presiones que se incluyen en esta categoría aunque no Estados de la Rabia se han repetido tanto son: alegre, risueño, contento y 6,00 4,49 4,49 4,43 3,57 3,32 4,00 3,19 divertido. Esta categoría alcanza 42 palabras en total. 2,00 Las emociones de baja vibración se atribuyen a las ex0,00 Total (Pre) Mujeres (Pre) Hombres (Pre) Total (Post) Mujeres (Post) Hombres (Post) presiones: cansado/a, agotado/a y exhausto/a que poFigura 17. Resultados del estado de la Rabia-Hostilidad (Factor A) de manera global y según el drían agruparse en la sensación que han tenido después género analizados antes (Pre) y después de la sesión (Post). Estados de la Fatiga Estados de la Tensión 10,00 10,00 5,18 5,00 6,07 4,72 5,44 Hombres (Pre) Total (Post) 6,29 5,00 8,12 7,54 8,43 6,82 5,93 7,28 5,00 0,00 0,00 Total (Pre) Mujeres (Pre) Mujeres (Post) Hombres (Post) Figura 15. Resultados del estado de la Tensión – Ansiedad (Factor T) de manera global y según el género analizados antes (Pre) y después de la sesión (Post). Retos, número 45, 2022 (3º trimestre) Total (Pre) Mujeres (Pre) Hombres (Pre) Total (Post) Mujeres (Post) Hombres (Post) Figura 18. Resultados del estado de la Fatiga-Inmovilidad (Factor F) de manera global y según el género analizados antes (Pre) y después de la sesión (Post). - 331 - Estados de la Vigor 15,00 11,04 10,00 7,50 6,93 12,14 10,46 6,63 5,00 0,00 Total (Pre) Mujeres (Pre) Hombres (Pre) Total (Post) Mujeres (Post) Hombres (Post) Figura 19. Resultados del estado de la Vigor-Actividad (Factor V) de manera global y según el género analizados antes (Pre) y después de la sesión (Post). Perfil General de Estado de Ánimo (PGEA) 130,00 128,89 128,00 126,70 127,73 127,43 127,13 126,31 126,00 124,00 Total (Pre) Mujeres (Pre) Hombres (Pre) Total (Post) Mujeres (Post) Hombres (Post) Figura 20. Perfil General de Estado de Ánimo (PGEA) de manera global y según el género analizados antes (Pre) y después de la sesión (Post). altos que en las mujeres. Aún así ambas poblaciones disminuyen su valor una vez transcurrida la clase (figura 16). La puntuaciónTotal(Pre) del estado de la Rabia-Hostilidad (Factor A) sumó 368, la Total(Post) 272, la AmujPre 124, la AmujPost 100, la AhomPre 244 y la AhomPost 172 (figura 17). Los valores obtenidos entre género son similares y disminuyen del Pre al Post. La pequeña diferencia que podría observarse es que la disminución del Pre al Post es algo más acentuada entre los hombres. La puntuación Total(Pre) del estado de la Fatiga-Inmovilidad (Factor F) sumó 666, la Total(Post) 559, la FmujPre 211, la FmujPost 166, la FhomPre 455 y la FhomPost 393 (figura 18). En este Factor, las mujeres obtienen valores más bajos que los hombres y disminuye la fatiga al terminar la sesión aunque en mayor medida entre las mujeres. La puntuaciónTotal(Pre) del estado deVigor-Actividad (Factor V) sumó 568, laTotal(Post) 905, laVmujPre 210, laVmujPost 340, la VhomPre 358 y la VhomPost 565 (figura 19). Los valores de Vigorosidad son más altos por parte de las mujeres. Se observa un gran cambio entre el comienzo y el cierre de la sesión, aumentando hasta 4 puntos los hombres y 5 las mujeres. Se obtuvieron los siguientes resultados del Perfil General de Estado de Ánimo (PGEA) PGEATotalPre 10389, PGEATotalPost 10474, la PGEAmujPre 3568, la PGEAmujPost 3609, la PGEAhomPre 6821 y la PGEAhomPost 6865 (figura 20). El valor medio PGEA en las mujeres está por encima que el de los hombres. En ambas poblaciones el valor medio PGEA es más alto al finalizar la sesión. Al finalizar la sesión algunos expresaron palabras como sentirse enérgicos/as, felices y motivados/as. Discusión Como se ha comentado en la introducción del estu- 332 - dio, uno de los modelos pedagógicos más estudiados e implementados en el área de EF, es el aprendizaje cooperativo donde el alumnado trabaja conjuntamente, en pequeños grupos heterogéneos, y que aprenden con y de sus compañeros/as mediante un planteamiento de enseñanza-aprendizaje que fomenta la interacción e interdependencia positivas, permitiendo al docente y a los estudiantes actuar como co-aprendices (SánchezMolina et al., 2021, p.735). Esta actividad rítmica que replica patrones rítmicos a través de la PC es indudablemente una actividad cooperativa donde todo el grupo reproduce sonidos conjuntamente y no puede darse, es decir, no pueden seguir jugando si se rompe el tempo propuesto inicialmente. Puede verse una similitud entre la propuesta didáctica de este estudio y las propuestas del método BAPNE como práctica rítmica de PC que tiene evidencias para desarrollar las habilidades socio-afectivas (Fabra-Brell & Romero-Naranjo, 2017; Jiménez-Molina, Vicedo-Cantó, Sayago-Martínez, & Romero-Naranjo, 2017; Moral-Bofill, Romero-Naranjo, Albiar-Aliaga, & Cid-Lamas, 2015; Sayago, Salerno, Di, & Arnau, 2021). Para interpretar los resultados obtenidos respecto a las emociones que emergen en esta sesión, se procede a comparar con los estudios de Muñoz, Lavega, Serna, Sáez, & March (2017) y los estudios 1 y 2 de Mateu, Garcías, Spadafora, Andrés, & Febrer (2021) ya que ambas investigaciones se realizaron con una muestra similar, estudiantes en la formación inicial del profesorado de EF, y utilizaron el mismo intrumento (cuestionario POMS) (Tabla 1 y Tabla 2). Los valores del factor Tensión-Ansiedad que se obtienen al inicio y al finalizar la sesión son muy similares. Sin embargo, en Mateu et al. (2021), los valores previos son mucho más altos que los de este estudio en 4 puntos por encima (9.87). Esta diferencia puede deberse a que el estudio de Mateu et al. (2021) está sometido bajo la presión de una evaluación. Los valores del factor Depresión-Abatimiento de este estudio se muestran algo por encima que en el estudio 1 y similares al del estudio 2 de Mateu et al. (2021). Los valores del factor R obtenidos en este estudio son más bajos que el Estudio 1 pero más altos que el Estudio 2 (Mateu et al., 2021). Esta disminución de Rabia se debe al acondicionamiento que se dio entre el Estudio 1 y el Estudio 2. Cosa que en esta investigación se analiza de manera puntual, corte transversal, y no puede verse si existe un cambio en estos valores de hostilidad. Los valores de Vigorosidad son similares al estudio 2 de Mateu et al.(2021) y las palabras de feliz, enérgico/a, activo/a y motivado/a reafirman estas respuesRetos, número 45, 2022 (3º trimestre) tas. Los valores del factor F y PGEA son los más elevados. La repetición de las palabras cansado/a, exhausto/ a y agotado/a reforzarían los datos registrados respecto a la fatiga. La Esku Dantza es una propuesta no competitiva y coincide con los valores facilitados por el estudio de Muñoz et al. (2017) aunque diferenciando los géneros podemos observar que esta práctica artística genera un PGEA mayor que los registrados tanto en competición como no competitivos, consiguiendo unos 20 puntos por encima en hombres y hasta 23 más en mujeres. Estos aspectos son clave para favorecer programas de coeducación que aseguren el bienestar afectivo tanto en el género masculino como en el femenino (Muñoz et al., 2017). Los valores del factor T de las mujeres se asemejan a los valores del factor T sin competición, mientras que los hombres obtienen valores hasta 3 puntos por debajo del estudio de Muñoz et al. (2017). Los valores del factor D en mujeres son más altos que en el estudio de Muñoz et al. (2017) pero en los hombres el valor previo es más elevado que el estudio de Muñoz et al. (2017) e incluso que las mujeres mientras que el Post es más bajo. La fatiga es mucho más elevada en este estudio y la vigorosidad no puede compararse porque Muñoz et al. (2017) analizaron únicamente los estados de ánimo que denomina negativos. Según Bores-García, Hortigüela-Alcalá, HernandoGarijo, & González-Calvo (2020), el profesorado de EF tiene varios conflictos para implementar la EC en sus clases. Siguen sin proponer retos colectivos, sus propuestas no generan satisfacción entre el estudiantado, las propuestas no siguen un criterio lógico de progresión y, por tanto, repercute en el aprendizaje asociado a este contenido. Sin embargo, la propuesta pedagógica que se ha llevado a cabo a través de la Esku Dantza, para desarrollar la percepción y ejecución del ritmo con PC, responde a estas limitaciones considerando que es un reto colectivo planteado con una progresión cognitiva y motriz desde la comprensión y el movimiento global corporal en el espacio y el tiempo y, posiblemente lo más importante, que ha generado una experiencia positiva ya que, después de la intervención, mostraron un aumento respecto a sentirse enérgico/a, lleno/a de energía, animado/a, vigoroso/a y activo/a; y, al mismo tiempo, una disminución respecto a sentirse furioso/a, molesto/a, luchador/a, irritable, amargado/a, enfadado/a, enojado/a, de mal genio, desamparado/a, deprimido/a, desdichado/a, triste e infeliz. La percepción de las sensaciones, emociones, actituRetos, número 45, 2022 (3º trimestre) des, retos, cogniciones y límites, propios y de sus compañeros/as, se estimulan con estas prácticas de forma que se produce una respuesta motriz y les hace más respetuosos con los demás (Valverde-Esteve, 2020). Así como también genera experiencias positivas vinculadas con la danza ya que la experiencia previa con la misma condiciona al maestro/a y/o profesor/a para afrontar este contenido e implementarlo en clase (Lafuente, 2022). Conlusiones La LOMLOE (2020) manifiesta utilizar la EF y el deporte para favorecer el desarrollo personal y social, afianzar los habitos de actividades fisico-deportivas para favorecer el bienestar fisico y mental, asi como medio de desarrollo personal y social. Esta materia obligatoria en secundaria se divide en bloques de los cuales la EC sigue implementándose en menor medida por parte del profesorado. Las dificultades vienen determinadas por miedos o vergüenzas mayoritariamente debido a la escasa formación inicial y continuada relacionada con este contenido. Sin embargo, se han encontrado estudios que confirman la utilización de la PC para desarrollar competencias relacionadas con la EC, el sentido del ritmo y la coordinación, entre otras capacidades y cualidades motrices. La propuesta pedagógica realizada a través de la Esku Dantza como danza colectiva que utiliza la PC se caracteriza por implementarse como una actividad que va de lo individual a lo colectivo, en todo momento cooperativa, de ejecución progresiva, de sencillo a complejo, en la que se relacionan con todos/as los/las compañeros/as del grupo clase a través de la PC, en el que se reflexiona entorno a la equidad, donde hay una transmisión de cultura de otra comunidad, se desarrolla la percepción y materialización del sentido del ritmo, se crea una coreografía en grupo y se escuchan y expresan las emociones, es decir, se facilita un clima de confianza para compartir qué va sucediendo en sus adentros a medida que trascurre la sesión. Como consecuencia se ha conseguido generar una experiencia positiva en EF relacionada con el contenido de EC gracias a que los/las practicantes disminuyeron su estado emocional de fatiga, rabia y depresión, al mismo tiempo que aumentaron, en gran medida, el estado emocional de vigorosidad y actividad. Por tanto, se concluye que, dependiendo de cómo se propone la Esku Dantza, el estudiantado puede sentir cambios emocionales que benefician su experiencia relacionada con la - 333 - EC disminuyendo los miedos que actualmente siente el profesorado de EF y puede que se sienta más capacitado para implementar este contenido. A pesar de haber observado algunas diferencias entre mujeres y hombres, cabe destacar que el género masculino ha experimentado positivamente la práctica propuesta. Por tanto, es otro hecho positivo ya que se cree de especial importancia proporcionar experiencias positivas relacionadas con la EC y la danza sobretodo para el género masculino que parece ser, aún, el que siente mayor limitación. Por tanto, se cree que es una propuesta pedagógica positiva, cooperativa y motivadora en la que el estudiantado se ayuda entre sí, sigue una adecuada progresión de los aprendizajes motrices, ajustada al nivel y a las posibilidades motrices del alumnado, teniendo en cuenta la heterogeneidad del grupo-clase e invita al debate, la reflexión y crítica de la misma por parte del estudiantado. Este estudio apoya la idea de que una propuesta pedagógica de estas características, la Esku Dantza, produce cambios en el estado de ánimo de los/las practicantes y se observa que los cambios actitudinales y emocionales son positivos, pudiendo crear un impacto que puede dar lugar a un aprendizaje significativo. Pese a que hay que seguir profundizando con nuevos estudios que confirmen estos resultados, se considera que con experiencias de este estilo el estudiantado se siente más atraído a esta práctica (materia de EC y Danza) y, por tanto, su relación con la misma mejora. Este puede ser un primer paso para que los futuros profesionales del ámbito de las actividades físicas y deportivas se atrevan a incorporar estas prácticas y concretamente, en el caso de futuros profesores/as obtener recursos e ideas que les permitan trabajar en pro de una EF de calidad. Limitaciones del estudio También destacar, como observación de una limitación entre el vocabulario del estudiantado, que preguntaban por el significado de desamparado/a, desdichado/a y vigoroso/a, mostrando el posible desconocimiento de ciertas emociones y por tanto podría desvirtuar los resultados. Se reconoce la limitación que puede suponer haber analizado una sola sesión. El estudio original incluye algunos aspectos cualitativos que no han sido utilizados en este artículo por razones de espacio, pero que posiblemente le darían mayor riqueza y significado. - 334 - Líneas de futuro La finalidad de esta sesión podría abarcarse desde diferentes perspectivas, como un poliedro analizando objetivos y/o aprendizajes asimilados por parte del estudiantado. Por ejemplo, se podría estudiar la relación intrapersonal como las emociones que afloran y cómo se gestionan, relación interpersonal (con el otro), el trabajo cooperativo para desarrollar una coreografía en grupo, la capacidad de percepción y ejecución (precisión) en los golpeos percusivos al ejecutar la danza, la interpretación expuesta durante la ejecución, etcétera. Agradecimientos Se agradece la participación en esta investigación a los/las estudiantes del Grado en CCAFyD del INEFC Lleida (curso 2020 – 2021) que han cursado la asignatura de EC y danza y que respondieron el cuestionario. Así como el apoyo de su profesora titular que ha facilitado la incorporación de este contenido en la materia. Esta publicación cuenta con el apoyo del Institut Nacional d’Educació Física de Catalunya (INEFC) de la Generalitat de Catalunya. Referencias Alonso-Sanz, A., Romero-Naranjo, F. J., Pons-Terrés, J., Carretero-Martínez, A., & Crespo-Colomino, N. (2015). La cultura visual desde el análisis multidisciplinar. Spot de Coca-cola al limón de Mayumana. En J.A. Bornay, F. J. Romero-Naranjo,V. J. Ruiz, & J. Vera (Eds.), Fronteras reales, fronteras imaginadas (pp. 117-129). Alicante: Ediciones Letra de Palo, S.L. Abilleira, M., & Fernández, M. (2017). Análisis de la formación del maestro / a de educación. EmásF. Revista Digital de Educación Física, (49), 36-59. Araolaza, O. (2020). Genero-identitatea euskal dantza tradizionalaren eraikuntzan. Donostia. Arias, J. R., Fernández, B., & San Emeterio, C. (2020). 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Retos, (20), 33-36. https://doi.org/ Retos, número 45, 2022 (3º trimestre) 10.47197/retos.v0i20.34621 García, M., Piqueres, I., Sánchez, E., Serna, M., Trives, E., & Romero-Naranjo, F. J. (2018). El uso de la percusión en las músicas urbanas. En Innovación y modelos de enseñanza-aprendizaje en la Educación Superior (Vol. 1). Garcías, S. (2021a). Impacto positivo de una master class online de percusión corporal para docentes. Retos, 42, 296-305. https://doi.org/10.47197/ retos.v42i0.87059 Garcías, S. (2021b). La Percusión Corporal: del escenario al aula. En S. Olivero (Ed.), El devenir de las civilizaciones: interacciones entre el entorno humano, natural y cultural (pp. 1588-1614). Madrid: Dykinson, S.L. Garcías, S. (2021c). La Percusión Corporal en la Formación Inicial del Profesorado de Educación Física. Satisfacción y/o Frustración de las Necesidades Psicológicas Básicas de los estudiantes universitarios. En O. Buzón-García, C. 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Conciencia corporal, reconocimietno y representación de los estados de ánimo: educación emocional a través de la danza. En J. Gil, C. Padilla, & C.Torrents (Eds.), Artes escénicas y creatividad para transformar la sociedad y la educación (pp. 167-180). Madrid: AFYEC (Actividad Física y Expresión Corporal). Hawkins, D. R. (2014). El Poder frente a la fuerza. Los determinantes ocultos de la conducta humana. Barcelona: El grano de mostaza. Jiménez-Molina, J. B., Vicedo-Cantó, E., SayagoMartínez, R., & Romero-Naranjo, F. J. (2017). Evaluating Attention, Socioemotional Factors and - 335 - Anxiety in Secondary School Students in Murcia (Spain) Using the BAPNE® Method. Research Protocol. Procedia - Social and Behavioral Sciences, 237, 1071-1075. https://doi.org/10.1016/ j.sbspro.2017.02.157 Lafuente, J. C. (2022). Valoración de los contenidos de Expresión Corporal por parte de los futuros maestros en la asignatura de Actividades Físicas Artístico-expresivas de la mención de Educación Física. 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https://openalex.org/W2597424048	https://archive.org/download/annualreport198081onta/annualreport198081onta_bw.pdf	English	null	The Fourteenth Annual Report	Australasian journal of optometry	1,925	public-domain	8,458	FOURTEENTH ANNUAL REPORT 1980-81 ONTARIO LAW REFORM COMMISSION Ontario Ministry of the Attorney General The Honourable James C. McRuer, the first Chairman of the Commission and Vice Chairman until February, 1977, resigned as a full-time Member of the Commission effective June, 1977. However, Mr. McRuer agreed to remain a Commissioner in relation to the Projects on the Law of Trusts and the Enforcement of Judgment Debts, in the development of which he has been much involved. FOURTEENTH ANNUAL REPORT 1980-81 ONTARIO LAW REFORM COMMISSION Ontario Ontario Ministry of the Attorney General The Ontario Law Reform Commission was established by section 1 of The Ontario Law Reform Commission Act for the purpose of reforming the law, legal procedures and legal institutions. The Commissioners are: Derek Mendes da Costa, q.c, ll.b., ll.m., s.j.d., ll.d., Chairman Derek Mendes da Costa, q.c, ll.b., ll.m., s.j.d., ll.d., Chairman Honourable George A. Gale, c.c, q.c, ll.d. Honourable Richard A. Bell, p.c, q.c. * Honourable James C. McRuer, o.c, ll.d., d.c.l. William R. Poole, q.c. Barry A. Percival, q.c. * Honourable James C. McRuer, o.c, ll.d., d.c.l. William R. Poole, q.c. Barry A. Percival, q.c. Barry A. Percival, q.c. M. Patricia Richardson, M.A., LL.B., is Counsel to the Commission. The Secretary of the Commission is Miss A. F. Chute, and its offices are located on the Sixteenth Floor at 18 King Street East, Toronto, Ontario, Canada M5C1C5. ISSN 0474-1862 TABLE OF CONTENTS Page Introduction 7 the programme: referred matters 8 (i) Witnesses Before Committees of the Legislature 8 (ii) Class Actions 9 THE PROGRAMME: PROJECTS INITIATED BY THE COMMISSION 9 1 Completed Projects 9 Enforcement of Judgment Debts and Related Matters. . . 9 2. Projects in Process 13 (i) The Law of Trusts 13 (ii) Administration of Estates of Deceased Persons 13 (iii) The Hague Convention Concerning the International Administration of the Estates of Deceased Persons 13 (iv) The Law of Mortgages 14 (v) Enforcement of Judgment Debts and Related Matters. . . 14 (vi) The Law of Standing 14 (vii) Powers of Entry 14 (viii) Law of Contract Amendment 15 (ix) Contribution Among Wrongdoers 15 Future Programme 15 General Activities 16 Visitors 17 Table of Implementation 17 Acknowledgments 17 Appendix A 19 Appendix B 27 Future Programme 15 General Activities 16 Visitors 17 Table of Implementation 17 Acknowledgments 17 Appendix A 19 Appendix B 27 [3] Ontario Law Reform Commission Ontario Law Reform Commission To The Honourable R. Roy McMurtry, Q.C. Attorney General for Ontario Dear Mr. Attorney: We have the honour to present the Fourteenth Annual Report of the Ontario Law Reform Commission, for the period April 1, 1980 to March 31,1981. [5] [5] INTRODUCTION This Report deals with the activities of the Commission during the period April 1, 1980 to March 31, 1981. During the past year the Commission has been engaged in a heavy research programme. We have completed much of the research involved in our Project on the Enforcement of Judgment Debts and Related Matters. The Commission decided that, due to the magnitude and the complexity of the subject, it was desirable to divide the Report into five Parts. We have now submitted Parts I, II and III of the Report, and have made substantial progress on Parts IV andV. The Commission records the enactment of The Occupiers' Liability Act, 1980, S.O. 1980, c. 14, which in large measure adopts the recommendations made by the Commission in its Report on Occupiers' Liability (1972). The past year has been productive and busy. During the year, much of the Commission's energy was devoted to the completion of the first three Parts of the Report on the Enforcement of Judgment Debts and Related Matters. We also have brought the Project on Class Actions close to conclusion. In addition, we have made progress on a Reference, Witnesses Before Committees of the Legislature. The Commission's Programme consists of eleven Projects. As we said in our last Annual Report, much balance and co-ordination is involved in the task of bringing all Projects forward in an orderly manner. Since the submission of Parts I, II and III of the Report on the Enforcement of Judgment Debts and Related Matters, the Commission and its research staff have been committed to the completion of research and preparation of the Report on Class Actions and the Report on Witnesses Before Committees of the Legislature, commitments that will continue into the coming year. We have not been able to develop, in the past year, two Projects: Basic Principles of Land Law and Declarations of Status. We recognize the importance of these Projects, and will return to them when time and resources permit. As in past years, the Commission has continued to receive helpful suggestions for additions to its Programme from members of the judiciary, the legal profession and the public. We welcome this interest in the work of the Commission, and wish to express our appreciation to all those who gave of their time to assist us in this way. THE PROGRAMME: REFERRED MATTERS Section 2(1 )(d) of The Ontario Law Reform Commission Act requires the Commission to inquire into and to consider any matter relating to any subject referred to it by the Attorney General. One new matter, Witnesses Before Committees of the Legislature, was referred to the Commission during the period covered by this Report. During the year, research on a prior Reference, the Class Actions Project, continued. [7] [7] THE PROGRAMME: PROJECTS INITIATED BY THE COMMISSION During the past year, no new Projects have been initiated by the Commission. (ii) Class Actions The terms of reference, and the scope and history of the Class Actions Project, are outlined in detail in previous Annual Reports. During the past year, substantial progress was made on the Project. In particular, internal research papers dealing with the important and controversial issues of damages, discovery, opting in and opting out, notice and res judicata have been prepared and considered by the Commission. We will discuss the remaining research papers, dealing with settlement, costs and procedural problems, in the spring and early summer of this year. It is hoped that the Commission's final Report on Class Actions, and an accompanying draft Class Actions Act, will be submitted to the Attorney General by the fall. (i) Witnesses Before Committees of the Legislature On June 5, 1980, in response to a recommendation made by the Standing Procedural Affairs Committee of the Ontario Legislative Assembly in its Report on Witnesses Before Committees, the Attorney General referred the subject of witnesses before legislative committees to the Commission. The Attorney General requested the Commission to conduct a thorough review of the subject, bearing in mind particularly the eleven questions stated for the Commission in Appendix A to the Report on Witnesses Before Committees. The Report on Witnesses Before Committees made it clear that both the law and practice in this area required clarification and rationalization. The Commission engaged Alistair Fraser, Esq., former Clerk of the House of Commons in Ottawa, to prepare a comprehensive internal research paper dealing with all aspects of the subject, including the following matters: the nature and functions of various types of legislative committees; the kinds of activities undertaken by committees; the power of committees to compel witnesses to appear and answer questions and produce documents at committee hearings; the power of the Legislative Assembly to punish witnesses who fail to appear and cooperate with committees; the obligations of witnesses to committees, and of committees to witnesses; the immunity or protection that extends or should extend to witnesses in respect of their oral testimony and written submissions; the protection of third parties whose names or activities are divulged or discussed at committee meetings; the nature and scope of the doctrine of Crown privilege in respect of oral testimony and the production of documents; the constitutional aspects of protecting witnesses from the use of their evidence in subsequent civil and criminal proceedings; and the right to counsel at committee meetings. The experience of legislative committees of the Parliament in Ottawa, as well as reports and relevant material from Australia and the United Kingdom, are discussed in the research paper. In addition, the Commission will review the operation of Congressional committees in the United States. We have completed our deliberations in respect of most of the material and recommendations contained in the research paper. The Commission is engaged in the preparation of its Report, which it will submit to the Attorney General at the earliest opportunity. Enforcement ofJudgment Debts and Related Matters As we have said, the Commission has submitted to the Attorney General Parts I, II and III of our projected five Part Report. The Commission's Project on the Enforcement of Judgment Debts and Related Matters is a response to administrative, substantive and procedural deficiencies in the present law and practice pertaining to debtor-creditor relations, and is an attempt to deal with these deficiencies comprehensively. Part I of our Report begins with a consideration of the methods by which a financially overcommitted debtor, and particularly a judgment debtor, may pay his debts by instalments and thereby avoid the imposition of traditional enforcement measures against him. Chapter 2 deals with Part III of the proposed new federal bankruptcy legislation (Bill C-12, First Session, Thirty-second Parliament, 29 Eliz. II, 1980) and with provincial consolidation orders, instalment payment plans and stays of enforcement proceedings. While we generally endorse the provisions of Part III of Bill C-12, we do make recommendations that differ from some of these provisions. In addition, Chapter 2 offers several proposals in respect of provincial instalment payment plans and stays of enforcement proceedings that fall outside the ambit of Bill C-12. With Chapter 3 of Part I of our Report, the Commission commences its study of the organizational and administrative aspects of enforcement. Chapter 3 recommends the creation of a new, integrated enforcement office for each county under the direction of a sheriff. We recommend that the new enforcement office should have overall responsibility for the enforce- ment of all money judgments, including support and maintenance orders, 10 from all courts, except "show cause" proceedings and expanded wage garnishment proceedings in the provincial court (family division). More- over, under our proposals, the enforcement of money judgments against a debtor would emanate from a single enforcement office, thereby eliminat- ing the existing fragmented enforcement system and ensuring the coordina- tion and integration of all enforcement activities against that debtor. from all courts, except "show cause" proceedings and expanded wage garnishment proceedings in the provincial court (family division). More- over, under our proposals, the enforcement of money judgments against a debtor would emanate from a single enforcement office, thereby eliminat- ing the existing fragmented enforcement system and ensuring the coordina- tion and integration of all enforcement activities against that debtor. Enforcement ofJudgment Debts and Related Matters Under our 11 proposals, for example, it would no longer be necessary to apply to a court for a garnishment order; rather, the new enforcement office, generally, would have carriage of garnishment proceedings. Chapter 4 of Part II deals with equitable execution, charging orders and stop orders. Essentially, it is proposed that the hitherto restrictive equitable execution remedy should be abolished and that the remedy of receivership should take its place. Under our proposals, the latter remedy would be fully integrated into the new enforcement regime as but another type of enforcement remedy to be employed by judgment creditors. Receivers would be permitted to sell property received by them and, in appropriate circumstances, they would be permitted to manage a debtor's property or commercial enterprise. With respect to charging orders, the Commission recommends the abolition of this remedy as redundant. Finally, recommendations are made to expand the stop order remedy to cover all property in court. Chapter 5, the last Chapter in Part II, deals with the resolution of disputes in enforcement proceedings. There is a consideration of the gaps and deficiencies in the interpleader rules of the Supreme Court of Ontario Rules of Practice, and proposals are made to render the interpleader rules more comprehensive. The Commission's most significant recommendations concern claims made by third parties to seized personal property. It is recommended, for example, that subsequent to a seizure, the sheriff should serve a "notice of seized property" on all persons who, to the knowledge or reasonable belief of the sheriff, may have some right, title or interest in the seized property. Generally speaking, the failure of a third party to make a claim to seized property within the prescribed limitation period would result in the extinguishment of that claim upon a sale of the property to a purchaser at an execution sale. However, provision is made for the filing of late claims and claims against the proceeds of sold property. Finally, recommendations are made in respect of the resolution of disputes in the context of garnishment proceedings — recommendations that basically mirror those made in the context of enforcement against personal property. Part III of our Report concerns the very important topic of enforcement against interests in land. Enforcement ofJudgment Debts and Related Matters In Chapter 4 of Part I, the Commission discusses the means by which a judgment creditor and the enforcement office may obtain information concerning a debtor's property. Proposals are made in respect of judgment debtor questionnaires and examinations, third party examinations and public register searches. In Part II of our Report, the Commission considers the traditional methods by which creditors may enforce their money judgments. Chapter 2 deals with the seizure and sale of personal property. Recommendations are made concerning, for example, the province-wide binding of a debtor's personal property by the proposed new writ of enforcement — a writ, more comprehensive than the present writ of fierifacias, that would be employed to initiate any and all traditional enforcement measures against a debtor. Other recommendations attempt, by overturning the restrictive common law, to ensure that all of a debtor's personal property is liable to seizure and sale, subject only to a meaningful set of exemptions designed to ensure that the debtor is left with the necessities of life. Yet further recommendations deal with specific problem areas relating, for example, to the seizure and sale of mortgages and other securities for money, negotiable instruments, shares, non-assignable or restrictively assignable interests, and the beneficial interest of a debtor under a personal property security agreement. Finally, Chapter 2 makes recommendations concerning the time, notice, location and manner of sale of seized personal property. Chapter 3 of Part II deals with the garnishment of a debtor's income and other debts owing to the debtor. As in the case of execution against personal property, recommendations are made to ensure that all debts owing to a debtor, including conditional, contingent and future debts, are subject to garnishment. The Commission also recommends that a judgment creditor should be entitled to a continuing garnishment order, that is, a garnishment order that remains effective until the amount specified in the order has been paid. In this way, the frustrating need to obtain a separate order each time wages, or a debt, are sought to be garnished would be obviated. In addition to extending the garnishment remedy, the Commission has been cognizant of the need to protect debtors' incomes. Accordingly, proposals are made for an expanded income exemption, beyond that now contained in The Wages Act. Finally, Chapter 3 makes recommendations designed to establish one uniform garnishment procedure for the enforcement of money judgments from all courts. 12 Special provisions are proposed in respect of a debtor's residence, including exemption provisions and provisions protecting the interests of creditors where a debtor's residence is classified in law as personal property and not land. Finally, recommendations respecting the rights of creditors of titled and non-titled spouses are made where the debtor's residence is a "matrimonial home" under The Family Law Reform Act, 1978. In Chapter 3, we consider one of the most frustrating and vexing areas of debtor-creditor law. After describing the manner by which enforcement law impinges on conveyancing law and practice, and after considering how the present enforcement regime serves to prejudice persons involved in the conveyancing process who are strangers to the debtor-creditor relationship, the Commission offers several long term and short term proposals for reform. Believing that the evils inherent in the present system stem from the operation of a writ of fieri facias as a general lien, binding a debtor's land without being registered directly against the title, the Commission, in its long term proposals, recommends the abolition of the writ as a general lien. In its stead, we propose that a creditor should be required to register his writ directly against the title to his debtor's land in order to bind that land. As a means by which creditors could discover land owned by their debtors, the Commission recommends the creation of an index of landholdings comprising all Registry Act and Land Titles Act land in Ontario. In the second portion of Chapter 3, the Commission addresses itself to several short term proposals that could be adopted pending the implementation of its long term proposals. It is recommended, for example, that writs, operating as general liens, should not bind land until the expiry of ten calendar days after filing. This delay in the binding effect of a writ would preclude the need for a subsearch for writs immediately prior to the closing of a real estate transaction; consequently there would be ten days within which to resolve "similar name" problems stemming from the discovery of a writ against a debtor with an identical or similar name to that of the vendor or mortgagor. In order to protect creditors, the Commission further recommends that creditors who know of land owned by their debtors should be entitled to register their writs directly against the title, with the land being bound immediately upon registration. Enforcement ofJudgment Debts and Related Matters Part III is divided into two major portions: Chapter 2 deals with certain substantive and procedural considerations pertaining to enforcement, and Chapter 3 canvasses the relationship between enforcement against land and the process by which land is sold or mortgaged. Under our proposals in Chapter 2, most of the existing restrictions on enforcement against land would be abolished. Although the mandatory delay period prior to a sale is retained, it would be reduced from twelve months to six months. Creditors would be given rights to make a claim against a surviving joint tenant where the debtor joint tenant has died leaving insufficient property to satisfy his judgment debts. (i) The Law of Trusts The preparation of the Report on the Law of Trusts and an Act to revise The Trustee Act has entered the final stages. Approximately one-half of the Report and the proposed revised Trustee Act has been approved by the Commission. We wish to acknowledge the assistance of Professor D.W.M. Waters of the Faculty of Law, University of Victoria, the Project Director, for whose scholarship and devotion, both in time and in energy, we are most grateful. So too, we have had the benefit of the great skill and long experience of L.R. MacTavish, Esq., Q.C., former Senior Legislative Counsel, in the preparation of the proposed revised Act. Although completion of the Report has been temporarily suspended, pending the conclusion of the Report on Class Actions, it is anticipated that work will resume in the fall, and that the Report and revised Trustee Act will be forwarded to the Attorney General before the end of the coming year. (ii) Administration of Estates ofDeceased Persons The basic research involved in this Project has been all but completed, and the Commission has considered the first Research Report, prepared by the Project Director, Professor George W. Alexandrowicz, of the Faculty of Law, Queen's University. The Advisory Committee of experts in the law governing estate administration, constituted under the chairmanship of Malcolm S. Archibald, Esq., Q.C., has met frequently, and has submitted many matters for consideration by the Commission. It is hoped that work will commence, during the coming year, on a proposed new Administration of Estates Act, which will bring together, in a revised and expanded form, relevant portions of The Trustee Act, The Devolution of Estates Act, and the provisions governing practice under The Surrogate Courts Act and Rules. A number of common law doctrines that now govern estate administration will be codified and revised in the proposed new Act. 13 execution certificate should be registered on title and that all person seeking to acquire any interest in the land should be entitled to rely on th representations made in the registered certificate. execution certificate should be registered on title and that all persons seeking to acquire any interest in the land should be entitled to rely on the representations made in the registered certificate. 12 Other short term proposals are made in Chapter 3 in an attempt to alleviate the "similar name" problem. For example, it is recommended that a writ should bind land only where the surname and at least one full given name on the writ are identical with the surname and one full given name on a document registered against the title to the land in question. Sheriffs would be required to report the existence of a writ only where the surname and at least one full given name on the writ are identical with the surname and one full given name on the request form delivered to the sheriff. Finally, in order to preclude the necessity for repeated historical searches for outstanding writs binding Registry Act land, with the attendant "similar name" problems, it is recommended that when land is conveyed a sheriff's (vi) The Law ofStanding The objective of the Project on the Law of Standing is to determine whether private individuals who wish to litigate in the public interest should be granted increased access to the courts. The law governing locus standi, or status to bring suit, has been frequently criticized, particularly as it relates to the right of a private individual to commence litigation concerning public rights. Generally speaking, the existing law restricts the right to litigate in the public interest to the Attorney General, either in his own name or by means of a relator action. In order to be granted standing, a private individual must establish that he or she has a special interest or has sustained special damages. During the initial stages of the Project, the internal legal staff prepared a background paper dealing with the present law and isolating reform issues. While priority has been accorded to the Minister's Reference on Class Actions, the Commission has commissioned a major research paper dealing with reform of the law of standing. It is anticipated that this paper will be received by the Commission soon and that, following completion of the References on Class Actions and Witnesses Before Committees of the Legislature, the Commission will consider the issues raised in the research paper. (v) Enforcement ofJudgment Debts and Related Matters Of the Commission's projected five Part Report on the Enforcement of Judgment Debts and Related Matters, two Parts remain to be completed. Part IV will consider prejudgment enforcement remedies and voidable transactions, and Part V of the Report will contain a review of creditors' relief legislation, including Crown priorities and the distribution to creditors of the proceeds of a sale of a debtor's property. Research on these topics has been completed. Part V also will consider the liability of the sheriff for acts or omissions in the course of his enforcement duties, and other miscellaneous matters pertaining to the enforcement of money judgments. As has been the case throughout this Project, we are very pleased to state that we will continue to have the benefit of the assistance of David E. Baird, Esq., Q.C. (iv) The Law ofMortgages With the appointment, during the last year, of Professor Richard H. McLaren, of the Faculty of Law, University of Western Ontario, as Project Director, research has commenced. A summary of mortgage remedies has been prepared, but has not yet been considered by the Commission. (Hi) The Hague Convention Concerning the International Administration of the Estates ofDeceased Persons The question as to whether the Hague Convention should be given effect in Ontario has been considered by the Commission. In relation to the administration of estates of foreign decedents, it is obvious that the Project is closely linked to our Project on the Administration of Estates of Deceased Persons. For this reason, the two Projects are being considered together. In due course we will decide whether to combine the Projects in one Report, or to submit two Reports. (viii) Law of Contract Amendment Our last Annual Report contained details of the content of this Project. Briefly stated, the Project is divided into three Phases, Phase I being concerned with the formational aspects of the law of contract, and the remaining two Phases with substantive and remedial issues. In the past year, the progress of this Project has been most satisfactory. An Advisory Group of experts, consisting of members of the judiciary and practising lawyers, under the chairmanship of James M. Spence, Esq., has been established, and the Commission has benefited greatly from its views and comments. A research report dealing with Phase I, prepared by the joint Project Directors, Professor Jacob S. Ziegel and Professor Stephen M. Waddams, both of the Faculty of Law, University of Toronto, has been considered by the Commission, and research on Phase II is well under way. It is likely that the Commission will issue separate Reports covering each Phase of the Project. In the coming year, should time permit, the Commission will commence the preparation of a draft Report on Phase I, and the consideration of the topics covered in Phase II. 15 York University, and Professor Stanley M. Makuch, of the Faculty of Law, University of Toronto, have completed their research, and the Commission has considered a draft Report concerning powers conferred by Ontario statutes and regulations to enter upon lands, buildings and private dwellings. The Commission is now engaged in the preparation of a proposed Powers of Entry Act, which will be appended to the Commission's Report. Depending upon the availability of time and resources, the Commission hopes to submit its Report in the coming year. (ix) Contribution Among Wrongdoers The purpose of this Project is to review the law relating to the allocation of loss between two or more persons who are responsible for the same injuries, and the law relating to contributory negligence. Professor John M. Evans, of Osgoode Hall Law School, York University, has been appointed Project Director, and the Commission has considered many difficult and complex issues, including: aspects of joint and several liability; extension of the right to contribution to all concurrent wrongdoers; elements of the right to contribution; the effect of a settlement upon the right to contribution; and defences to the right to contribution. The remaining research should be completed in the coming year. (vii) Powers ofEntry Excellent progress has been made on this Project during the past year. The Project Directors, Professor Alan Grant, of Osgoode Hall Law School, 16 several of our Projects have been brought to, or near to, the stage when a draft Report can be prepared. However, the preparation of the Report on Class Actions and the Report on Witnesses Before Committees of the Legislature will dominate the attention of the Commission in the near future. several of our Projects have been brought to, or near to, the stage when a draft Report can be prepared. However, the preparation of the Report on Class Actions and the Report on Witnesses Before Committees of the Legislature will dominate the attention of the Commission in the near future. FUTURE PROGRAMME In recent years, the Commission has been required to formulate priorities and to allocate limited resources among the Projects that constitute our research Programme. Last year was no exception, and it is clear that the issue will be with us again in the coming year. As we have said, 16 TABLE OF IMPLEMENTATION Attached to this Report as Appendix A is a list of the Reports that have been prepared and submitted by the Commission since its inception in 1964, together with a table setting out the extent to which legislation concerning our proposals has been enacted. VISITORS As in the past, the Commission has endeavoured to maintain a mutually beneficial working relationship with other law reform agencies. It is always a great pleasure to welcome to our offices representatives of these agencies, and to hear of trends and developments in their jurisdictions. The visitors whom we were pleased to receive, included: Bruce M. Debelle, Esq., Commissioner, The Law Reform Commission, Australia; Dr. P.M. North, Commissioner, The Law Commission, England; and Denis Gressier, Esq., Commissioner, Law Reform Commission, New South Wales. Other visitors whom we were privileged to welcome included: The Honourable Mr. Justice R.S. Watson, Senior Judge, Family Court of Australia; and, Mrs. Gloria Cumper, a distinguished member of the legal profession of Jamaica. 17 year students on law reform in Ontario, and spoke to students at the Forest Hill Collegiate Institute. year students on law reform in Ontario, and spoke to students at the Forest Hill Collegiate Institute. As part of the March Special Lecture Series 1981 , of the Department of Continuing Education of the Law Society of Upper Canada, the Chairman delivered a paper entitled "The Enforcement of Judgments: Proposals for Reform", that had been prepared by the Chairman and by Eric Gertner, Esq., one of the Legal Research Officers of the Commission. GENERAL ACTIVITIES The Chairman and Counsel, on behalf of the Commission, attended a meeting of the Canadian Law Reform Agencies at Charlottetown in August, 1980. This meeting provided an opportunity to exchange information with representatives of the other law reform agencies across Canada, and to be informed of the research programmes of these agencies. Immediately following the meeting, the Chairman represented the Commission at the Sixty-second Annual Meeting of the Uniform Law Conference of Canada, in the same city. In our last Annual Report, we stated that the Uniform Law Section of the Uniform Law Conference of Canada had established a Committee on the Sale of Goods to consider the need for uniform sale of goods legislation, and to assess the utility of the Ontario Law Reform Commission's Report on Sale of Goods as a basis for such a uniform law. Our Chairman was appointed Chairman of this Committee, which, during the past year, met on six occasions in Toronto. The Committee has made great progress, and expects that its Report will be completed by the summer of 1981 In the year under review, the Commission has been pleased to renew its collaboration with the Law Reform Committee of the Ontario Branch of the Canadian Bar Association. On May 22, 1980, the Chairman, Vice Chairman and Counsel met with the Committee, under the chairmanship of Mrs. Judith M. Oyen, Q.C., and with Julian H. Huffer, Esq., C.A.E., Executive Director of the Ontario Branch of the Canadian Bar Association, to discuss methods of future liaison between the Commission and the Committee. The Commission wishes to express its gratitude to the Committee for the help and cooperation that was so willingly offered. We will take full advantage of this offer, and are in touch with the Committee concerning the establishment of an Advisory Committee to the Project on the Law of Mortgages. During the past year, the Chairman was called upon to represent the Commission on many occasions. These included an address to the Metropolitan Toronto Legal Secretaries Association of Canada, and an address to the Family Law II Conference on the Matrimonial Property Act, sponsored by the Continuing Legal Education Society of Nova Scotia. The Chairman also visited the Law School of McGill University to speak to first 17 ACKNOWLEDGMENTS Appendix B consists of a list of the officers and permanent staff of the Commission. As will be noted, the past year has seen no change in the ranks of the Commissioners or the legal staff. However, we regret the loss of several members of our administrative staff, to whom we extend our best wishes for success in their new endeavours: Mrs. Roslynne F. Mains, B.A., Ms. Grace C. Novakowski, B.A., and Mrs. E.M. Renda. We warmly welcome those who have joined our administrative staff during the past year: Mrs. J. A. Brown, B.A., Mrs. Toni Farrace, and Ms. Victoria Van Asperen, B.Sc. 18 Our sincere thanks are also extended to the Secretary, Miss A.F. Chute, and to the administrative staff for all they have done to assist the Commission during the year. To you, Mr. Attorney, and to the officers of the Ministry, we extend our sincere appreciation and thanks for the manner in which we have been sustained and encouraged in our work. anks for the manner in which we have been work. submitted. >. fouler CU^fl-^ * — Derek Mendes da Costa, Chairman George A. Gale Vice Chairman Richard A. Bell Commissioner James C. McRuer Commissioner William R. Poole Commissioner ^^-^- Barry A. Percival Commissioner All of which is respectfully submitted. ^ci>. fouler CU^fl-^ * — Derek Mendes da Costa, Chairman George A. Gale Vice Chairman Richard A. Bell Commissioner James C. McRuer Commissioner William R. Poole Commissioner ^^-^- March 31, 1981 Barry A. Percival Commissioner All of which is respectfully submitted. ^ci>. fouler CU^fl-^ * — Derek Mendes da Costa, Chairman George A. Gale Vice Chairman James C. McRuer Commissioner William R. Poole Commissioner Barry A. Percival Commissioner Barry A. Percival Commissioner Barry A. Percival Commissioner March 31, 1981 March 31, 1981 10. The Execution Act: Exemption of Goods from Seizure REPORTS MADE BY THE ONTARIO LAW REFORM COMMISSION Title Date of Report Legislation Concerning Commission Proposals 1. No. 1 The Rule Against Perpetuities February 1, 1965 The Perpetuities A ct, S.O. 1966, c. 113 2. No. 1A Supplementary Report on the Rule Against Perpetuities March 1,1966 do. 3. No. 2 The Wages Act; Assignment of Wages March 3, 1965 The Wages Amendment Act, S.O. 1968, c. 142 4. No. 3 Personal Property Security Legislation May 28, 1965 The Personal Property Security Act, S.O. 1967, c. 72 5. No. 3A Supplementary Report on Personal Property Security Legislation May 18, 1966 do. 6. The Evidence Act; Admissibility of Business Records February 16, 1966 The Evidence Amendment Act, S.O. 1966, c. 51, s. 1 7. The Mechanics' Lien Act February 22, 1966 The Mechanics * Lien Act, S.O. 1968-69, c. 65 8. Supplementary Report on The Mechanics' Lien Act May 26, 1967 do. 9. Proposed Extension of Guarantor's Liability on Construction Bonds May 30, 1966 See The Mechanics' Lien A mendment Act, S.O. 1975, c.43 The Ministry of Transportation and Communica- tions Creditors Payment Act, S.O. 1975, c. 44 The Public Works Creditors Payment Repeal Act, S.O. 1975, c.45 10. The Execution Act: Exemption of Goods from Seizure December 9, 1966 The Execution A mend- ment Act, S.O. 1967, c. 26 Title Date of Report Legislation Concerning Commission Proposals 1. No. 1 The Rule Against Perpetuities February 1, 1965 The Perpetuities A ct, S.O. 1966, c. 113 2. No. 1A Supplementary Report on the Rule Against Perpetuities March 1,1966 do. 3. No. 2 The Wages Act; Assignment of Wages March 3, 1965 The Wages Amendment Act, S.O. 1968, c. 142 4. No. 3 Personal Property Security Legislation May 28, 1965 The Personal Property Security Act, S.O. 1967, c. 72 5. No. 3A Supplementary Report on Personal Property Security Legislation May 18, 1966 do. 6. The Evidence Act; Admissibility of Business Records February 16, 1966 The Evidence Amendment Act, S.O. 1966, c. 51, s. 1 7. The Mechanics' Lien Act February 22, 1966 The Mechanics * Lien Act, S.O. 1968-69, c. 65 8. Supplementary Report on The Mechanics' Lien Act May 26, 1967 do. 9. Proposed Extension of Guarantor's Liability on Construction Bonds May 30, 1966 See The Mechanics' Lien A mendment Act, S.O. 1975, c.43 [19] Title 11. The Law of Condominium 13 14. 15. 16. 17. 18, 19. 20. REPORTS MADE BY THE ONTARIO LAW REFORM COMMISSION Basis for Compensation on Expropriation The Limitation Period for Actions under The Sandwich, Windsor and Amherstburg Railway Act, 1930 Annual Report 1967 Certain Aspects of the Proposed Divorce Legislation contained in Bill C-l 87 (Can.) The Proposed Adoption in Ontario of The Uniform Wills Act The Protection of Privacy in Ontario The Insurance Act, R.S.O. 1960, c. 190, s. 183, as amended by S.O. 1961-62, c. 63, s. 4 (commutation) Trade Sale of New Houses and the Doctrine of Caveat Emptor Interim Report on Landlord and Tenant Law Applicable to Residential Tenancies Date of Report March 6, 1967 September 21, 1967 January 8, 1968 January 15, 1968 January 19, 1968 February 5, 1968 September 10, 1968 October 3, 1968 October 4, 1968 December 10, 1968 Legislation Concerning Commission Proposals The Condominium Act, S.O. 1967, c. 13 See now The Condo- minium Act, S.O. 1978, c. 84 The Expropriations A ct, S.O. 1968-69, c. 36 The Sandwich, Windsor andA mherstburg Railway A mendment Act, S.O. 1968, c. 120 Divorce Act, S.C. 1967 68, c. 24, s. 26 The Succession Law Reform Act, S.O. 1977, c.40 See The Registry Amendment Act, S.O. 1978, c. 8, s. 1 See The Consumer Reporting Act, S.O. 1973, c. 97 See The Ontario New Home Warranties Plan Act, S.O. 1976, c. 52 The Landlord and Tenant Amendment Act, S.O. 1968-69, c. 58 The Trustee Amend- ment Act, S.O. 1971, c. 32, s. 2 Title [20] [20] Date of Report Legislation Concerning Commission Proposals See The Highway Traffic Amend- ment Act (No. 2), S.O. 1975, c. 37 The Fatal A cciden ts A mend- mentAct, S.O. 1975, c. 38 See now The Family Law Reform Act, S.O. 1978, c. 2, s. 60(4) The Trustee A mend- mentAct, S.O. 1975, c. 39 22. Annual Report 1968 23. The Age of Majority and Related Matters 24. Status of Adopted Children 25 . Report on Family Law: Part I — Torts 26. 27. 28. 29. Report on Section 20 of The Mortgages Act Report on Family Law: Part II — Marriage Annual Report 1969 Report on Actions Against Representatives of Deceased Persons April 7, 1969 June 3, 1969 June 3, 1969 November 4, 1969 March 12, 1970 April 6, 1970 April 20, 1970 November 30, 1970 , c. 39 The Age ofMajority and A ccountability A ct, S.O. 1971, c. 98 The Child Welfare Amendment Act, S.O. 1970, c. 96, s. 23 See now The Child Welfare Act, S.O. 1978, c. 85 The Family Law Reform Act, S.O. 1978, c. 2 (partial implementation) The Mortgages A mend- mentAct, S.O. 1970, c. 54, s. 1 The Civil Rights Statute Law Amendment Act, S.O. 1971, c. 50, s. 55 (partial implementation) See now The Marriage Act, S.O. 1977, c. 42 (partial implemen- tation) The Trustee Amend- ment Act, S.O. 1971, c. 32, s. 2 [21] The Age ofMajority and A ccountability A ct, S.O. 1971, c. 98 The Age ofMajority and A ccountability A ct, S.O. 1971, c. 98 The Child Welfare Amendment Act, S.O. 1970, c. 96, s. 23 See now The Child Welfare Act, S.O. 1978, c. 85 The Child Welfare Amendment Act, S.O. 1970, c. 96, s. 23 See now The Child Welfare Act, S.O. 1978, c. 85 25 . Report on Family Law: Part I — Torts 26. 27. Report on Section 20 of The Mortgages Act Report on Family Law: Part II — Marriage November 4, 1969 March 12, 1970 April 6, 1970 The Family Law Reform Act, S.O. 1978, c. 2 (partial implementation) The Mortgages A mend- mentAct, S.O. 1970, c. 54, s. 1 The Civil Rights Statute Law Amendment Act, S.O. 1971, c. 50, s. 55 (partial implementation) See now The Marriage Act, S.O. 1977, c. 42 (partial implemen- tation) See now The Marriage Act, S.O. 1977, c. Title 42 (partial implemen- tation) April 20, 1970 November 30, 1970 The Trustee Amend- ment Act, S.O. 1971, c. 32, s. 2 [21] 30. Title The Coroner System in Ontario 3 1 Sunday Observance Legislation 32. Land Registration 33. Annual Report 1970 34. The Change of Name Act 35. Section 16, The Mortgages Act 36. Development Control 37 Powers of Attorney 38. Occupiers' Liability 39. Consumer Warranties and Guarantees in the Sale of Goods 40. Review of Part IV of The Landlord and Tenant Act 41. Annual Report 1971 42. The Non-Possessory Repairman's Lien Date of Report Legislation Concerning Commission Proposals January 25, 1971 The Coroners A ct, S.O. 1972, c. 98 See The Coroners Amendment Act, S.O. 1978, c. 38 February 26, 1971 The Retail Business Holidays Act, S.O. 1975 (2nd Session), c. 9 March 23, 1971 See The Corporations Tax Amendment Act (No. 2), S.O. 1979, c. 89 March 31, 1971 — May 31, 1971 The Change ofName Amendment Act S.O. 1972, c. 44 See The Change of Name Amend- ment Act, S.O. 1978, c. 28 June 18, 1971 September 28, 1971 January 11, 1972 January 11, 1972 March 3 1,1972 March 31, 1972 March 3 1,1972 October 4, 1972 The Planning A mend- mentAct, S.O. 1973 c. 168, s. 10 See now The Planning Amendment Act, S.O. 1979, c. 59 The Powers ofA ttorney Act, S.O. 1979, c. 107 The Occupiers ' Liability Act, S.O. 1980, c. 14 The Landlord and Tenan t A mendmen t Act, S.O. 1972, c. 123 Title 1977, c.40 (partial implementation) The Family Law Reform Act, S.O. 1978, c. 2 (partial implementation) See The Land Titles Amendment Act, S.O. 1978, c. 7 The Registry Amendment Act, S.O. 1978, c. 8 51. Report on Family Law: Part V—Family Courts February 8, 1974 See The Unified Family Court Act, S.O. 1976, c. 85 The Unified Family Court Amendment Act, S.O. 1978, c. 68 The Children 's Probation Act, S.O. 1978, c. 41 (partial implementation) 52. Annual Report 1973 May 6, 1974 — 53. International Convention Providing a Uniform Law on the Form of the International Will July 3, 1974 The Succession Law Reform Act, S.O. 1977, c. 40, s. 42 54. Annual Report 1974 March 31, 1975 — 55. Report on Family Law: Part VI — Support Obligations April 18, 1975 The Succession Law Reform Act, S.O. 1977, c.40 (partial implementation) The Family Law Reform 2 Date of Report Title [22] Title Date of Report Legislation Concerning Commission Proposals 43. Administration of Ontario Courts, Part I February 26, 1973 See The Administra- tion of Courts Project Act, S.O. 1975, c. 31 The Judicature Amendment Act (No. 2), S.O. 1977, c. 51, s. 9 44. Annual Report 1972 March 3 1,1973 — 45. Administration of Ontario Courts, Part II May 23, 1973 See The Administra- tion of Courts Project Act, S.O. 1975, c. 31 46. Report on Family Law: Part III—Children September 25, 1973 The Child Welfare Amendment Act, S.O. 1975, c. 1 (partial implementation) See now The Child Welfare Act, S.O. 1978, c. 85 The Succession Law Reform Act, S.O. 1977, c. 40 (partial implementation) The Children 's Law Reform Act, S.O. 1977, c. 41 (partial implementation) 47 Report on The Solicitors Act 48. Report on Motor Vehicle Accident Compensation 49. Administration of Ontario Courts, Part III September 28, 1973 November 6, 1973 December 17, 1973 The JudicatureA mend- mentAct, S.O. 1975, c. 30 (partial implementation) Title Date of Report Legislation Concerning Commission Proposals 43. Administration of Ontario Courts, Part I February 26, 1973 See The Administra- tion of Courts Project Act, S.O. 1975, c. 31 The Judicature Amendment Act (No. 2), S.O. 1977, c. 51, s. 9 44. Annual Report 1972 March 3 1,1973 — 45. Administration of Ontario Courts, Part II May 23, 1973 See The Administra- tion of Courts Project Act, S.O. 1975, c. 31 46. Report on Family Law: Part III—Children September 25, 1973 The Child Welfare Amendment Act, S.O. 1975, c. 1 (partial implementation) See now The Child Welfare Act, S.O. 1978, c. 85 The Succession Law Reform Act, S.O. 1977, c. 40 (partial implementation) The Children 's Law Reform Act, S.O. 1977, c. 41 (partial implementation) 47 Report on The Solicitors Act 48. Report on Motor Vehicle Accident Compensation 49. Administration of Ontario Courts, Part III September 28, 1973 November 6, 1973 December 17, 1973 The JudicatureA mend- mentAct, S.O. 1975, c. 30 (partial implementation) See The Administra- tion of Courts Project Act S O 47 Report on The Solicitors Act 48. Report on Motor Vehicle Accident Compensation 49. Administration of Ontario Courts, Part III September 28, 1973 November 6, 1973 December 17, 1973 [23] Title Date of Report Legislation Concerning Commission Proposals 50. Report on Family Law: Part IV — Family Property Law February 8, 1974 The Succession Law Reform Act, S.O. Title (partial implementation) The Family Law Reform Act, S.O. 1978, c. 2 (partial implementation) See The Land Titles Amendment Act, S.O. 1978, c. 7 The Registry Amendment Act, S.O. 1978, c. 8 February 8, 1974 51. Report on Family Law: Part V—Family Courts 51. Report on Family Law: Part V—Family Courts 52. Annual Report 1973 May 6, 1974 53. International Convention Providing a Uniform Law on the Form of the International Will July 3, 1974 54. Annual Report 1974 March 31, 1975 55. Report on Family Law: Part VI — Support Obligations April 18, 1975 [24] 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. Title Mortmain, Charitable Uses and Religious Institutions Date of Report February 27, 1976 Landlord and Tenant Law The Law of Evidence Annual Report 1975 Report on Changes of Name Report on The Impact of Divorce on Existing Wills Annual Report 1976 Annual Report 1977 Report on Sale of Goods Annual Report 1978 Report on Products Liability Annual Report 1979 Report on the Enforcement of Judgment Debts and Related Matters, Part I Report on the Enforcement of Judgment Debts and Related Matters, Part II Report on the Enforcement of Judgment Debts and Related Matters, Part III March 15, 1976 March 29, 1976 March 31, 1976 August 16, 1976 February 28, 1977 March 31, 1977 March 31, 1978 March 30, 1979 March 30, 1979 November 16, 1979 March 31, 1980 February 20, 1981 March 31, 1981 March 31, 1981 Legislation Concerning Commission Proposals The Religious Organiza- tions' Lands Act, S.O. 1979, c. 45 The Anglican Church of Canada Act, S.O. 1979, c.46 The Registry Amendment Act, S.O. 1979, c. 94, s. 17 The Vital Statistics Amendment Act, S.O. 1978, c. 81, s. 1 (partial implementation) The Succession Law Reform Act, S.O. 1977, c. 40, s. 17(2) 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. February 8, 1974 Title Mortmain, Charitable Uses and Religious Institutions Date of Report February 27, 1976 Landlord and Tenant Law The Law of Evidence Annual Report 1975 Report on Changes of Name Report on The Impact of Divorce on Existing Wills Annual Report 1976 Annual Report 1977 Report on Sale of Goods Annual Report 1978 Report on Products Liability Annual Report 1979 Report on the Enforcement of Judgment Debts and Related Matters, Part I Report on the Enforcement of Judgment Debts and Related Matters, Part II Report on the Enforcement of Judgment Debts and Related Matters, Part III March 15, 1976 March 29, 1976 March 31, 1976 August 16, 1976 February 28, 1977 March 31, 1977 March 31, 1978 March 30, 1979 March 30, 1979 November 16, 1979 March 31, 1980 February 20, 1981 March 31, 1981 March 31, 1981 Legislation Concerning Commission Proposals The Religious Organiza- tions' Lands Act, S.O. 1979, c. 45 The Anglican Church of Canada Act, S.O. 1979, c.46 The Registry Amendment Act, S.O. 1979, c. 94, s. 17 The Vital Statistics Amendment Act, S.O. 1978, c. 81, s. 1 (partial implementation) The Succession Law Reform Act, S.O. 1977, c. 40, s. 17(2) 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. Landlord and Tenant Law The Law of Evidence Annual Report 1975 Report on Changes of Name Report on The Impact of Divorce on Existing Wills Annual Report 1976 Annual Report 1977 Report on Sale of Goods Annual Report 1978 Report on Products Liability Annual Report 1979 Report on the Enforcement of Judgment Debts and Related Matters, Part I Report on the Enforcement of Judgment Debts and Related Matters, Part II Report on the Enforcement of Judgment Debts and Related Matters, Part III March 15, 1976 March 29, 1976 March 31, 1976 August 16, 1976 February 28, 1977 March 31, 1977 March 31, 1978 March 30, 1979 March 30, 1979 November 16, 1979 March 31, 1980 February 20, 1981 March 31, 1981 March 31, 1981 1979, c. 94, s. 17 The Vital Statistics Amendment Act, S.O. 1978, c. 81, s. 1 (partial implementation) The Succession Law Reform Act, S.O. 1977, c. 40, s. 17(2) Many of the Commission's earlier Reports are no longer in print. Those that are still in print may be ordered from Publications Services, Ministry of Government Services, 5th Floor, 880 Bay Street, Toronto, Ontario, Canada, M7A1N8. [25] APPENDIX B OFFICERS AND PERMANENT STAFF ONTARIO LAW REFORM COMMISSION Chairman Vice Chairman Commissioners Derek Mendes da Costa, Q.C., LL.B., LL.M.,S.J.D.,LL.D. Counsel Secretary and Administrative Officer Legal Research Officers Administrative Assistant Secretary to Chairman Secretary to Vice Chairman Secretary to Counsel Secretary to Administrative Officer Secretaries to Legal Research Officers Receptionist Honourable George A. Gale, C.C., Q.C., LL.D. Honourable Richard A. Bell, P.C., Q.C. Honourable James C. McRuer, O.C., LL.D.,D.C.L. William R. Poole, Q.C. Barry A. Percival, Q.C. M. Patricia Richardson, B.A., M.A., LL.B. Miss A. F. Chute M. A. Springman, B.A., M.A., M.Sc, LL.B. Eric Gertner, LL.B., B.C.L. (Oxon) Ann M. Merritt, B.A., LL.B. L. M. Fox, LL.B. Pamela M. Gibson, B.A., LL.B. Mrs. J. A. Brown, B.A. Mrs. Stephanie Hlynka Mrs. E. N. Page Mrs. D. M. Halyburton Mrs. B. G. Woodley Mrs. ToniFarrace Ms. Victoria Van Asperen, B.Sc. Miss Mary M. O'Hara Chairman Derek Mendes da Costa, Q.C., LL.B., Derek Mendes da Costa, Q.C., LL.B., Derek Mendes da Costa, Q.C., LL.B., LL.M.,S.J.D.,LL.D. Honourable George A. Gale, C.C., Q.C., LL.D. Honourable Richard A. Bell, P.C., Q.C. Honourable James C. McRuer, O.C., LL.D.,D.C.L. William R. Poole, Q.C. Barry A. Percival, Q.C. M. Patricia Richardson, B.A., M.A., LL.B. LL.M.,S.J.D.,LL.D. LL.M.,S.J.D.,LL.D. Honourable George A. Gale, C.C., Q.C., LL.D. Honourable Richard A. Bell, P.C., Q.C. William R. Poole, Q.C. M. Patricia Richardson, B.A., M.A., LL.B. Miss A. F. Chute M. A. Springman, B.A., M.A., M.Sc, LL.B. Eric Gertner, LL.B., B.C.L. (Oxon) Ann M. Merritt, B.A., LL.B. L. M. Fox, LL.B. Pamela M. Gibson, B.A., LL.B. Mrs. J. A. Brown, B.A. Mrs. Stephanie Hlynka Mrs. E. N. Page Mrs. D. M. Halyburton Mrs. B. G. Woodley Mrs. ToniFarrace Ms. Victoria Van Asperen, B.Sc. Miss Mary M. O'Hara [27]
https://openalex.org/W4362517776	https://zenodo.org/record/7675763/files/14.pdf	Latin	null	Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	5,566	Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine Niekrasova L.A., Polishchuk A.S. DOI: 10.15276/EJ.04.2022.2 DOI: 10.5281/zenodo.7675763 UDC: 336.71:338.28 JEL: G24, M13, O33 INNOVATIVE TECHNOLOGIES OF THE DIGITAL ECONOMY AS A DIRECTION OF INVESTMENT IN THE DEVELOPMENT OF UKRAINE ІННОВАЦІЙНІ ТЕХНОЛОГІЇ ЦИФРОВОЇ ЕКОНОМІКИ ЯК НАПРЯМОК ІНВЕСТУВАННЯ У РОЗВИТОК УКРАЇНИ Liubov A. Niekrasova, DEcon, Professor Odesa Polytechnic National University, Odesa, Ukraine ORCID: 0000-0002-4305-7547 Email: l.a.nekrasova@op.edu.ua Liubov A. Niekrasova, DEcon, Professor Odesa Polytechnic National University, Odesa, Ukraine ORCID: 0000-0002-4305-7547 Email: l.a.nekrasova@op.edu.ua Anastasiia S. Polishchuk Odesa Polytechnic National University, Odesa, Ukraine ORCID: 0000-0002-4300-447X Email: polishchuk.8864664@stud.op.edu.ua krasova L.A., Polishchuk A.S. Innovative Technologies of the Digital Economy as a Direction of Investment in the elopment of Ukraine. Scientific and methodical article. Niekrasova L.A., Polishchuk A.S. Innovative Technologies of the Digital Economy as a Direction of Investment in Development of Ukraine. Scientific and methodical article. The article examines the issue of innovative financial technologies in the context of the digital economy. The leading foreign experience of investing in fintech companies was analyzed, and the dynamic development of financial technologies was proven based on the analysis of statistical data. The main features of fintech companies are revealed. Quantitative and qualitative methods (the method of discounted cash flows, the first Chicago method and the Berkus method) of company value estimation, which are better than others, are able to take into account the peculiarities of the fintech industry, are considered. It has been proven that the optimal valuation method for mature fintech companies is the discounted cash flow method and the first Chicago method. A qualitative assessment of the Ukrainian fintech startup was also carried out using the Berkus method. Keywords: digital economy innovations financial technologies Internet cost estimation methods investment Keywords: digital economy, innovations, financial technologies, Internet, cost estimation methods, investment he pervasion of the Internet into all spheres of life is a global trend today. The transformation of economic activity under the influence of the rapid development of online technologies led to the formation of the digital economy. Innovative technologies of the digital economy contribute to the opening of new opportunities – optimization of business processes, costs, open up new areas of attracting capital and promotion of business projects. One area in which the impact of technology has become particularly significant is finance. As a result of the integration of technology and finance, a new industry called fintech has emerged. Fintech helps to speed up and simplify money-related transactions, and also creates new opportunities. T T pp In the field of financial technologies, radical changes are taking place at the current stage, which are associated with increasing the level of automation, openness and consumer orientation. The importance of this innovative sector is very great, as the global implementation of financial technologies grows by 15-20% annually. In addition, in the conditions of the spread of the coronavirus infection, quarantine and forced isolation, the relevance of the use of digital technologies in the financial sphere began to increase throughout the world. Received 26.11.2022 Некрасова Л.А., Поліщук А.С. Інноваційні технології цифрової економіки як напрямок інвестування у розвиток України. Науково-методична стаття. У статті розглянуто питання інноваційних фінансових технологій у контексті цифрової економіки. Проаналізовано провідний зарубіжний досвід інвестування у фінтех-компанії, доведено на основі аналізу статистичних даних динамічний розвиток фінансових технологій. Виявлено основні особливості фінтех-компаній. Розглянуто кількісні та якісні методи (метод дисконтованих грошових потоків, перший Чиказький метод та метод Беркуса) оцінки вартості компанії, які краще за інших здатні врахувати особливості фінтех-галузі. Доведено, що для зрілих фінтех-компаній оптимальним методом оцінки є метод дисконтованих грошових потоків та перший Чиказький метод. Також було проведено якісну оцінку українського фінтех-стартапу методом Беркуса. у у у у у Ключові слова: цифрова економіка, інновації, фінансові технології, Інтернет, методи оцінки вартості, інвестиції The main part However, already in 2021, the region was revived and continues to grow rapidly [6]. In order to present a complete picture of the development of the fintech industry, it is worth looking at the quantitative indicators of the age of fintech companies in the world. With the help of the CrunchBase platform, which contains information about public and private organizations, a database of fintech companies with the date of their creation was prepared. In total, information was received on 7.6 thousand operating fintech companies of various industries. In figure 2 presents the distribution of fintech companies by year of foundation. According to the data analysis, the average age of fintech companies created in the period from 2012 to 2021 is 4 years, the median is 5 years. Young companies, whose age is up to 5 years, occupy 60% of the total market of fintech companies, while mature companies make up less than 15% of the total number [6]. Stable growth in the number of fintech companies is observed during the entire period under study, however, the global pandemic of COVID- 19 also affected the total number of companies in 2020 [7]. As can be seen from the chart, despite a temporary decrease in the number of investments in fintech companies in 2016 and 2017, the number of transactions continues to grow, and in 2018-2019 the volume of investments shows significant growth. 2020 sees a decline in investment and work due to the global crisis during the COVID-19 pandemic. However, already in 2021, the region was revived and continues to grow rapidly [6]. In order to present a complete picture of the development of the fintech industry, it is worth looking at the quantitative indicators of the age of fintech companies in the world. With the help of the CrunchBase platform, which contains information about public and private organizations, a database of fintech companies with the date of their creation was prepared. In total, information was received on 7.6 thousand operating fintech companies of various industries. In figure 2 presents the distribution of fintech companies by year of foundation. According to the data analysis, the average age of fintech companies created in the period from 2012 to 2021 is 4 years, the median is 5 years. Analysis of recent researches and publications The work (Lee, D.K.C. et al, 2015) [1] shows that fintech companies are divided into 2 fundamentally different groups: start-ups and developing ones. Depending on which group a company belongs to, there are reasonable methods and approaches to determine the future profitability of a company. Methods for quantitative analysis of fintech companies are justified in the articles (Rothman & Tiran, 2020) [2] and (Kotova, 2014) [3]. The rationale for the qualitative method in fintech companies is described in (Berkus, 2012) [4]. 14 ISSN:2521-6384 (Online), ISSN: 2521-103X (Print) Economic journal Odessa polytechnic university №4(22), 2022 The aim of the article. The purpose of this work is to analyze the leading foreign experience of investing in financial technologies of the digital economy, to identify the optimal method for evaluating fintech companies as an investment object, and to conduct a qualitative assessment of a Ukrainian fintech startup. The main part The term "Fintec" refers to the process of combining technology and finance, which leads to numerous innovations such as online banking, robo-advising, peer-to-peer lending, cryptocurrencies, and so on. The "financial technology market" refers to a rapidly growing market segment where existing and emerging technologies simplify and improve business processes, products and services in the financial sector [5]. The vast majority of fintech companies are located in the US or China due to the country's developed and open approach to innovation. In view of the openness of reporting information on the financial results of companies, the American company Square was chosen as an example of calculating the cost of a fintech company. A large number of unique fintech companies are created daily. Moreover, due to globalization and the Internet, which creates an ease of international communication, fintech companies are a global trend. According to statistics, approximately 30% of people in the world have access to traditional banking services. As of the end of 2018, 50% of the population has access to the Internet, and more than 56% of the population already use smartphones. The indicators continue to grow, therefore, the study of the fintech industry is extremely necessary, because fintech contributes to an increase in the volume of economic integration in the world and reduces inequality. Thus, this industry is currently attracting many investors. According to the KPMG FinTech Report, global investments in fintech companies in 2018 reached $148 billion across 2.196 transactions. The growth dynamics of investments in fintech companies from 2012 to 2021 can be seen in figure 1. Figure 1. Investments in fintech companies and the number of transactions Source: compiled by the authors on materials [6-7] 6,1 18,9 45,4 67,1 63,4 59,2 148,6 215,1 127,7 226,5 0 500 1000 1500 2000 2500 3000 0 50 100 150 200 250 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Figure 1. Investments in fintech companies and the number of transactions Source: compiled by the authors on materials [6-7] As can be seen from the chart, despite a temporary decrease in the number of investments in fintech companies in 2016 and 2017, the number of transactions continues to grow, and in 2018-2019 the volume of investments shows significant growth. 2020 sees a decline in investment and work due to the global crisis during the COVID-19 pandemic. The main part Young companies, whose age is up to 5 years, occupy 60% of the total market of fintech companies, while mature companies make up less than 15% of the total number [6]. Stable growth in the number of fintech companies is observed during the entire period under study, however, the global pandemic of COVID- 19 also affected the total number of companies in 2020 [7]. The main source of funding for fintech projects in the world is venture capital, its share in the total amount of funding is more than 70%. An analysis of the current state of the global market of financial technologies showed that the following sectors received the majority of investments: money transfers and payments - 20%, lending and banking technologies – 39%, financial planning – 10%, etc. Global trends in fintech include mobile financial services, financial and account management, money transfers, robot advisors, insurance technologies, crowdfunding, P2P lending, blockchain and cryptocurrencies. In Ukraine, the market of financial technologies is at the initial stage of formation. Today, more than 100 companies work in the field of financial technologies in Ukraine, most of which work in the field of payment/money transfer (38 companies), technology and 15 Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine Niekrasova L.A., Polishchuk A.S. infrastructure (36 companies), and mobile wallets (22 companies). According to the information provided in the Ukrainian Fintech catalog 2019, about 70% of fintech companies work without attracting money from third- party investors, of which 61% rely exclusively on their own funds, another 9% – attract money from friends and relatives, 30% use external investments, mainly private investors and angel investments [8]. Figure 2. Distribution of fintech companies by year of foundation Source: compiled by the authors on materials [6, 7] 88 611 879 946 963 1071 991 984 862 1093 0 200 400 600 800 1000 1200 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Number of fintech companies Figure 2. Distribution of fintech companies by year of foundation Source: compiled by the authors on materials [6, 7] The crisis situation in the innovative sphere of the Ukrainian economy is caused primarily by the lack of it innovative investment model of market strategy enterprises in the management of innovative activities. The main part Value of Square by DCF Period number 1 2 3 4 5 6 7-10 11 12 Stable growth Projected revenue growth rate 52% 56% 47% 43% 35% 32% 26.4% -10% 6% 4% 2% FCFF 742 (127) (50) 101 405 842 7866 3361 3299 91593 Reduced FCFF 662 (101) (36) 65 236 440 3069 1022 900 24984 Square's value in millions of dollars $ 31240 Source: compiled by the authors on materials [10] Table 2. Discount rate for Square Index Growth Slowdown in growth Stability WACC 12.0% 11.4% 7.1% Re 21.8% 20.6% 10.7% Beta 3.20 3.00 1.35 Rd 5.5% E/V 43.7% D/V 56.3% Corporate tax 21% Source: compiled by the authors on materials [10] Table 3. Value of Square by DCF Period number 1 2 3 4 5 6 7-10 11 12 Stable growth Projected revenue growth rate 52% 56% 47% 43% 35% 32% 26.4% -10% 6% 4% 2% FCFF 742 (127) (50) 101 405 842 7866 3361 3299 91593 Reduced FCFF 662 (101) (36) 65 236 440 3069 1022 900 24984 Square's value in millions of dollars $ 31240 Source: compiled by the authors on materials [10] Table 2. Discount rate for Square Index Growth Slowdown in growth Stability WACC 12.0% 11.4% 7.1% Re 21.8% 20.6% 10.7% Beta 3.20 3.00 1.35 Rd 5.5% E/V 43.7% D/V 56.3% Corporate tax 21% Source: compiled by the authors on materials [10] p Source: compiled by the authors on materials [10] Table 3. Value of Square by DCF Period number 1 2 3 4 5 6 7-10 11 12 Stable growth Projected revenue growth rate 52% 56% 47% 43% 35% 32% 26.4% -10% 6% 4% 2% FCFF 742 (127) (50) 101 405 842 7866 3361 3299 91593 Reduced FCFF 662 (101) (36) 65 236 440 3069 1022 900 24984 Square's value in millions of dollars $ 31240 Source: compiled by the authors on materials [10] Table 3. Value of Square by DCF The first Chicago Method. Since the company is relatively young and historically has not yet begun to turn a profit, but its revenue is growing rapidly, the probability of an optimistic scenario was estimated at 80%, realistic at 15%, and pessimistic at 5%. Table 4 contains scenario projections. p Calculation results in tables 5 and 6. Table 4. The main part Methodology for evaluating fintech companies 16 ISSN:2521-6384 (Online), ISSN: 2521-103X (Print) Economic journal Odessa polytechnic university This paper considers such approaches to valuation of developed fintech companies as cash flow discounting methods and the first Chicago method. But quantitative methods are not suitable for evaluating start-up fintech companies, because these companies have no financial reporting, that is why the Berkus qualitative method was chosen to evaluate a Ukrainian fintech start-up company Zeely. The analysis of Square. 1. Discount method. The company has a short history. According to the financial statements presented in Appendix 3, it can be seen that over the 10 years of its existence, the company has not managed to reach the level of positive net profit1. However, by the end of 2018, the value began to approach zero more and more. At the same time, the company's revenue growth rate is increasing annually and reaches 41% in 2018, which tells us that the company is in a stage of rapid growth. It is expected that the growth rate will increase and then begin to decline to the level of economic growth. Accordingly, to calculate the value of Square, build a forecast for 12 years, assuming that after 22 years of its existence (2030), the company will enter the stage of stable growth. In the first step, the expected growth rate will increase from 52% in 2019 to 56% in 2020, and then gradually decrease to 3.7% by 2030. To calculate the terminal value of the company, the growth rate of a stable fintech company engaged in electronic payments is used, equal to 2% [10]. The cash flow discount rates will differ across the three stages as the company's risk decreases over time. The results of calculating the company's value using the discounted cash flow method are presented in tables 2 and 3, in US dollars: Table 2. Discount rate for Square Index Growth Slowdown in growth Stability WACC 12.0% 11.4% 7.1% Re 21.8% 20.6% 10.7% Beta 3.20 3.00 1.35 Rd 5.5% E/V 43.7% D/V 56.3% Corporate tax 21% Source: compiled by the authors on materials [10] Table 3. The main part According to researchers, the main goal of innovation strategy in Ukraine is to prevent the disintegration of the scientific and innovative sphere and to create prerequisites for the rapid and effective introduction of technical and technological innovations in all spheres of the economy activities, ensuring structural and technological prerequisites at the enterprise level [9]. p q p Valuation of companies is a widely researched issue. Of the many ways to assess the value of companies, those that are best able to take into account the peculiarities of the fintech industry were chosen (table 1). When analyzing articles by various authors, special attention was paid to methods for evaluating innovative, young and fast-growing companies [2-4]. Table 1. Methodology for evaluating fintech companies Method name The essence of the methodology Formula Discounted cash flow method The components of the method are cash flows, which can be defined as a series of expected periodic cash flows from the business. The value of a company using this method is calculated by discounting the company's capital at a discount rate equal to the cost of raising equity, and then adding the market value of the debt to the resulting value. Cost of own capital= ( ) ( ) ( ) , First Chicago Method This method involves the calculation of a financial forecast for each of the three scenarios for the company's development: success (optimistic scenario); survival (realistic scenario), failure (pessimistic scenario). The value of the firm under each scenario is calculated using the discounted cash flow method. The last step is an expert assessment of the probabilities of occurrence of each of the scenarios and the summation of the weighted average cost of all scenarios according to the formula. alu α* 1 β* 2 γ* 3, α β γ , where: α, β, γ – the estimated probabilities of each of the scenarios, V1, V2, V3 – estimated discounted cash flows for the three scenarios. The Berkus method Dave Berkus proposed the concept of firm valuation, in which the creator must answer one question, will the company achieve $20 million in revenue after 5 years of operation. If the answer is yes, each of the following characteristics must be assessed in the range from $0 to $500.000. Charasteristics: idea, prototype, qualified management team, strategic communications, product promotion Source: compiled by the authors on materials [2-4] Table 1. The main part Calculating the value of Square by the first Chicago method Projected growth rate Optimistic Realistic Pessimistic 1 2 3 4 2019 52% 42% 25% 2020 56% 47% 30% 2021 47% 35% 18% 2022 43% 30% 16% 2023 35% 24% 12% 2024 32% 21% 10% 2025 26% 17% 7% 2026 21% 13% 5% 2027 16% 14% 4% 2028 10% 8% 3% 2029 6% 4% 2% 2030 4% 3% 2% Post-forecast period 2% 2% 1% Table 4. Calculating the value of Square by the first Chicago method 17 Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine Niekrasova L.A., Polishchuk A.S. Continuation of Table 4 1 2 3 4 Discounted Cash Flows FCFF 2019 662 684 718 FCFF 2020 (101) (73) (31) FCFF 2021 (36) (1) 35 FCFF 2022 65 87 83 FCFF 2023 236 201 138 FCFF 2024 440 316 186 FCFF 2025 703 441 227 FCFF 2026 662 387 187 FCFF 2027 789 418 187 FCFF 2028 915 472 189 FCFF 2029 1 022 517 195 FCFF 2030 900 446 165 FCFF in the post-forecast period 24984 12228 3680 Company value 31240 16121 5959 Scenario probabilities 80% 15% 5% Total value of Square $27 708 Source: compiled by the authors on materials [10] Table 5. Results of Square's valuation using the first Chicago method Scenarios Optimistic Realistic Pessimistic Company value 31240 16121 5959 Scenario probabilities 80% 15% 5% The total cost $27708 Source: compiled by the authors on materials [10] Table 5. Results of Square's valuation using the first Chicago method Table 6. Summary of obtained results in billion U.S. dollars Method Result DCF 31.2 The first Chicago method 27.7 Net value 31.94 Source: compiled by the authors on materials [10] Thus, the most accurate assessment of the company was obtained using the discounted cash flow method, because when using the first Chicago method, there is a subjective component in the assessment of experts, which can affect the results. Qualitative evaluation of a fintech company using the Berkus method. On the example of the Ukrainian fintech company Zeely, the application of the Berkus method will be considered. This is a growth marketing app that increases business income for entrepreneurs. The information needed for the evaluation was obtained from the startup website [11]. The main part As part of the Berkus method, it is assumed that after 5 years of operation, Zeely will be able to achieve a revenue of $20 million. Next, the quality characteristics of the company were assessed. The first characteristic evaluated is the idea. Similar platforms already exist, however, they often require a higher fee, unlike the startup in question. The next characteristic is the prototype, indicating the presence of unique, well- developed technologies. It is the process of automating the process of creating a sales-oriented website, sales funnels, automatic launch of Facebook and Google online advertising, integration with a CRM system and analytics in just a few clicks that is unique. The third characteristic involves the assessment of the company's management team. The CEO & Founder has experience in startups and spent 3 years at Google building and improving company websites. However, the rest of the team of co-founders of the project do not have experience in working with similar projects because they are students at Ukrainian universities. Regarding strategic connections, the company's CEO & Founder is a founder/employee of other fintech companies, which greatly reduces market risk due to the company's management's awareness of the industry, as well as the assumed presence of the necessary connections. The last characteristic evaluates how well the company promotes its services and whether the company has started its activities. To date, Zeely is just starting to gain a customer base with the help of the possibility of registering on the site. Internet sources were analyzed for company information and advertising, and only social networks and a website were found. Accordingly, this characteristic receives a low rating. The evaluation results are presented in the table 7 in US dollars: 18 Economic journal Odessa polytechnic university ISSN:2521-6384 (Online), ISSN: 2521-103X (Print) №4(22), 2022 Table 7. Berkus Valuation of Zeely Zeely qualitative evaluation Value 1. Idea $400000 2. Prototype $400000 3. Qualified management team $100000 4. Strategic connections $150000 5. Promoting a product or launching an activity $100000 Zeely company value $1150000 Source: authors' own development Table 7. Berkus Valuation of Zeely The total cost of the company according to the Berkus method is estimated at 1.150 million US dollars. The method includes some characteristics of fintech companies and is able to evaluate them, however, because of the 2.5 million limit, however, it may not reflect the real value of fintech startups. Abstract Innovative technologies of the digital economy contribute to the opening of new opportunities - optimization of business processes, costs, open up new areas of attracting capital and promotion of business projects. As a result of the integration of technology and finance, a new industry called fintech has emerged. The importance of this innovative sector is very great, as the global implementation of financial technologies grows by 15-20% annually. The purpose of this work is to analyze the leading foreign experience of investing in financial technologies of the digital economy, to identify the optimal method for evaluating fintech companies as an investment object, and to conduct a qualitative assessment of a Ukrainian fintech startup. Within this framework, the concept of a fintech company was revealed, trends in the development of the industry were analyzed, forecasts were made for the development of the industry as a whole. The vast majority of fintech companies are located in the US or China due to the country's developed and open approach to innovation. Fintech contributes to an increase in the volume of economic integration in the world and reduces inequality. Thus, this industry is currently attracting many investors. In Ukraine, the market of financial technologies is at the initial stage of formation. In the empirical part of the work, a quantitative analysis was carried out using the discounted cash flow method and the first Chicago method for developed fintech companies. Thus, the most accurate assessment of the company was obtained using the discounted cash flow method, because when using the first Chicago method, there is a subjective component in the assessment of experts, which can affect the results. The Berkus method was used to evaluate Ukrainian start-up fintech companies without financial results. Based on the calculations made and comparisons with the market value, the relevance of these research methods was confirmed. Since the popularity of fintech companies is growing at a noticeable pace, and universal methods for valuing such companies still do not exist, this may be a prerequisite for further research on the issue. The main part Within this framework, the concept of a fintech company was revealed, trends in the development of the industry were analyzed, forecasts were made for the development of the industry as a whole. In the empirical part of the work, a quantitative analysis was carried out using the discounted cash flow method and the first Chicago method for developed fintech companies, while the Berkus method was used to evaluate start-up fintech companies without financial results. Based on the calculations made and comparisons with the market value, the relevance of these research methods was confirmed. Since the popularity of fintech companies is growing at a noticeable pace, and universal methods for valuing such companies still do not exist, this may be a prerequisite for further research on the issue. It is likely that over the years, when more mature fintech companies appear on the market, it will be possible to build more accurate valuation models that, in addition to assumptions and theory, will be based on average historical data. 1. Lee, D.K.C., & TEO, G.S.Z.J. Emergence of FinTech and the LASIC Principles. Journal of Financial P sp c iv s. 0 5. ( ), . р. -24. . Rothman, Tiran. Valuation Methods: The First Chicago Venture Method and the Use of Real Option Valuations of Early-Stage Companies and Disruptive Technologies. 2020. p. 10-12. 3. Kotova M.V. The theoretical and methodological basis of startups valuation. Economy: realities of time. 2014. № 1 (11). p. 107-112. 1. Lee, D.K.C., & TEO, G.S.Z.J. Emergence of FinTech and the LASIC Principles. Journal of Financial P sp c iv s. 0 5. ( ), . р. -24. 2. Rothman, Tiran. Valuation Methods: The First Chicago Venture Method and the Use of Real Options. Valuations of Early-Stage Companies and Disruptive Technologies. 2020. p. 10-12. 3. Kotova M.V. The theoretical and methodological basis of startups valuation. Economy: realities of time. 2014. № 1 (11). p. 107-112. 1. Lee, D.K.C., & TEO, G.S.Z.J. Emergence of FinTech and the LASIC Principles. Journal of Financial P sp c iv s. 0 5. ( ), . р. -24. 2. Rothman, Tiran. Valuation Methods: The First Chicago Venture Method and the Use of Real Options. Valuations of Early-Stage Companies and Disruptive Technologies. 2020. p. 10-12. 3. Kotova M.V. The theoretical and methodological basis of startups valuation. Economy: realities of time 2014 №1 (11) p 107 112 Reference a Journal Article: Niekrasova L.A. Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine / L.A. Niekrasova, A.S. Polishchuk // Economic journal Odessa polytechnic university. – 2022. – № 4 (22). – P. 14-20. – Retrieved from https://economics.net.ua/ejopu/2022/No4/14.pdf. DOI: 10.15276/EJ.04.2022.2. DOI: 10.5281/zenodo.7675763. 4. Berkus D. The Berkus Method: Valuing an Early Stage Investment. [Електронний ресурс] – Режим доступу: http://berkonomics.com/?p=1214. Список літератури: 19 Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine Niekrasova L.A., Polishchuk A.S. 4. Berkus D. The Berkus Method: Valuing an Early Stage Investment. [Електронний ресурс] – Режим доступу: http://berkonomics.com/?p=1214. 5. Buckley, Ross & Arner, Douglas & Barberis, Janos. The Evolution of Fintech: A New Post-Crisis Paradigm? Georgetown Journal of International Law. 0 6. 47. р. 7 -1319. . Офіційний сайт u chBas . [Електронний ресурс] – Режим доступу: https://www.crunchbase.com Офіційний сайт Yahoo i a c [Електронний ресурс] Режим доступу: https://finance yahoo com Офіційний сайт u chBas . [Електронний ресурс] – Режим доступу: https://www.crunchbase.com. Офіційний сайт Yahoo i a c . 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The process of forming the innovative investment strategy of the enterprise and its problems. Scientific Bulletin of Kherson State University. Series "Economic Sciences", 5/2, 204-207 [in Ukraine]. 10. Official site of the Square. Retrieved from: https://squareup.com [in English]. 10. Official site of the Square. Retrieved from: https://squareup.com [in English of the Zeely. Retrieved from: https://zeely.app [in En This is an open access journal and all published articles are licensed under a Creative Commons "Attribution" 4.0. This is an open access journal and all published articles are licensed under a Creative Commons "Attribution" 4.0. Niekrasova L.A. Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine / L.A. Niekrasova, A.S. Polishchuk // Економічний журнал Одеського політехнічного університету. – 2022. – № 4(22). – С. 14-20. – Режим доступу до журн.: https://economics.net.ua/ejopu/2022/No4/14.pdf. DOI: 10.15276/EJ.04.2022.2. DOI: 10.5281/zenodo.7675763. 8. Шевченко О.М., Рудич Л.В. Розвиток фінансових технологій в умовах цифровізації економіки України. Ефективна економіка. 0 0. № 7. [Електронний ресурс] – Режим доступу: http://www. economy.nayka.com.ua/?op=1&z=8053. Посилання на статтю: Посилання на статтю: Niekrasova L.A. Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine / L.A. Niekrasova, A.S. Polishchuk // Економічний журнал Одеського політехнічного університету. – 2022. – № 4(22). – С. 14-20. – Режим доступу до журн.: https://economics.net.ua/ejopu/2022/No4/14.pdf. DOI: 10.15276/EJ.04.2022.2. DOI: 10.5281/zenodo.7675763. Niekrasova L.A. Innovative Technologies of the Digital Economy as a Direction of Investment in the Development of Ukraine / L.A. Niekrasova, A.S. Polishchuk // Економічний журнал Одеського політехнічного університету. – 2022. – № 4(22). – С. 14-20. – Режим доступу до журн.: https://economics.net.ua/ejopu/2022/No4/14.pdf. DOI: 10.15276/EJ.04.2022.2. DOI: 10.5281/zenodo.7675763. Reference a Journal Article: 20
https://openalex.org/W2889326210	https://journalbipolardisorders.springeropen.com/track/pdf/10.1186/s40345-018-0127-7	English	null	Internet use by older adults with bipolar disorder: international survey results	International journal of bipolar disorders	2,018	cc-by	6,715	Abstract Background: The world population is aging and the number of older adults with bipolar disorder is increasing. Digi‑ tal technologies are viewed as a framework to improve care of older adults with bipolar disorder. This analysis quanti‑ fies Internet use by older adults with bipolar disorder as part of a larger survey project about information seeking. Background: The world population is aging and the number of older adults with bipolar disorder is increasing. Digi‑ tal technologies are viewed as a framework to improve care of older adults with bipolar disorder. This analysis quanti‑ fies Internet use by older adults with bipolar disorder as part of a larger survey project about information seeking. Methods: A paper-based survey about information seeking by patients with bipolar disorder was developed and translated into 12 languages. The survey was anonymous and completed between March 2014 and January 2016 by 1222 patients in 17 countries. All patients were diagnosed by a psychiatrist. General estimating equations were used to account for correlated data. Results: Overall, 47% of older adults (age 60 years or older) used the Internet versus 87% of younger adults (less than 60 years). More education and having symptoms that interfered with regular activities increased the odds of using the Internet, while being age 60 years or older decreased the odds. Data from 187 older adults and 1021 younger adults were included in the analysis excluding missing values. Conclusions: Older adults with bipolar disorder use the Internet much less frequently than younger adults. Many older adults do not use the Internet, and technology tools are suitable for some but not all older adults. As more health services are only available online, and more digital tools are developed, there is concern about growing health disparities based on age. Mental health experts should participate in determining the appropriate role for digital tools for older adults with bipolar disorder. © The Author(s) 2018. 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. Correspondence: michael.bauer@uniklinikum‑dresden.de 1 Department of Psychiatry and Psychotherapy, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany Mark Zetin: Deceased in 2017 Full list of author information is available at the end of the article Internet use by older adults with bipolar disorder: international survey results Rita Bauer1, Tasha Glenn2, Sergio Strejilevich3, Jörn Conell1,4, Martin Alda5, Raffaella Ardau6, Bernhard T. Baune7, Michael Berk8,9,10,11,12, Yuly Bersudsky13, Amy Bilderbeck14, Alberto Bocchetta15, Angela M. Paredes Castro8,9, Eric Y. W. Cheung16, Caterina Chillotti6, Sabine Choppin17, Alessandro Cuomo18, Maria Del Zompo15, Rodrigo Dias19, Seetal Dodd8,9,10, Anne Duffy20, Bruno Etain21, Andrea Fagiolini18, Miryam Fernández Hernandez22, Julie Garnham5, John Geddes14, Jonas Gildebro23, Michael J. Gitlin24, Ana Gonzalez‑Pinto22, Guy M. Goodwin14, Paul Grof25,26, Hirohiko Harima27, Stefanie Hassel28, Chantal Henry21,29, Diego Hidalgo‑Mazzei30, Anne Hvenegaard Lund23, Vaisnvy Kapur31, Girish Kunigiri32, Beny Lafer19, Erik R. Larsen33,34, Ute Lewitzka1, Rasmus W. Licht35,36, Blazej Misiak37, Patryk Piotrowski37, Ângela Miranda‑Scippa38, Scott Monteith39, Rodrigo Munoz40, Takako Nakanotani41, René E. Nielsen35, Claire O’Donovan5, Yasushi Okamura27, Yamima Osher13, Andreas Reif42, Philipp Ritter1, Janusz K. Rybakowski43, Kemal Sagduyu44, Brett Sawchuk20, Elon Schwartz45, Claire Slaney5, Ahmad H. Sulaiman46, Kirsi Suominen47, Aleksandra Suwalska43, Peter Tam48, Yoshitaka Tatebayashi41, Leonardo Tondo49,50, Julia Veeh42, Eduard Vieta30, Maj Vinberg51, Biju Viswanath52, Mark Zetin53, Peter C. Whybrow24 and Michael Bauer1 Abstract Bauer et al. Int J Bipolar Disord (2018) 6:20 https://doi.org/10.1186/s40345-018-0127-7 RESEARCH Internet use by older adults with bipolar disorder: international survey results Rita Bauer1, Tasha Glenn2, Sergio Strejilevich3, Jörn Conell1,4, Martin Alda5, Raffaella Ardau6, Bernhard T. Baune7, Michael Berk8,9,10,11,12, Yuly Bersudsky13, Amy Bilderbeck14, Alberto Bocchetta15, Angela M. Paredes Castro8,9, Eric Y. W. Cheung16, Caterina Chillotti6, Sabine Choppin17, Alessandro Cuomo18, Maria Del Zompo15, Rodrigo Dias19, Seetal Dodd8,9,10, Anne Duffy20, Bruno Etain21, Andrea Fagiolini18, Miryam Fernández Hernandez22, Julie Garnham5, John Geddes14, Jonas Gildebro23, Michael J. Gitlin24, Ana Gonzalez‑Pinto22, Guy M. Goodwin14, Paul Grof25,26, Hirohiko Harima27, Stefanie Hassel28, Chantal Henry21,29, Diego Hidalgo‑Mazzei30, Anne Hvenegaard Lund23, Vaisnvy Kapur31, Girish Kunigiri32, Beny Lafer19, Erik R. Larsen33,34, Ute Lewitzka1, Rasmus W. Licht35,36, Blazej Misiak37, Patryk Piotrowski37, Ângela Miranda‑Scippa38, Scott Monteith39, Rodrigo Munoz40, Takako Nakanotani41, René E. Nielsen35, Claire O’Donovan5, Yasushi Okamura27, Yamima Osher13, Andreas Reif42, Philipp Ritter1, Janusz K. Rybakowski43, Kemal Sagduyu44, Brett Sawchuk20, Elon Schwartz45, Claire Slaney5, Ahmad H. Sulaiman46, Kirsi Suominen47, Aleksandra Suwalska43, Peter Tam48, Yoshitaka Tatebayashi41, Leonardo Tondo49,50, Julia Veeh42, Eduard Vieta30, Maj Vinberg51, Biju Viswanath52, Mark Zetin53, Peter C. Whybrow24 and Michael Bauer1* Open Access Bauer et al. Int J Bipolar Disord (2018) 6:20 https://doi.org/10.1186/s40345-018-0127-7 RESEARCH Internet use by older adults with bipolar disorder: international survey results Rita Bauer1, Tasha Glenn2, Sergio Strejilevich3, Jörn Conell1,4, Martin Alda5, Raffaella Ardau6, Bernhard T. Baune7, Michael Berk8,9,10,11,12, Yuly Bersudsky13, Amy Bilderbeck14, Alberto Bocchetta15, Angela M. Paredes Castro8,9, Eric Y. W. Cheung16, Caterina Chillotti6, Sabine Choppin17, Alessandro Cuomo18, Maria Del Zompo15, Rodrigo Dias19, Seetal Dodd8,9,10, Anne Duffy20, Bruno Etain21, Andrea Fagiolini18, Miryam Fernández Hernandez22, Julie Garnham5, John Geddes14, Jonas Gildebro23, Michael J. Gitlin24, Ana Gonzalez‑Pinto22, Guy M. Goodwin14, Paul Grof25,26, Hirohiko Harima27, Stefanie Hassel28, Chantal Henry21,29, Diego Hidalgo‑Mazzei30, Anne Hvenegaard Lund23, Vaisnvy Kapur31, Girish Kunigiri32, Beny Lafer19, Erik R. Larsen33,34, Ute Lewitzka1, Rasmus W. Licht35,36, Blazej Misiak37, Patryk Piotrowski37, Ângela Miranda‑Scippa38, Scott Monteith39, Rodrigo Munoz40, Takako Nakanotani41, René E. Nielsen35, Claire O’Donovan5, Yasushi Okamura27, Yamima Osher13, Andreas Reif42, Philipp Ritter1, Janusz K. Rybakowski43, Kemal Sagduyu44, Brett Sawchuk20, Elon Schwartz45, Claire Slaney5, Ahmad H. Sulaiman46, Kirsi Suominen47, Aleksandra Suwalska43, Peter Tam48, Yoshitaka Tatebayashi41, Leonardo Tondo49,50, Julia Veeh42, Eduard Vieta30, Maj Vinberg51, Biju Viswanath52, Mark Zetin53, Peter C. Whybrow24 and Michael Bauer1* Open Access Bauer et al. Int J Bipolar Disord (2018) 6:20 https://doi.org/10.1186/s40345-018-0127-7 Open Access Backgroundh was approved by institutional review boards according to local requirements. The patients who completed the survey resided in 17 countries. The survey was translated into 12 local languages: Chinese, Danish, Finnish, French, German, Hebrew, Italian, Japanese, Polish, Portuguese, Spanish, and English (versions for US/Canada, UK and Australia). The 1222 surveys were received from patients in Australia (N = 22), Brazil (N = 100), Canada (N = 109), Denmark (N = 209), Finland (N = 16), France (N = 50), Germany (N = 82), Hong Kong (N = 91), India (N = 30), Israel (N = 46), Italy (N = 80), Japan (N = 35), Malaysia (N = 25), Poland (N = 125), Spain (N = 82), UK (N = 50), and the US (N = 70).h The world’s population is living longer, with the percent- age of people over age 60 years expected to nearly dou- ble from 12 to 22% between 2015 and 2050 (WHO 2015). Today, up to 25% of the population with bipolar disorder is age 60 years or older (Sajatovic et al. 2015). Older adults with bipolar disorder differ in the disease onset and clini- cal course, and most have multiple medical comorbidities especially endocrine, respiratory and cardiovascular con- ditions (Lala and Sajatovic 2012). Digital technology pro- vides a framework to improve care for older adults with bipolar disorder by enabling remote visits, online psy- chological interventions, health monitoring, information seeking, peer support groups and self-management tools (Gliddon et al. 2017; Hidalgo-Mazzei et al. 2015; Torous et al. 2016). The survey questions and methodology were published previously (Bauer et al. 2016; Bauer R et al. 2017; Conell et al. 2016). Since paper-based surveys were used, dupli- cate data entry was performed to minimize data entry errors. A model to evaluate the differences in Internet use by those age 60 years and older was estimated using the generalized estimating equation (GEE) statistical technique to accommodate imbalances in the number of responses from collection sites, and correlation in survey responses within collection sites. Variables significant at the 0.05 level in univariate analyses were included in the multivariate model estimates. SPSS version 24.0 was used for all analyses. In addition to providing help with bipolar disor- der, Internet use by older adults in the community may decrease loneliness, and increase social support (Fors- man and Nordmyr 2017; Heo et al. 2015). Bauer et al. Int J Bipolar Disord (2018) 6:20 Page 2 of 7 Page 2 of 7 Results 1222 patients completed the survey. The patients were 62% female, had a mean age of 44 years (SD 13.8) rang- ing between 17 and 86 years, and completed 14 (SD 3.2) years of education. The demographic characteristics are shown in Table 1. Of the 1222 patients, 81% used the Internet (976 of 1212 valid responses) (Bauer et al. 2016). There were 1208 valid responses to both the ques- tions on age and “Do you use the Internet?” Of the 1208 patients, 187 were 60 years or older and of these 88 (47%) used the Internet. Of the 1021 younger adults, 884 (87%) used the Internet. Table 2 shows the best fitting model to assess differences in Inter- net use between the older adults and younger adults. The model includes variables for age 60 years or older, years of education, and if bipolar disorder sometimes or frequently interfered with regular activities. The estimated coefficients suggest that if age was 60 years or older, the odds of using the Internet will decrease by 86%, a 1 year increase in education will increase the odds of using the Internet by 30%, and if bipolar disor- der interferes with regular activities, the odds of using the Internet will increase by 76%. 1222 patients completed the survey. The patients were 62% female, had a mean age of 44 years (SD 13.8) rang- ing between 17 and 86 years, and completed 14 (SD 3.2) years of education. The demographic characteristics are shown in Table 1. Of the 1222 patients, 81% used the Internet (976 of 1212 valid responses) (Bauer et al. 2016). To gain insight into online information seeking by patients with bipolar disorder, we previously surveyed 1222 adult outpatients with bipolar disorder living in 17 countries between March 2014 and January 2016 (Bauer et al. 2016; Conell et al. 2016). Of the patients in the sur- vey, 81% used the Internet, a percentage similar to that of the general public (Bauer et al. 2016). The purpose of this analysis was to compare Internet use between the older adults, defined as 60 years or older, and younger adults less than 60 years, who completed this survey. Backgroundh Internet use may also contribute to maintaining health literacy, or the ability to read, understand and act on health informa- tion (Kobayashi et al. 2015; Andrus and Roth 2002). Gov- ernment and health care providers increasingly use the Internet as the primary form of communication about health and social services (Chang et al. 2015). Digital technologies are viewed as a means to maximize inde- pendence and facilitate aging in place, including for those with disabilities (Agree 2014; Reeder et al. 2013; Schulz et al. 2015), and to provide cost-effective care for the growing elderly population (Deloitte 2015). Most studies of Internet use involve community dwelling older adults and do not focus on mental illness. As the role of online services and monitoring technologies increases, more understanding of Internet use by older adults with bipo- lar disorder is needed. Methodsh The 39-question survey was anonymous, and took about 20 min to complete. The survey was paper based to maxi- mize participation including of those who do not use the Internet. All participants were recruited locally by their psychiatrist with no online recruitment. The study Of those who used the Internet, 689/880 (78%) of younger adults and 59/88 (67%) of older adults looked for information on bipolar disorder. While this appears Bauer et al. Int J Bipolar Disord (2018) 6:20 Page 3 of 7 Table 1 Patient demographics (N = 1222)a Table 1 Patient demographics (N = 1222)a Table 1 Patient demographics (N = 1222)a a 14 patients were missing responses to questions on age or “Do you use the Internet”? Methodsh All missing values were excluded Age 60 or older (N = 187) Age 59 or younger (N = 1021) All ages (N = 1208) N % N % N % Diagnosis BP I 107 58 657 65 764 63 BP II 70 38 308 30 378 32 BP NOS 8 4 48 5 56 5 Gender Female 120 64 637 62 757 62 Male 68 36 390 38 458 38 Employment status Full-time 31 17 529 52 560 47 Not full-time 156 83 482 48 638 53 Marital status Married or living with partner 112 60 478 47 590 49 Not married 76 40 543 53 619 51 Income group Upper income 14 8 66 7 80 7 Middle income 105 57 487 48 592 49 Lower income 65 35 468 45 533 44 Live alone Yes 54 29 245 24 299 25 No 131 71 777 76 908 75 Mood in last 6 months Mostly normal 118 63 460 45 578 48 Mostly not normal 69 37 561 55 630 52 BP disorder interfered with regular activities Frequently or sometimes 89 47 676 66 765 63 Rarely or never 99 53 347 34 446 37 Confident managing living Very confident 89 48 363 36 452 38 Not very confident 98 52 659 64 754 62 Confident knowing when to see physician Very confident 117 62 578 57 695 57 Not very confident 71 38 445 43 516 43 N Mean (SD) N Mean (SD) N Mean (SD) Years of education 184 13 (3.6) 1010 14 (3.1) 1194 14 (3.2) Age of onset 186 36 (13.6) 1011 25 (9.4) 1197 27 (10.9) research on older adults not specific to bipolar disor- der. Internet use by community dwelling older adults is increasing, with studies reporting percentages between 36 and 67%, but remains considerably lower than for younger adults (Levine et al. 2016; Friemel 2016; Yu et al. 2016; Anderson and Perrin 2017; Chang et al. 2015). As in prior research, more education and expe- riencing symptoms were associated with increased similar, insufficient data were available on the older adults for a more detailed statistical analysis. Discussion Older adults with bipolar disorder used the Internet much less frequently than younger adults. Overall, 47% of older adults used the Internet versus 87% of younger adults. This finding is consistent with prior Bauer et al. Int J Bipolar Disord (2018) 6:20 Page 4 of 7 Table 2 Explanatory model based on responses from the patients who use the Internet (N = 1208) a Patients with missing values were not included b 187 patients were age 60 years or older at time of study Independent variablesa Parameter Significance OR 95% CI Intercept < 0.001 0.136 0.053, 0.349 Age 60 years or olderb < 0.001 0.141 0.082, 0.241 Bipolar disorder sometimes or frequently interferes with regular activities 0.001 1.764 1.246, 2.497 Years of education < 0.001 1.302 1.229, 1.380 Table 2 Explanatory model based on responses from the patients who use the Internet (N = 1208) psychiatrist (Hallett et al. 2013). Some older adults do not trust the Internet as a source of health information (Sbaffi and Rowley 2017; Zulman et al. 2011). In studies of patients with a mean age ≥ 50 years, those with a strong therapeutic relationship with a physician were less likely to search for health information on the Internet (Hou and Shim 2010), and more likely to defer decision mak- ing to the physician (Park et al. 2014). In this survey, the primary reason why Internet users of all ages did not seek information about bipolar disorder was because they pre- fer to rely on information from a physician (Bauer et al. 2016). Regardless of age, most patients who did not use the Internet in this survey lacked technical skills (Bauer et al. 2016). One option to increase technology use by older adults is to provide training, but there are many serious concerns with novice Internet users. The elderly are such frequent targets for financial fraud that it is considered a public health problem in the US (Burnes et al. 2017; CDC 2015), and the scams targeting older adults have moved online (FBI 2014; Carlson 2007). In 2016, in the US, adults over age 60 were the largest group of victims of Internet crime, and suffered the largest monetary losses (FBI 2016). Factors that increase vulnerability to online fraud include low technical skills, individual traits, cognitive impairment, and depression in older adults (Monteith and Glenn 2016; Lichtenberg et al. 2016). Discussion Many older adults are not knowledgeable about Inter- net security hazards and measures to protect privacy (Grimes et al. 2010; Home Instead 2017; Holtfreter et al. 2015; White et al. 2017). Furthermore, many people of all ages have little understanding of privacy issues related to digital technology. For example, in this survey, 43% of patients of all ages searched the Internet for information about bipolar disorder because they mistakenly thought they were anonymous online (Conell et al. 2016). Internet use (Yu et al. 2016; Gell et al. 2015; Powell and Clarke 2006; Gallagher and Doherty 2009; Flynn et al. 2006), although symptoms of depression, and cogni- tive decline may decrease use in older adults (Choi and Dinitto 2013; Levine et al. 2018). Smartphone use by community dwelling older adults is even lower than Internet use, at about 40% (Anderson and Perrin 2017). In this survey, considering Internet users of all ages, 89% accessed the Internet to find information about bipolar disorder from a computer compared with 11% from a smartphone or tablet (Conell et al. 2016). Older adults are diverse, differing in age, education, income, living situation, employment, and experience with technology. Notably, in a US survey of 567 adults age 60 or older, those who used the Internet were often com- fortable doing so, and may have a job requiring computer use (Chang et al. 2015). In an international survey, infor- mation technology professionals over age 50 experienced less trouble working with multiple devices than younger workers (Patrizio 2016). However, as in this study, many older adults with or without bipolar disorder do not use the Internet. The reasons are complex and include dif- ficulty learning technical skills, high costs of comput- ers, mobile devices and broadband services, attitudes towards technology, increasing age, language issues for immigrants, cognitive decline, preference for traditional media, low health literacy, and relocation to a nursing home (Kuerbis et al. 2017; Fischer et al. 2014; Levine et al. 2018; Levy et al. 2015; Nimrod 2017; Chang et al. 2015). Some older adults are concerned that technology use will reduce face-to-face interactions, including contact with health care providers, and increase isolation (Kang et al. 2010; Kuerbis et al. 2017). In addition to financial fraud, older adults may fall vic- tim to risky online medical activities. Author details There are some limitations to this report. The survey was not designed to study technology habits of older adults. The study participants do not reflect the demo- graphic composition of the countries. People with bipo- lar disorder who did not seek professional help did not participate. People who did not understand the local language may not have participated. All data were self- reported and there was no follow-up discussion of responses. Many issues related to digital technology use by older adults were not discussed. These include the complex ethical challenges, quality of web sites and valid- ity of digital tools for bipolar disorder (Bauer M et al. 2017), physiological effects of blue light exposure from digital devices (Bauer et al. 2018), and the potential for digital assistive tools to erode skills, decrease motivation, and promote a false sense of security (Schulz et al. 2015). 1 Department of Psychiatry and Psychotherapy, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. 2 ChronoRecord Association, Fullerton, CA, USA. 3 Bipolar Disorder Program, Neuroscience Insti‑ tute, Favaloro University, Buenos Aires, Argentina. 4 AMEOS Klinika Holstein, Neustadt, Germany. 5 Department of Psychiatry, Dalhousie University, Halifax, NS, Canada. 6 Unit of Clinical Pharmacology, University Hospital of Cagli‑ ari, Cagliari, Italy. 7 Discipline of Psychiatry, School of Medicine, University of Adelaide, Adelaide, SA, Australia. 8 School of Medicine, IMPACT Strategic Research Centre, Deakin University, Geelong, VIC, Australia. 9 University Hospi‑ tal Geelong, Barwon Health, Geelong, VIC, Australia. 10 Department of Psy‑ chiatry, The University of Melbourne, Parkville, VIC, Australia. 11 Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia. 12 Orygen Youth Health Research Centre and the National Centre of Excellence in Youth Mental Health, Parkville, VIC, Australia. 13 Department of Psychiatry, Faculty of Health Sciences, Ben Gurion University of the Negev; Beer Sheva Mental Health Center, Beer Sheva, Israel. 14 Department of Psychiatry, University of Oxford, Warneford Hospital, Oxford, UK. 15 Section of Neurosciences and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, Cagliari, Sardinia, Italy. 16 Department of General Adult Psychiatry, Castle Peak Hospital, Hong Kong, China. 17 AP–HP, Hôpitaux Universitaires Henri-Mondor, Créteil, France. 18 Department of Molecular Medicine and Department of Mental Health (DAI), University of Siena and University of Siena Medical Center (AOUS), Siena, Italy. 19 Bipolar Disorder Research Program, Depart‑ ment of Psychiatry, University of São Paulo Medical School, São Paulo, Brazil. Author details 20 Department of Psychiatry, University of Calgary, Calgary, Canada. 21 AP–HP, Hôpitaux Universitaires Henri‑Mondor, INSERM U955 (IMRB), Université Paris Est, Créteil, France. 22 Department of Psychiatry, University Hospital of Alava, University of the Basque Country, CIBERSAM, Vitoria, Spain. 23 Department of Affective Disorders, Q, Mood Disorders Research Unit, Aarhus University Hospital, Aarhus, Denmark. 24 Department of Psychiatry and Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles (UCLA), Los Angeles, CA, USA. 25 Mood Disorders Center of Ottawa, Ottawa, Canada. 26 Department of Psychiatry, Univer‑ sity of Toronto, Toronto, ON, Canada. 27 Department of Psychiatry, Tokyo Metropolitan Matsuzawa Hospital, Setagaya, Tokyo, Japan. 28 Department of Psychiatry, Cumming School of Medicine, University of Calgary, Calgary, Canada. 29 Unité Perception et Mémoire, Institut Pasteur, F‑75015 Paris, France. 30 Bipolar Disorders Program, Hospital Clinic, University of Barcelona, IDIBAPS, CIBERSAM, Barcelona, Catalonia, Spain. 31 Department of Clinical Psychology, NIMHANS, Bangalore 560029, India. 32 Leicestershire Partnership NHS Trust, Leicester, UK. 33 Institute of Clinical Research, Research Unit of Psychiatry, Uni‑ versity of Southern Denmark, Odense, Denmark. 34 Department of Psychiatry, Psychiatry in the Region of Southern Denmark, Odense, Denmark. 35 Aalborg University Hospital, Psychiatry, Aalborg, Denmark. 36 Department of Clinical Medicine, Aalborg University, Aalborg, Denmark. 37 Department of Psychiatry, Wroclaw Medical University, Wroclaw, Poland. 38 Department of Neuroscience and Mental Health, Federal University of Bahia, Salvador, Brazil. 39 Michigan State University College of Human Medicine, Traverse City Campus, Traverse City, MI, USA. 40 Department of Psychiatry, University of California San Diego, San Diego, CA, USA. 41 Affective Disorders Research Project, Tokyo It is important to remember that technology will keep evolving (Arthur 2010). There will be disparities in the adaption of the new products and services, leaving digi- tal equality a continuously moving target (Hilbert 2014, 2016). Young adults of today who are very comfortable using smartphones will continue to use smartphones as they age, and struggle with the new technologies avail- able when they are seniors. The need to respect genera- tional differences in the preferred means to access health information, minimize the burden of new technologies on older adults, and implement programs that expand access and lessen the negative impacts of digital inequali- ties will remain in the future. Authors’ contributions JC RB TG d MB d i JC, RB, TG and MBa designed the study. MA, RA, RB, MB, YB, AB, APC, EYWC, CC, SC, JC, AC, RD, SD, AD, BE, AF, JG, JG, JG, MJG, AG, GMG, PG, HH, SH, CH, DH-M, VK, GK, BL, CL, ERL, UL, RW, AHL, ÂM-S, BM, SM, RM, TN, REN, CO’D, YO, YO, PP, AR, PR, JKR, KS, BS, ES, CS, SS, AHS, KS, AS, PT, YT, LT, JV, EV, MV, BV, and MZ were involved with data collection. TG provided data analysis. RB, JC, TG, PCW and MBa were involved in the draft manuscript and initial review. All authors read and approved the final manuscript. Discussion Older adults who are seeking to save money by purchasing expensive pre- scription drugs online will primarily be presented with rogue pharmacies that do not require a prescription (Monteith and Glenn 2017; Monteith et al. 2016). Some of the risks of using rogue pharmacies include counter- feit drugs, low-quality drugs, unapproved drugs, substi- tutions of strengths and formulations, drug interactions, adverse reactions, and financial fraud (Mackey and Nay- yar 2016; Mackey and Liang 2011; GAO 2014). Another problem area involves the online advertising of unneces- sary or inappropriate medical screening tests that are not included in evidence-based guidelines (Lovett et al. 2012; Lovett and Mackey 2013). Regardless of Internet use, older adults view health care professionals as the primary and most trusted source of information (Hall et al. 2015; Medlock et al. 2015). Patients of all ages would prefer to learn about a serious mental illness by direct conversation with their Older adults with physical limitations use the Inter- net less frequently than healthier older adults (Gell et al. Bauer et al. Int J Bipolar Disord (2018) 6:20 Page 5 of 7 2015; Levine et al. 2018). Many older adults have vision, hearing, and dexterity impairments. Assistive tech- nologies offer innovative options to get connected such as low-vision software for oversized monitors, speech amplification phones using landlines, and tremor stabi- lizing mouse controls (BT 2013; Watanabe et al. 2015; Fischer et al. 2014). More emphasis is needed on finding the optimal individualized approach for older adults to use digital technology rather than focusing on standard mobile devices (Fischer et al. 2014; Kuerbis et al. 2017). Additionally, technology approaches that combine data from those with and without Internet access, such as interactive voice response (IVR), should be considered for projects involving older adults (Verma et al. 2014; Pie- tte et al. 2013).hh remember that many older adults do not use the Inter- net. As the population is aging and more health services are only available online, there is concern about growing health disparities for older adults with bipolar disorder. Mental health experts should contribute to defining the appropriate role for technologies in the care of older adults with bipolar disorder. Competing interests Michael Berk is supported by a National Health and Medical Research Council (NHMRC) Senior Principal Research Fellowship (Grant Number 1059660). René E. Nielsen has received research grants from H. Lundbeck and Otsuka Pharmaceuticals for clinical trials, received speaking fees from Bristol- Myers Squibb, Astra Zeneca, Janssen and Cilag, Lundbeck, Servier, Otsuka Pharmaceuticals, and Eli Lilly and has acted as advisor to Astra Zeneca, Eli Lilly, Lundbeck, Otsuka Pharmaceuticals, Takeda, and Medivir. Michael Berk is supported by a National Health and Medical Research Council (NHMRC) Senior Principal Research Fellowship (Grant Number 1059660). Burnes D, Henderson CR Jr, Sheppard C, Zhao R, Pillemer K, Lachs MS. Reva‑ lence of financial fraud and scams among older adults in the United States: a systematic review and meta-analysis. Am J Public Health. 2017;107:e13–21. Carlson EL. Phishing for elderly victims: as the elderly migrate to the Internet fraudulent schemes targeting them follow. Elder Law J. 2007;14:423–52. CDC. CDC Brief addressing financial exploitation among people living with cognitive impairment and their caregivers: role of the public health and aging services networks. 2015. https://www.cdc.gov/aging/pdf/exploitati on-cognitive-impairment-brief-july-2015.pdf. Accessed 30 Mar 2018. Rasmus W. Licht has received research grant from Glaxo Smith Kline, honoraria for lecturing from Pfizer, Glaxo Smith Kline, Eli Lilly, Astra-Zeneca, Bristol-Myers Squibb, Janssen Cilag, Lundbeck, Otsuka, Servier and honoraria from advisory board activity from Glaxo Smith Kline, Eli Lilly, Astra-Zeneca, Bristol-Myers Squibb, Janssen Cilag, and Sunovion. The other authors declare that they have no competing interests. Chang J, McAllister C, McCaslin R. Correlates of, and barriers to, Internet use among older adults. J Gerontol Soc Work. 2015;58:66–85. Choi NG, Dinitto DM. Internet use among older adults: association with health needs, psychological capital, and social capital. J Med Internet Res. 2013;15:e97. Acknowledgements British Telecommunications. Communication choices for deaf or hard of hear‑ ing people. 2013. https://btplc.com/inclusion/HelpAndSupport/Docum entsandDownloads/Communicationchoices/Fordeaforhardofhearingpeo ple/Communication_Choices_Deaf_Hard_Hearing.pdf. Accessed 30 Mar 2018. We thank Daniela Jany for preparing submission files and assisting with manuscript submission. We thank Daniela Jany for preparing submission files and assisting with manuscript submission. Funding We acknowledge support by the Open Access Publication Funds of the SLUB/ TU Dresden (Grant No. IN-1502335). Flynn KE, Smith MA, Freese J. When do older adults turn to the Internet for health information? Findings from the Wisconsin longitudinal study. J Gen Intern Med. 2006;21(12):1295–301. 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Accessed 30 Mar 2018.f Author details In conclusion, the finding that many older adults with bipolar disorder do not use the Internet confirms the need for further investigation of technology habits, the efficacy of digital tools, and how best to determine who will use these tools appropriately and safely. Today, tech- nology based tools and treatments are suitable for some but not all older adults with bipolar disorder. With gov- ernment and health care providers increasingly rely- ing on electronic communication, it is important to Bauer et al. Int J Bipolar Disord (2018) 6:20 Page 6 of 7 Bauer et al. 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https://openalex.org/W2893156485	https://transmed.almazovcentre.ru/jour/article/download/381/293	Russian	null	ANALYSIS OF THE REASONS OF LATER DIAGNOSTICS OF MYOCARDITIS	Translâcionnaâ medicina	2,018	cc-by	4,917	Для цитирования: Иванова С.В., Титов В.А., Митрофанова Л.Б., Моисеева О.М. Анализ причин позд- ней диагностики миокардита. Трансляционная медицина. 2018; 5 (2): 5–14. Резюме Верификация диагноза «миокардит», несмотря на активное внедрение современных методов обследо- вания, по-прежнему сложна, что приводит не только к поздней постановке заболевания, но и ухудшению прогноза пациентов. Цель настоящего исследования – выявить причины поздней диагностики воспалительных заболева- ний миокарда, оценить факторы, влияющие на течение заболевания, и эффективность проводимой тера- пии у пациентов с морфологически документированным миокардитом. В исследовании приняли участие 50 пациентов с морфологически документированным миокардитом. Длительность наблюдения составила 3,5±1,6 года. В 59% случаев диагноз «миокардит» верифициро- ван в сроки, превышающие 6 месяцев от первых клинико-инструментальных проявлений. Отдаленный прогноз пациентов в отношении глобальной сократительной способности левого желудочка зависел от сроков верификации диагноза (c² = 6,9; р <0,05) и клинического варианта дебюта миокардита (c² = 13,7; р <0,01). Отмечено достоверное ухудшение прогноза пациентов, имеющих в дебюте заболевания клини- ку сердечной недостаточности и нарушения ритма, а также пациентов с поздней диагностикой заболева- ния вследствие ошибочной его трактовки. Сделан вывод о том, что наличие идиопатических нарушений ритма должно рассматриваться как повод для исключения миокардита у пациентов с уже диагностиро- ванными болезнями сердца. Доказано улучшение прогноза на фоне комбинированной терапии сердеч- ной недостаточности (иАПФ, бета-адреноблокаторы и диуретики) (c² = 9,28; p <0,01) и приема статинов (c² = 6,04; р <0,05). Трансляционная медицина / Translyatsionnaya meditsina / Translational Medicine Трансляционная медицина / Translyatsionnaya meditsina / Translational Medicine ISSN 2311-4495 ISSN 2410-5155 (Online) УДК 616.127.07 ISSN 2311-4495 ISSN 2410-5155 (Online) УДК 616.127.07 Иванова С.В., Титов В.А., Митрофанова Л.Б., Моисеева О.М. Иванова С.В., Титов В.А., Митрофанова Л.Б., Моисеева О.М. Контактная информация Иванова Софья Вадимовна ФГБУ «НМИЦ им. В. А. Алмазова» Минздрава России ул. Аккуратова, д. 2, Санкт-Петербург, Россия, 197341. E-mail: sonya.spbgmu16@gmail.com Статья поступила в редакцию 16.05.2018 и принята к печати 13.06.2018. Федеральное государственное бюджетное учреждение «Национальный медицинский исследовательский центр им. В. А. Алмазова» Минздрава России, Санкт-Петербург, Россия Ключевые слова: миокардит, диагностика, прогноз, терапия Ключевые слова: миокардит, диагностика, прогноз, терапия Для цитирования: Иванова С.В., Титов В.А., Митрофанова Л.Б., Моисеева О.М. Анализ причин позд- ней диагностики миокардита. Трансляционная медицина. 2018; 5 (2): 5–14. 5 5 том 5 №2 / 2018 Сердечно-сосудистые заболевания / Cardiovascular medicine ДКМП – дилатационная кардиомиопатия, СН – сердечная недостаточность, ЭМБ – эндомиокарди- альная биопсия, ЭХО-КГ – эхокардиография ДКМП – дилатационная кардиомиопатия, СН – сердечная недостаточность, ЭМБ – эндомиокарди- альная биопсия, ЭХО-КГ – эхокардиография заболеваниях [1]. Наряду с этиопатогенетическим определением миокардита, все чаще классифи- кация миокардита строится преимущественно по морфологическим критериям, что подтверждает значимость эндомиокардиальной биопсии (ЭМБ) в его диагностике. Применение иммуногистохими- ческого анализа существенно повысило чувстви- тельность морфологических методов диагностики воспалительных заболеваний миокарда. Однако су- ществует явное несоответствие между количеством больных с предполагаемым миокардитом и возмож- ностью проведения ЭМБ, особенно когда речь идет ANALYSIS OF THE REASONS OF LATER DIAGNOSTICS OF MYOCARDITIS Corresponding author: Sofya. V. Ivanova Almazov National Medical Research Centre Akkuratova str. 2, Saint Petersburg, Russia, 197341 E-mail: sonya.spbgmu16@gmail.com Received 16 May 2018; accepted 13 June 2018 Ivanova S.V., Titov V.A., Mitrofanova L.B., Moiseeva O.M. Almazov National Medical Research Centre, Saint Petersburg, Russia Abstracti Abstracti The verification of the diagnosis of “myocarditis”, despite the active introduction of modern sur- vey methods, is still complicated, which leads not only to late diagnosis, but also to worsening the progno- sis of patients. The purpose of this study was to identify the causes of late diagnosis of inflammatory dis- eases of the myocardium, evaluate the factors influencing the course of the disease, and the effectiveness of the therapy in patients with morphologically documented myocarditis. The study involved 50 patients with morphologically documented myocarditis. The duration of follow-up was 3.5±1.6 years. In 59% of cas- es, myocarditis was diagnosed within a period exceeding 6 months of the first clinical and instrumen- tal manifestations. Long-term prognosis of patients with regard to global contractility of the left ven- tricle depended on the timing of verification of the diagnosis (c² = 6.9, p <0.05) and the clinical variant of the debut of the myocarditis (c² = 13.7, p <0.01). There was a significant deterioration in the prognosis in patients who have a heart failure clinic and rhythm disorders in the onset of the disease, as well as in patients with late diagnosis of the disease due to erroneous treatment. It was concluded that the presence of idiopathic rhythm disturbances should be considered as an excuse for excluding myocarditis in patients with already diag- nosed heart diseases. The study proved an improvement in the prognosis on the background of combined therapy for heart failure (ACE inhibitors, beta-blockers and diuretics) (c² = 9.28, p <0.01), and statin therapy (c² = 6.04, p <0.05). Key words: myocarditis, diagnosis, prognosis, therapy For citation: For citation: Ivanova S.V., Titov V.A., Mitrofanova L.B., Moiseeva O.M. Analysis of the reasons of later diagnostics of myocarditis. Translyatsionnaya meditsina= Translational Medicine. 2018; 5 (2): 5–14 pages. (In Russ.) For citation: Ivanova S.V., Titov V.A., Mitrofanova L.B., Moiseeva O.M. Analysis of the reasons of later diagnostics of myocarditis. Translyatsionnaya meditsina= Translational Medicine. 2018; 5 (2): 5–14 pages. (In Russ.) Введение Миокардит − это поражение сердечной мышцы воспалительного характера, обусловленное непо- средственным или опосредованным через иммун- ные механизмы воздействием инфекции, парази- тарной или протозойной инвазии, химических или физиологических факторов, а также поражение, возникшее при аллергических и аутоиммунных том 5 №2 / 2018 6 Сердечно-сосудистые заболевания / Cardiovascular medicine Таблица 1. Клиническая характеристика больных миокардитом Таблица 1. Клиническая характеристика больных миокардитом Пациенты с миокардитом n = 50 Возраст, лет 44,6 ± 13,9 Пол, м : ж 29 : 21 Индекс массы тела, кг/м² 26,7 ± 5,2 Сахарный диабет, % (n) 2% (1) Курение, % (n) 46% (23) Артериальная гипертензия, % (n) 50% (25) Гистологическое и иммуногистохимическое подтверждение диагноза Фиброз, % (n) 80% (40) Некроз кардиомиоцитов, % (n) 22,5 % (9) СD3+ Т-лимфоциты 18,0 ± 13,7 СD8+ Т-лимфоциты 8,8 ± 1,0 СD68+ лейкоциты 13,9 ± 6,3 СD45+ лейкоциты 22,2 ± 17,1 HLA-DR, баллы от 0 до 4 3 (2;3) Липоматоз, % (n) 34% (17) Вирусный геном, % (n) 34% (17) Эхокардиография исходно в динамики Левое предсердие, мм 42 ± 8 50 ± 16 КДО ЛЖ, мл 172 ± 96 147 ± 76 КСО ЛЖ, мл 109 ± 88 82 ± 67 ФВ по Симпсону, % 44 ± 19 50 ± 16 МЖП, мм 9 ± 2 10 ± 3 ЗС ЛЖ, мм 9 ± 2 9 ± 2 Правый желудочек, мм 37 ± 8 35 ± 6 РСДЛА, мм 34 ± 11 31 ± 7 ЗС – задняя стенка левого желудочка, КДО – конечный диастолический объём, КДР – конечный диастолический размер, КСО – конечный систолический объём, КСР – конечный систолический размер, ЛЖ – левый желудочек, МЖП – межжелудочковая перегородка, РСДЛА – расчетное систолическое давление в легочной артерии, ФВ – фракция выброса. ЗС – задняя стенка левого желудочка, КДО – конечный диастолический объём, КДР – конечный диастолический размер, КСО – конечный систолический объём, КСР – конечный систолический размер, ЛЖ – левый желудочек, МЖП – межжелудочковая перегородка, РСДЛА – расчетное систолическое давление в легочной артерии, ФВ – фракция выброса. о пациентах с легким течением заболевания. Кроме того, чувствительность ЭМБ ограничена объемом получаемого материала, локальным характером воспаления миокарда и трудностями интерпретации данных у пациентов с сопутствующей патологией. Традиционные неинвазивные методы диагностики воспалительных заболеваний миокарда, такие как электрокардиография, эхокардиография (ЭХО-КГ), о пациентах с легким течением заболевания. Кроме того, чувствительность ЭМБ ограничена объемом получаемого материала, локальным характером воспаления миокарда и трудностями интерпретации данных у пациентов с сопутствующей патологией. Введение Традиционные неинвазивные методы диагностики воспалительных заболеваний миокарда, такие как электрокардиография, эхокардиография (ЭХО-КГ), радионуклидные и лабораторные методы, к сожале- нию, имеют достаточно низкую специфичность [2]. В связи с этим актуальной задачей является разра- ботка комплексного подхода к оценке неинвазивных клинических, лабораторных и инструментальных признаков, позволяющих верифицировать диагноза «миокардит», на основе клинико-морфологических сопоставлений с данными ЭМБ. радионуклидные и лабораторные методы, к сожале- нию, имеют достаточно низкую специфичность [2]. В связи с этим актуальной задачей является разра- ботка комплексного подхода к оценке неинвазивных клинических, лабораторных и инструментальных признаков, позволяющих верифицировать диагноза «миокардит», на основе клинико-морфологических сопоставлений с данными ЭМБ. 7 7 том 5 №2 / 2018 Сердечно-сосудистые заболевания / Cardiovascular medicine Рисунок 1. Распределение пациентов в исследуемой выборке по основным морфологическим вариантам миокардита Сложности возникают не только в поиске дока- зательств воспалительного повреждения миокарда, но и в оценке вероятности наличия такого заболе- вания, что служит причиной задержки в проведе- нии необходимых диагностических мероприятий и, соответственно, позднего назначения адекватной терапии. В связи с этим цель настоящего исследо- вания – выявить причины поздней диагностики воспалительных заболеваний миокарда, факторы, влияющие на течение заболевания, и оценить эф- фективность проводимой терапии у пациентов с морфологически документированным миокарди- том в процессе динамического наблюдения. за кардиомиоцитов расценивалась как погранич- ный миокардит [4]. Морфологическим признаком хронизации воспалительного процесса служило наличие фиброзной ткани в биоптатах миокарда. Всем пациентам выполнялось ЭХО-КГ на ап- парате Vivid 7 (GE, США) по стандартному про- токолу. В зависимости о времени постановки предполагаемого диагноза «миокардит» пациенты разделены на 2 подгруппы: первая подгруппа – ме- нее 6 месяцев от дебюта заболевания, вторая под- группа – более 6 месяцев. Статистический анализ данных, полученных в ходе исследования, проведен с использованием при- кладных статистических программ SPSS Statistica for Windows ver. 23.0. Для показателей, имеющих приближенно нормальное распределение, результа- ты представлены в виде среднего арифметическо- го значения (М), среднеквадратичного отклонения (σ) и количества признаков в группе (n). Критерий значимости устанавливался на уровне р <0,05. Кор- реляционные связи между парами количественных переменных оценивали, используя непараметриче- ский критерий Спирмена. Для выявления независи- мого влияния на количественные показатели каче- ственных факторов была использована процедура однофакторного дисперсионного анализа (ANOVA). Материалы и методы В основную группу включены 50 пациентов с морфологически документированным миокарди- том, проходивших обследование в ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России в период с 2010 по 2017гг. Для включения в исследование мы использовали критерии диагностики воспали- тельного заболеваниями миокарда, предложенные экспертной группой Европейского общества кар- диологов [3], а именно наличие ≥ 14 лейкоцитов/ мм2 биоптатов миокарда, в том числе CD3 положи- тельных Т-лимфоцитов ≥ 7 клетов/мм2 и до 4 мо- ноцитов/мм2 по данным иммуногистохимического анализа [3]. При наличии воспалительного инфиль- трата и признаков некроза кардиомиоцитов без явных ишемических изменений диагностировали активный миокардит. Наличие воспалительной ин- фильтрации без гистологических признаков некро- Результаты Среди включенных в исследование пациентов в возрасте от 19 до 70 лет с морфологически до- кументированным лимфоцитарным миокардитом преобладали мужчины (1,4:1) (Табл. 1). 8 8 том 5 №2 / 2018 Сердечно-сосудистые заболевания / Cardiovascular medicine Таблица 2. Клинико-инструментальная характеристика пациентов в зависимости от времени верификации диагноза Постановка диагноза до 6 месяцев Постановка диагноза более 6 месяцев Кол-во пациентов, n (%) 20 (40) 30 (60) Возраст, лет 45,5 ± 12,7 44,0 ± 14,9 Одышка, % (n) 95% (19) 67% (20)* Болевой синдром, % (n) 35 % (7) 43% (13) Отеки ног, % (n) 30% (6) 7% (2)* Сердцебиение, % (n) 50% (10) 57% (17) Перебои, % (n) 45% (9) 83% (25)** Синкопальные состояния, % (n) 30% (6) 50% (15) Желудочковая экстрасистолия, % (n) 40% (8) 70% (21)* Фибрилляция предсердий, % (n) 30% (6) 50% (15) Желудочковая тахикардия, % (n) 40% (8) 54% (16) Левое предсердие, мм 42,8 ± 9,4 40,9 ± 9,1 КДО ЛЖ, мл 211,9 ± 106,7 172,4 ± 95,6* КСО ЛЖ, мл 155,2 ± 89,7 108,61 ± 87,6 ФВ по Симпсону, % 30,8 ± 15,5 53,5 ± 14,6 МЖП, мм 9,4 ± 1,9 9,2 ± 2,0 ЗС ЛЖ, мм 9,7 ± 1,9 9,0 ± 1,7 Правый желудочек, мм 39,5 ± 7,7 34,6 ± 7,5 РСДЛА, мм 40,3 ± 12,3 30,1 ± 8,4 линико-инструментальная характеристика пациентов в зависимости от времени верификации диагноза ЗС – задняя стенка левого желудочка, КДО – конечный диастолический объём, КДР – конечный диастолический размер, КСО – конечный систолический объём, КСР – конечный систолический размер, ЛЖ – левый желудочек, МЖП – межжелудочковая перегородка, РСДЛА – расчетное систолическое давление в легочной артерии, ФВ – фракция выброса. Данные представлены в виде среднего арифметического значения (М), среднеквадратичного отклонения (σ) и количества признаков в группе (n). Корреляция значима на уровне - р<0,05, * – p<0,01. кардиалгия – 40% (n = 20), перебои в работе серд- ца – 68% (n = 34), синкопальные состояния – 42% (n = 21). Распределение пациентов в исследуемой вы- борке по основным морфологическим вариантам миокардита представлено на рисунке 1. Большин- ство обследованных имели признаки хронического миокардита, на что указывало наличие фиброзных изменений в биоптатах. Активный миокардит диа- гностирован у 22,5% (n = 9) пациентов. Вирусный геном, представленный в 34% случаев (n = 17), в основном в виде комбинации парвовируса В19 с энтеровирусом (n = 8), вирусом герпеса человека типа 6 и вирусом Эпштейн-Барр (n = 3) (Табл. 1). Результаты АРА – антагонисты рецепторов альдостерона II типа, иАПФ – ингибиторы ангиотензин-превращающего фермента. и клинического варианта дебюта заболевания (c² = 13,7; р <0,01) (Рис. 3). В подгруппе пациентов со смешанным вариантом дебюта миокардита (арит- мия и синдром ДКМП) у каждого четвертого па- циента при динамическом наблюдении регистри- ровалось снижение глобальной сократительной способности миокарда. ПФ – ингибиторы ангиотензин-превращающего фермента. и клинического варианта дебюта заболевания (c² = 13,7; р <0,01) (Рис. 3). В подгруппе пациентов со смешанным вариантом дебюта миокардита (арит- мия и синдром ДКМП) у каждого четвертого па- циента при динамическом наблюдении регистри- ровалось снижение глобальной сократительной способности миокарда. ПФ – ингибиторы ангиотензин-превращающего фермента. что более чем в 40% случаев, данные подгруппы имели смешанные клинические проявления. и клинического варианта дебюта заболевания (c² = 13,7; р <0,01) (Рис. 3). В подгруппе пациентов со АРА – антагонисты рецепторов альдостерона II типа, иАПФ – ингибиторы ангиотензин-превращающего фермента. В подгруппе отмечалось преобладание па- циентов, у которых диагноз «миокардит» вери- фицирован в сроки, превышающие 6 месяцев от первых клинико-инструментальных проявлений (Рис. 2). Среди большинства пациентов с ранней диагностикой миокардита преобладали больные с клиникой СН (одышка, отечный синдром) и пато- логическими отклонениями по данным ЭХО-КГ (сниженная фракция выброса, дилатация камер). У пациентов с поздней верификацией заболева- ния в большинстве случаев структурных изме- нений по данным ЭХО-КГ не зарегистрировано. Однако с большей частотой встречались различ- ные нарушения ритма и проводимости (Табл. 2). Указания в анамнезе на инфекцию, перенесенную в течении последних 6 месяцев, выявлена в более чем половине случаев (56%, n = 28), однако в 41% случаев данные анамнеза не учитывались врачами при поставке диагноза. Данные о характере медикаментозной терапии и интервенционных вмешательствах отражены в таблице 4. По клиническим показаниям у 60% (n = 30) пациентов выполнена радиочастотная абла- ция зон нарушений ритма. В 18% (n = 9) случаев проведена имплантация внутрисердечных вспомо- гательных устройств (электрокардиостимуляторы, кардиовертердефибриллятор, ресинхронизирую- щая терапия). Трансплантация сердца выполнена у 2 пациентов. Интервенционные методы лечения лишь в 45% случаев привели к увеличению фрак- ции выброса, тогда как у 27% она осталась без из- менений, а у 27% больных снизилась. Выявлена взаимосвязь между динамикой фракции выброса и приемом статинов (c² = 6,04; р <0,05), а также комплексной терапией с применением трех препа- ратов: ингибиторы ангиотензин-превращающего фермента, бета-адреноблокаторы и диуретики (c² = 9,28; p <0,01). Средняя длительность наблюдения в исследова- нии составила 3,5 ± 1,6 года. Результаты При анализе первоначальных предполагаемых диагнозов мы сгруппировали пациентов для оцен- ки диагностических масок миокардита: аритмия – 45% случаев, синдром дилатационной кардиомио- патии (ДКМП) (кардиомегалия, клиника сердечной недостаточности) – 12% случаев, совокупность аритмического варианта с явлениями сердечной недостаточности (СН) – 23%, ишемическая бо- лезнь сердца – 10%, миоперикардит – 2% и другие заболевания (бронхиальная астма, тиреотоксикоз, эпилепсия) – 8% случаев. Однако стоит заметить, В исследовании в качестве основных первич- ных симптомов миокардита представлены: одыш- ка – 78% (n = 39), сердцебиение – 54% (n = 27), В исследовании в качестве основных первич- ных симптомов миокардита представлены: одыш- ка – 78% (n = 39), сердцебиение – 54% (n = 27), 9 9 том 5 №2 / 2018 Сердечно-сосудистые заболевания / Cardiovascular medicine Таблица 3. Медикаментозное и интервенционное лечением пациентов с миокардитом Медикаментозная терапия Бета-адреноблокаторы, % (n) 84% (42) иАПФ или АРА, % (n) 62% (31) Диуретики, % (n) 40% (20) Антагонисты альдостерона, % (n) 36% (18) Бета-адреноблокаторы + иАПФ, % (n) 58% (29) Бета-адреноблокаторы + иАПФ + Диуретики, % (n) 36% (18) Статины, % (n) 34% (17) Глюкокортикостероиды, % (n) 16% (8) Цитостатические средства, % (n) 12% (6) Интервенционные методы лечения Радиочастотная аблация, % (n) 60% (30) Электрокардиостимулятор, % (n) 12% (6) Кардиовертер-дефибриллятор, % (n) 6% (3) Ресинхронизирующая терапия, % (n) 12% (6) Трансплантация сердца, % (n) 4% (2) Таблица 3. Медикаментозное и интервенционное лечением пациентов с миокардитом что более чем в 40% случаев, данные подгруппы имели смешанные клинические проявления. В подгруппе отмечалось преобладание па- циентов, у которых диагноз «миокардит» вери- фицирован в сроки, превышающие 6 месяцев от первых клинико-инструментальных проявлений (Рис. 2). Среди большинства пациентов с ранней и клинического варианта дебюта заболевания (c² = 13,7; р <0,01) (Рис. 3). В подгруппе пациентов со смешанным вариантом дебюта миокардита (арит- мия и синдром ДКМП) у каждого четвертого па- циента при динамическом наблюдении регистри- ровалось снижение глобальной сократительной способности миокарда. АРА – антагонисты рецепторов альдостерона II типа, иАПФ – ингибиторы ангиотензин-превращающего фермента. что более чем в 40% случаев, данные подгруппы имели смешанные клинические проявления. В подгруппе отмечалось преобладание па- циентов, у которых диагноз «миокардит» вери- фицирован в сроки, превышающие 6 месяцев от первых клинико-инструментальных проявлений (Рис. 2). Среди большинства пациентов с ранней и клинического варианта дебюта заболевания (c² = 13,7; р <0,01) (Рис. 3). В подгруппе пациентов со смешанным вариантом дебюта миокардита (арит- мия и синдром ДКМП) у каждого четвертого па- циента при динамическом наблюдении регистри- ровалось снижение глобальной сократительной способности миокарда. Результаты Установлено, что из- менения глобальной сократительной способности левого желудочка в процессе наблюдения зависели от сроков верификации диагноза (c² = 6,9; р <0,05) 10 том 5 №2 / 2018 Сердечно-сосудистые заболевания / Cardiovascular medicine Рисунок 2. Периоды постановки диагноза в исследуемой выборке Рисунок 2. Периоды постановки диагноза в исследуемой выборке Обсуждение ассоциированы с неблагоприятным для больного прогнозом [6]. По данным отечественных публикаций первым проявлением миокардита может быть одышка – 68,6%, сердцебиение 32,8%, повышение температуры 29,8%, кардиалгия 22,3%, перебои в работе сердца 16,4 %, головокружение 2,9%, синкопальные состояния 1,5% [7]. Следует отметить, что патогномоничных симптомов для данного диагноза не существует. Миокардит следует подозревать во всех случаях необъяснимой СН, тахикардии и аритмии, особенно, когда рутинные методы кардиологического обследования не выявляют причину. Так выявляемый по данным ЭМБ у лиц с нарушениями ритма эндомиокардиальный или интерстициальный фиброз, трактуемый как постмиокардитический, ставит под сомнение существование так называемых идиопатических аритмий [8]. Кроме того, миокардит может сопровождать и такие широко распространенные заболевания, как ИБС и пороки сердца, утяжеляя их течение [9]. По результатам исследований аритмического варианта миокардита О.В. Благовой с соавторами сделаны выводы, что у подавляющего большинства пациентов нет четких анамнестических оснований подозревать миокардит, что, возможно, связано с отсутствием патогномоничных симптомов заболевания [10]. Если учесть, что при эхокардиографических исследованиях бессимптомная дисфункция левого желудочка встречается в популяции в 0,9−12,9% в зависимости от пороговых значений фракции выброса (от 30 до 54%, соответственно), Несмотря на значительный прогресс, достиг- нутый в последнее десятилетие в диагностике миокардита, верификация диагноза по-прежнему остается достаточно сложной проблемой, что при- водит к длительному отсутствию клинического ди- агноза. Большинство экспертов подтверждают, что своевременная диагностика миокардита чрезвы- чайно важна для принятия решения в отношении лечебной тактики и позитивно влияет на отдален- ные результаты, так как на фоне неконтролируемо- го воспалительного процесса в миокарде быстро формируются фиброзные изменения со стойкими нарушениями ритма, патологическим ремоделиро- ванием камер сердца и снижением систолической функции [1]. Полиморфизм клинических проявлений миокардита, “размытость” его клинической картины и нечеткость основных симптомов у большинства пациентов служат основной причиной поздней верификации диагноза. В отечественной классификации миокардита наиболее полно отражается разнообразие клинических вариантов: малосимптомный, псевдокоронарный, декомпенсационный, аритмический, псевдоклапанный, тромбоэмболический, смешанный [5]. Зарубежные исследователи выделяются только три основных варианта дебюта заболевания: инфарктоподобный (27−31%), вариант с развитием жизнеопасных нарушений ритма и проводимости (18%), и с клиникой СН (31−69%). Два последних варианта, как правило, 11 том 5 №2 / 2018 Сердечно-сосудистые заболевания / Cardiovascular medicine Рисунок 3. Распределение пациентов в исследуемой выборке по характеру изменения фракции выброса (ФВ) и клинического течения миокардита. ДКМП – дилатационная кардиомиопатия Рисунок 3. Распределение пациентов в исследуемой выборке по характеру изменения фракции выброса (ФВ) и клинического течения миокардита. ДКМП – дилатационная кардиомиопатия Рисунок 3. Распределение пациентов в исследуемой выборке по характеру изменения фракции выброса (ФВ) и клинического течения миокардита. ДКМП – дилатационная кардиомиопатия то очевидна актуальность своевременной диагностики заболеваний, ведущих к развитию сердечной недостаточности [11, 12]. Однако в большинстве случаев диагноз миокардит по- прежнему верифицируется только ретроспективно. статистического анализа среди лиц с более поздней верификацией диагноза было достоверно больше пациентов с сохранной фракцией выброса и «иди- опатическими» аритмиями. Наличие упорного и необъяснимого аритмического синдрома должно рассматриваться как повод для исключения мио- кардита у пациентов с уже диагностированными болезнями сердца. то Гендерные и возрастные характеристики паци- ентов, включенных в нашу работу, существенно не отличались от данных ранее опубликованных ис- следований [13]. В отличии от данных отечествен- ных публикаций в нашей выборке больные чаще предъявляли жалобы на перебои в работе сердца, а также синкопальные состояния [7]. Вероятно, это обусловлено более частым выполнением ЭМБ пациентам, направляемым на интервенционные вмешательства по поводу тахиаритмий. В ходе работы выделено несколько основных вариантов дебюта миокардита: «идиопатические» аритмии, синдром ДКМП, а также их сочетание, что соот- ветствует данным, представленным О.В. Благовой с соавторами [10]. Также следует отметить, что у 44% пациентов с подтвержденным диагнозом «ми- окардит» отсутствовала связь дебюта клинических проявлений заболевания с перенесенной в течение предшествующих 6 месяцев инфекцией. Возмож- но, для углубленного поиска связи с инфекцией следует расширить рассматриваемый промежуток до 1 года. По данным ЭМБ у каждого четвертого обнару- жены фиброзные изменения в миокарде. Однако при сравнении пациентов по морфологическим группам (активный/пограничный и острый/хро- нический миокардит) не выявлено связи структур- ных изменений миокарда с временным интервалом от появления первых симптомов до постановки диагноза. Частота выделения вирусного генома в миокарде по данным настоящего исследования со- ставляла 34%, что в пределах процентных интерва- лов в ранее опубликованным исследованиям [14]. Сроки постановки диагноза существенно влия- ют на отдаленный прогноз заболевания. Так в под- группе пациентов у которых диагноз «миокардит» выставлен по прошествии не менее пяти лет отме- чался наибольший процент снижения фракции вы- броса за период наблюдения. Динамика фракции выброса зависит и от клинического варианта де- бюта заболевания. Поэтому с учетом неблагопри- ятного прогноза при смешанном варианте дебюта миокардита (аритмия и синдром ДКМП) пациент должен находится под наблюдением в специализи- рованном центре. Для оценки причин поздней верификации диа- гноза в этом исследовании проводилось сравнение между пациентами с ранней и поздней постанов- кой диагноза (менее 6 месяцев и более). По данным 12 том 5 №2 / 2018 Сердечно-сосудистые заболевания / Cardiovascular medicine 3. Caforio AL, Pankuweit S, Arbustini E, et al. Рисунок 3. Распределение пациентов в исследуемой выборке по характеру изменения фракции выброса (ФВ) и клинического течения миокардита. ДКМП – дилатационная кардиомиопатия Current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the Euro- pean Society of Cardiology Working Group on Myocardial and Pericardial Diseases. European Heart J. 2013; 34(33): 2636−2648. В отличии от ожидаемого положительного вли- яния бета-адреноблокаторов и ингибиторов АПФ на отдаленные результаты глобальной сократи- тельной способности левого желудочка у больных с миокардитом, неожиданным для нас оказался эффект статинов. Однако в ранее опубликован- ных исследованиях показано, что эффект стати- нов может быть связан с подавлением продукции Th1-клетками провоспалительных цитокинов, не- гативно влияющих на процессы апоптоза карди- омиоцитов, структурно-функциональное и элек- трофизиологическое ремоделирование миокарда как основу сердечной недостаточности и наруше- ний ритма [15, 16]. Напротив, в работе не выявлено влияния интервенционной терапии на отдаленный прогноз больных с миокардитом, что, вероятно, связано с присутствием в исследуемой выборке больных с активным миокардитом, при котором эффективность радиочастотной аблации зон арит- могенеза значительно ниже [17]. 4. Leone O, Veinot JP, Angelini A, et al. 2011 consensus statement on endomyocardial biopsy from the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology. Cardiovascular Pathology. 2012; 21(4): 245–274. 5. Paleyev NR, Paleyev FN. Non-coronary myocardial disease and its classification. Rossiyskiy kardiologiches- kiy zhurnal=Russian journal of cardiology. 2009; 3: 5-9. In Russian. [Палеев Н. Р., Палеев Ф. Н. Некоронарогенные заболевания миокарда и их классификация. Российский кардиологический журнал. 2009; 3: 5−9]. 6. Anzini M, Merlo M, Sabbadini G, et al. Long-term evolution and prognostic stratification of biopsy-proven active myocarditis. Circulation. 2013; 128(22): 2384−2394. 7. Tereshchenko SN, Arutyunov GP, Gilyarevsky SR. Clinical guidelines. Diagnosis and treatment of myocarditis. Evraziyskiy kardiologicheskiy zhurnal=Eurasian heart journal. 2015; 3: 3−17. In Russian. [Терещенко С. Н., Ару- тюнов Г. П., Гиляревский С. Р. Диагностика и лечение миокардитов. Клинические рекомендации. Евразийский кардиологический журнал. 2015; 3: 3−17]. Заключение Таким образом, в качестве основных причин поздней диагностики миокардита следует назвать плохой сбор анамнеза, отсутствие настороженно- сти врачей в отношении данного диагноза, оши- бочная трактовка идиопатических аритмий. Позд- няя диагностика смешанного варианта дебюта миокардита (аритмия и синдром ДКМП), как пра- вило, ассоциирована с неблагоприятным прогно- зом. Комбинированная терапия бета-адренобло- каторами и ингибиторами АПФ подтвердила свою эффективность в отношении прогноза больных с миокардитом. Дополнительно нуждается в про- ведении клинических исследований целесообраз- ность назначения статинов у больных с воспали- тельными заболеваниями миокарда. 8. Antonova TV, Zhevnerova NS. Viral myocarditis: etiology and pathogenesis, problems of diagnostics. Zhur- nal Infektologii=Journal of infections. 2013; 5(2): 13−21. In Russian. [Антонова Т. В., Жевнерова Н. С. Вирусные миокардиты: этиология и патогенез, проблемы диагностики. Журнал Инфектологии. 2013; 5(2): 13−21]. 9. Mitrofanova LB, Kovalskiy GB. Myocardi- al morphological changes in atrial fibrillation. Arkh- iv patologii=Archives of parhologies. 2011; 6: 10−14. In Russian. [Митрофанова Л. Б., Ковальский Г. Б. Морфологические изменения миокарда при фибрилляции предсердий. Архив патологии. 2011; 6: 10−14]. 9. Mitrofanova LB, Kovalskiy GB. Myocardi- al morphological changes in atrial fibrillation. Arkh- iv patologii=Archives of parhologies. 2011; 6: 10−14. In Russian. [Митрофанова Л. Б., Ковальский Г. Б. Морфологические изменения миокарда при фибрилляции предсердий. Архив патологии. 2011; 6: 10−14]. 10. Blagova OV, Nedostup AV. Contemporary masks of the myocarditis (from clinical signs to diagnosis). Rossiyskiy kardiologicheskiy zhurnal=Russian journal of cardiology. 2014; 5: 13−22. In Russian. [Благова О. В., Недоступ А. В. Современные маски миокардита (от клинических синдромов к диагнозу). Российский кардиологический журнал. 2014; 5: 13−22]. 10. Blagova OV, Nedostup AV. Contemporary masks of the myocarditis (from clinical signs to diagnosis). Rossiyskiy kardiologicheskiy zhurnal=Russian journal of cardiology. 2014; 5: 13−22. In Russian. [Благова О. В., Недоступ А. В. Современные маски миокардита (от клинических синдромов к диагнозу). Российский кардиологический журнал. 2014; 5: 13−22]. 14. Dennert R, Crijns HJ, Heymans S. Acute viral myo- carditis. European Heart J. 2008; 29(17): 2073−2082.f Информация об авторах: Иванова Софья Вадимовна, ординатор, ФГБУ «НМИЦ им. В. А. Алмазова»; Титов Владислав Андреевич, врач-кардиолог, ФГБУ «НМИЦ им. В. А. Алмазова»; Митрофанова Любовь Борисовна, д.м.н., зав НИЛ патоморфологии ФГБУ «НМИЦ им. В. А. Алмазова»; Митрофанова Любовь Борисовна, д.м.н., зав НИЛ патоморфологии ФГБУ «НМИЦ им. В. А. Алмазова»; Моисеева Ольга Михайловна, д.м.н., заведующая на- учно-исследовательским отделом некоронарогенных за- болеваний сердца, ФГБУ «НМИЦ им. В. А. Алмазова»; Моисеева Ольга Михайловна, д.м.н., заведующая на- учно-исследовательским отделом некоронарогенных за- болеваний сердца, ФГБУ «НМИЦ им. В. А. Алмазова»; Моисеева Ольга Михайловна, д.м.н., заведующая на- учно-исследовательским отделом некоронарогенных за- болеваний сердца, ФГБУ «НМИЦ им. В. А. Алмазова»; Конфликт интересов / Conflict of interest Авторы заявили об отсутствии потенциального конфликта интересов. / The authors declare no conflict of interest. Авторы заявили об отсутствии потенциального конфликта интересов. / The authors declare no conflict of interest. 11. Birnie DH, Kandolin R, Nery PB, et al. Cardiac manifestations of sarcoidosis: diagnosis and management. European Heart J. 2017; 38(35): 2663−2670. 12. Kouranos V, Tzelepis GE, Rapti A, et al. Comple- mentary Role of CMR to Conventional Screening in the Diagnosis and Prognosis of Cardiac Sarcoidosis. JACC Car- diovasc Imaging. 2017; 10(12): 1437−1447.f Список литературы / References 1. Moiseeva OM. Myocarditis: the basic principles of di- agnosis and treatment. Kardiologiya: novosti, mneniya, obu- cheniye= Cardiology: news, opinions, training. 2016; 1(8): 50−64. In Russian. [Моисеева О.М. Миокардит: основные принципы диагностики и лечения. Кардиология: новости, мнения, обучение. 2016; 1(8): 50−64]. 13. Kytö V, Sipilä J, Rautava P. The effects of gender and age on occurrence of clinically suspected myocarditis in adulthood. Heart. 2013; 99(22): 1681-1684. 14. Dennert R, Crijns HJ, Heymans S. Acute viral myo- carditis. European Heart J. 2008; 29(17): 2073−2082.f 2. Runov A, Kurchakova E, Khaschevskaya D, et al. Viral reseptor gene expression and myocarditis develop- ment. Medical Virology. 2016; 30(1): 39−47. In Russian. [Рунов А., Курчакова Е., Хащевская Д. и др. Экспрессия вирусных рецепторов и развитие миокардита. Медицинская вирусология. 2016; 30(1): 39−47]. 15. Liu W, Li WM, Gao C, et al. Effects of atorvasta- tin on the Th1/Th2 polarization of ongoing experimental autoimmune myocarditis in Lewis rats. J. of autoimmunity. 2005; 25(4): 258-263. 16. Lazzerini PE, Capecchi PL, Laghi-Pasini F. Statins 13 том 5 №2 / 2018 Сердечно-сосудистые заболевания / Cardiovascular medicine as a New Therapeutic Perspective in Myocarditis and Post- myocarditis Dilated Cardiomyopathy. Cardiovascular Drugs and Therapy. 2013; 27(5): 365-369. py ; ( ) 17. Syrovnev VA, Lebedev DS, Mitrofanova LB, et al. Interventional treatment in patients with myocarditis: Pro and Contra. Kardiologiya=Cardiology. 2017; 57(3): 49-56. In Russian [Сыровнев В. А., Лебедев Д. С., Митрофанова Л. Б., и др. Инвазивная тактика лечения желудочковых нарушений ритма у больных с миокардитом: за и про- тив. Кардиология 2017; 57(3): 49-56.] 17. Syrovnev VA, Lebedev DS, Mitrofanova LB, et al. Interventional treatment in patients with myocarditis: Pro and Contra. Kardiologiya=Cardiology. 2017; 57(3): 49-56. In Russian [Сыровнев В. А., Лебедев Д. С., Митрофанова Л. Б., и др. Инвазивная тактика лечения желудочковых нарушений ритма у больных с миокардитом: за и про- тив. Кардиология 2017; 57(3): 49-56.] Author information: Sofya V. Ivanova, medical resident, Almazov National Medical Research Centre; Sofya V. Ivanova, medical resident, Almazov National Medical Research Centre; Vladislav A. Titov, MD, Almazov National Medical Research Centre; Vladislav A. Titov, MD, Almazov National Medical Research Centre; Lubov` B. Mitrofanova, PhD, prof., Head of the laboratory of pathomorphology, Almazov National Medical Research Centre; Lubov` B. Mitrofanova, PhD, prof., Head of the laboratory of pathomorphology, Almazov National Medical Research Centre; Olga M. Moiseeva, MD, PhD, prof., Head of the Department of Noncoronary Diseases, Almazov National Medical Research Centre. Olga M. Moiseeva, MD, PhD, prof., Head of the Department of Noncoronary Diseases, Almazov National Medical Research Centre. Olga M. Moiseeva, MD, PhD, prof., Head of the Department of Noncoronary Diseases, Almazov National Medical Research Centre. 14 14 том 5 №2 / 2018
https://openalex.org/W3127469629	https://www.frontiersin.org/articles/10.3389/fped.2020.619853/pdf	English	null	Mitochondrial Fission-Mediated Lung Development in Newborn Rats With Hyperoxia-Induced Bronchopulmonary Dysplasia With Pulmonary Hypertension	Frontiers in pediatrics	2,021	cc-by	7,485	Keywords: bronchopulmonary dysplasia, pulmonary hypertension, mitochondrial ﬁssion, Mdivi-1, Drp1, echocardiography, pulmonary vascular resistance Specialty section: This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics Specialty section: This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics Results: Pulmonary artery resistance of the hyperoxide-induced neonatal rat model of BPD increased after it entered normoxic convalescence. During the critical stage of alveolar development in neonatal rats exposed to high oxygen levels for an extended period, the expression and phosphorylation of Drp1 increased in lung tissues. When Drp1 expression was inhibited, small pulmonary vessel development improved and PH was relieved. Received: 21 October 2020 Accepted: 29 December 2020 Published: 28 January 2021 ORIGINAL RESEARCH published: 28 January 2021 doi: 10.3389/fped.2020.619853 1 Department of Neonatology, The Second Afﬁliated Hospital, Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang, China, 2 Department of Neonatology, The Children’s Hospital of Fudan University, Shanghai, China Edited by: Sylvia Knapp, Medical University of Vienna, Austria Edited by: Sylvia Knapp, Medical University of Vienna, Austria Background: Bronchopulmonary dysplasia (BPD) is the most common chronic respiratory disease in premature infants. Oxygen inhalation and mechanical ventilation are common treatments, which can cause hyperoxia-induced lung injury, but the underlying mechanism is not yet understood. Mitochondrial ﬁssion is essential for mitochondrial homeostasis. The objective of this study was to determine whether mitochondrial ﬁssion (dynamin-related protein 1, Drp1) is an important mediator of hyperoxia lung injury in rats. Reviewed by: MaryAnn Volpe, Tufts University School of Medicine, United States Jonathan Michael Davis, Tufts University, United States Correspondence: Chao Chen chen6010@163.com Shangqin Chen chensq5725@163.com Correspondence: Chao Chen chen6010@163.com Shangqin Chen chensq5725@163.com Methods: The animal model of BPD was induced with high oxygen (80–85% O2). Pulmonary histological changes were observed by hematoxylin-eosin (HE) staining. Pulmonary microvessels were observed by immunoﬂuorescence staining of von Willebrand Factor (vWF). Protein expression levels of Drp1 and p-Drp1 (Ser616) were observed using Western Blot. We used echocardiography to measure pulmonary artery acceleration time (PAT), pulmonary vascular resistance index (PVRi), peak ﬂow velocity of the pulmonary artery (PFVP), pulmonary arteriovenous diameter, and pulmonary vein peak velocity. Mitochondrial division inhibitor-1 (Mdivi-1) was used as an inhibitor of Drp1, and administered through intraperitoneal injection (25 mg/kg). †These authors have contributed equally to this work and share ﬁrst authorship Specialty section: This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics Hyperoxia-Induced Lung Injury All animal experiments were performed in accordance with the policies and guidelines of the Laboratory Animal Ethics Committee of Wenzhou Medical University. A total of 10 pregnant Sprague Dawley rats were purchased from the Experimental Animal Center of Wenzhou Medical University. The dams were maintained in humidity- and temperature- controlled rooms on a 12:12-h light-dark cycle and were allowed food and water ad libitum. On the ﬁnal day of pregnancy, the dams delivered naturally (120 pups). Seventy-two pups from six pregnant rats were pooled, randomized, and returned to the nursing dams and then divided into two groups: the control (n = 36) and hyperoxia (n = 36) groups. The hyperoxia group of pups was exposed to 80–85% oxygen in a sealed Plexiglass box for 14 days, while the control group was maintained in room air (21% oxygen). Over the 14 days, the nursing dams were exchanged between the two groups every 24 h to avoid oxygen toxicity. The oxygen level of the Plexiglass box was monitored continuously using an oxygen analyzer. g ( ) It is generally believed that mitochondrial dynamics play a vital role in mitochondrial homeostasis (9). Mitochondrial ﬁssion is mediated mainly by dynamin-related protein 1 (Drp1), a GTPase associated with cytoplasmic dynamin- related proteins, which belongs to the dynamin-related family and was the ﬁrst ﬁssion protein discovered (10). When activated, cytoplasmic Drp1 is transported to the mitochondrial outer membrane, where GTPase is hydrolyzed and polymerized (9). Accumulating data also suggest that Drp1 is a key molecule in mitochondrial dynamics that controls mitochondrial fusion and ﬁssion (11), and abnormal expression of it may lead to abnormal changes in chronic lung diseases such as PH and lung cancer (12, 13). Besides, post-translational modiﬁcation of Drp1, such as phosphorylation at Ser616, is an important mechanism for modulating mitochondrial ﬁssion (14). Recent studies have found that hypoxia can lead to mitochondrial ﬁssion of pulmonary artery smooth muscle (15), but the changes in pulmonary vascular mitochondrial dynamics induced by excessive oxygen have not been studied. The pups from the other four pregnant rats (48 pups) were experimentally divided into four groups: control + vehicle (n = 12), control + Mdivi-1 (n = 12), hyperoxia + vehicle (n = 12), and hyperoxia + Mdivi-1 (n = 12). Mdivi-1 (25 mg/kg) was given to the pups on days 7–14 by intraperitoneal injection. Hyperoxia-Induced Lung Injury The pups in the control + vehicle and hyperoxia + vehicle groups were injected with the same volume of vehicle (corn oil, Sohrab Biotechnology, Beijing, China). INTRODUCTION useful treatment strategy for hyperoxia-associated pulmonary endothelial injury and related diseases. Bronchopulmonary dysplasia (BPD) is a chronic lung disease that occurs in preterm infants who require respiratory support and oxygen therapy at birth (1). It is caused by a variety of molecular factors such as genetic predisposition, oxygen toxicity, and inﬂammatory injury, whose complex interactions are still not fully understood; and the prevention and treatment strategies for BPD are still limited (2–4). Impaired intrauterine lung development and post-partum injury can impair angiogenesis and alveolar formation, resulting in simpliﬁcation of the distal alveoli. These characteristic histological changes of BPD clinically manifest as persistent respiratory diseases, requiring long-term oxygen supplementation (5), and pulmonary hypertension (PH). Approximately 15–25% of BPD cases will develop PH (6). Among severe cases of BPD, the incidence of PH is higher (7). Furthermore, the existence of PH is closely related to adverse outcomes of BPD, and the mortality rate of BPD combined with PH is as high as 48% (8). Citation: Dai Y, Yu B, Ai D, Yuan L, Wang X, Huo R, Fu X, Chen S and Chen C (2021) Mitochondrial Fission-Mediated Lung Development in Newborn Rats With Hyperoxia-Induced Bronchopulmonary Dysplasia With Pulmonary Hypertension. Front. Pediatr. 8:619853. doi: 10.3389/fped.2020.619853 Conclusion: Our study shows that excessive mitochondrial ﬁssion is an important mediator of hyperoxia-induced pulmonary vascular injury, and inhibition of mitochondrial ﬁssion may be a useful treatment for hyperoxia-induced related pulmonary diseases. January 2021 \| Volume 8 \| Article 619853 Frontiers in Pediatrics \| www.frontiersin.org 1 Mitochondrial Fission-Mediated BPD With PH Dai et al. Lung Histology and Morphometric Analyses y After 14 days, all hyperoxia groups (hyperoxia alone, hyperoxia + vehicle, and hyperoxia + Mdivi-1) were maintained in room air. Sixty pups in total from the control and hyperoxia groups were sacriﬁced on days 3, 7, 14, 21, and 28 by injection of 1% pentobarbital. The left lungs were removed and ﬁxed in 4% paraformaldehyde for 48 h. The sections were then embedded into paraﬃn and sliced into 4-µm sections for hematoxylin-eosin (HE) staining (Sohrab Biotechnology, Beijing, China). At the same time, the right lungs were stored at −80◦C for western blot. The radial alveolar count (RAC, the number of alveoli contained in the terminal respiratory unit), which reﬂects the degree of alveolation, and Mean alveolar diameter (MAD) was the average alveolar diameter (19). And they were important indicators for the evaluation of non-development. Brieﬂy, six lung sections were taken for HE staining on days 3, 7, 14, 21, and 28 from the control and hyperoxia groups, ﬁve ﬁelds were randomly selected for imaging under a 100x magniﬁcation lens, and the number of alveoli passing from the center of the respiratory bronchioles to the nearest interpleural line were counted as the RAC. The MAD was measured by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). After 14 days, all hyperoxia groups (hyperoxia alone, hyperoxia + vehicle, and hyperoxia + Mdivi-1) were maintained in room air. Sixty pups in total from the control and hyperoxia groups were sacriﬁced on days 3, 7, 14, 21, and 28 by injection of 1% pentobarbital. The left lungs were removed and ﬁxed in 4% paraformaldehyde for 48 h. The sections were then embedded into paraﬃn and sliced into 4-µm sections for hematoxylin-eosin (HE) staining (Sohrab Biotechnology, Beijing, China). At the same time, the right lungs were stored at −80◦C for western blot. Echocardiography is a common method of PH examination in adults and children (16). Echocardiography can show direct signs of PH due to increased tricuspid regurgitation. However, the tricuspid regurgitation velocity used in adult pulmonary artery pressure estimations cannot be used in children, particularly infants, because it is diﬃcult to obtain a good and clear image, and this measurement may not have good agreement with the data measured using a cardiac catheter (17). Therefore, indirect signs, such as changes in right ventricular function and changes in pulmonary artery acceleration time, are indispensable. Frontiers in Pediatrics \| www.frontiersin.org Lung Histology and Morphometric Analyses The radial alveolar count (RAC, the number of alveoli contained in the terminal respiratory unit), which reﬂects the degree of alveolation, and Mean alveolar diameter (MAD) was the average alveolar diameter (19). And they were important indicators for the evaluation of non-development. Brieﬂy, six lung sections were taken for HE staining on days 3, 7, 14, 21, and 28 from the control and hyperoxia groups, ﬁve ﬁelds were randomly selected for imaging under a 100x magniﬁcation lens, and the number of alveoli passing from the center of the respiratory bronchioles to the nearest interpleural line were counted as the RAC. The MAD was measured by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). In this study, we hypothesized that hyperoxia would induce mitochondrial ﬁssion and thus impact lung development, resulting in the occurrence of BPD combined with PH. We found that after excessive oxygen stimulation, alveolar simpliﬁcation, PH, and p-Drp1 mitochondrial translocation increased mitochondrial ﬁssion. Mdivi-1 is a Drp1 inhibitor that decreases mitochondrial fragmentation (18). Our results also suggest that inhibition of mitochondrial ﬁssion may be a January 2021 \| Volume 8 \| Article 619853 2 Mitochondrial Fission-Mediated BPD With PH Dai et al. Doppler sampling point was placed at the junction to measure the pulmonary vein ﬂow velocity. RESULTS On days 14, 21, 28, and 42, six pups from the control and hyperoxia groups and 24 rats from other four groups on days 21 and 28 were prepared for ultrasonic imaging. The rats were anesthetized with isoﬂurane using a small animal respiratory anesthesia machine (R620-S1, RWD life science, Shenzhen, China). After full chest hair removal, the rats received continuous isoﬂurane anesthesia, were ﬁxed in the supine position on the examination table, and connected with electrocardiogram electrodes. Their chests were coated with coupling agent. Statistical Analysis The experiments were performed in triplicate and repeated at least three times. The data are presented as the mean ± SD or SEM and were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test (equal variance) or Dunnett T3’s post-hoc test (unequal variance) for multiple comparisons. Correlation analyses were performed using Spearman’s rank correlation. Statistical analysis was carried out using SPSS Statistics 19.0 (SPSS Inc., Chicago, USA) or GraphPad 6.0 (GraphPad Software, San Diego, USA). Values of P < 0.05 were considered statistically signiﬁcant. Immunoﬂuorescence On day 14, the lung tissue sections from 24 rats [control + vehicle (n = 6), control + Mdivi-1(n = 6), hyperoxia + vehicle (n = 6), and hyperoxia + Mdivi-1 (n = 6)] were dried overnight at 37◦C and then hydrated in xylene and an ethanol gradient series. The sections were then heated in a microwave in 10 mM citric acid buﬀer (pH 6.0) for 20 min for antigen retrieval. The sections were then incubated in 5% bovine serum albumin at 37◦C for 1 h. The sections were incubated at 4◦C overnight with a rabbit polyclonal anti-vWF (AF3000; 1:200 dilution; Aﬃnity Biosciences. OH. USA), while the negative control group was incubated with phosphate-buﬀered saline. The sections were then incubated with Alexa Fluor-594 sheep anti-rabbit IgG (AB150076; diluted 1:500; Abcam) at room temperature for 4 h and subsequently with 4 ′,6- diamidino-2-phenylindole (DAPI). Dual immunophotography images were acquired using a scanning microscope (C1; Nikon, Tokyo, Japan). To measure the hemodynamics of the pulmonary arteries and veins, the short-axis view of the aortic valve was obtained ﬁrst, and the pulmonary artery was identiﬁed using a color Doppler instrument. The diameter of the pulmonary artery was measured at the attachment point of the pulmonary valve. The pulsed Doppler gate was placed at the proximal end of the pulmonary valve at an incident angle <20◦to maximize laminar ﬂow. Pulmonary acceleration time (PAT), pulmonary ejection time (PET), and peak ﬂow velocity of the pulmonary artery (PFVP) were measured. PAT was measured as the time from the beginning of systolic blood ﬂow to the peak ﬂow rate, while PET was measured as the time from the beginning of systolic blood ﬂow to the completion of pulmonary blood ﬂow. The pulmonary vascular resistance index (PVRi) was calculated as the ratio of PET to PAT. Similarly, the size of the left atrial pulmonary vein junction was measured as the value of the pulmonary vein diameter, and the peak velocity of the pulmonary vein was measured according to the pulmonary vein ﬂow velocity curve. To measure right ventricular load, short-axis views of the right and left ventricles were obtained at the level of the distal mitral valve. Frontiers in Pediatrics \| www.frontiersin.org Western Blotting g Protein was extracted from frozen lung tissue samples from 36 rats in the control and hyperoxia groups on days 3, 7, and 14, and mixed with the loading buﬀer. Equal amounts of protein were separated by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis at 100 V for 3 h and transferred to polyacrylamide diﬂuoride membranes at 100 V for 50 min. Membranes were then blocked in 5% skimmed milk for 3 h at room temperature (20–25◦C). Membranes were then incubated with rabbit monoclonal anti-Drp1 (ab184247; 1:500 dilution; Abcam, Cambridge, UK) or rabbit Phospho- Drp1 (Ser616) Antibody (#3455; 1:1,000 dilution; Cell Signaling Technology, Boston, USA) and gently shaken at 4◦C overnight. The next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:5,000 dilution; Cell Signaling Technology, Boston, USA) for 2 h after being washed three times in Tris- buﬀered saline and tween-20, and developed using enhanced chemiluminescence reagents (Thermo Scientiﬁc Pierce; Thermo Fisher Scientiﬁc, Waltham, MA, USA). Densitometry values of each sample were calculated by Image Lab 5.0 software (Bio- Rad, Hercules, CA, USA) for all bands and standardized relative to β-actin. Right and left ventricular diastolic area (RVEDA and LVEDA, respectively) were measured by manual endocardial boundary tracing. The ratio between RVEDA and LVEDA (RVEDA/LVEDA) was calculated as the measurement index of the right ventricular load (20). Hyperoxia Stunts Alveolar Development, Which Is Restored After Recovery in Room Air Hyperoxia causes substantial morphological simpliﬁcation in the lung tissue, including a visible decrease in alveolar numbers and increase in alveolar size. These negative changes in the hyperoxia group can be quantiﬁed by the decrease in RAC and MAD, an important indicator of lung development. Signiﬁcant histological diﬀerences between the hyperoxia and control groups were found in day 3, 7, 14, 21, and 28 (Figure 1A). This result indicated that chronic exposure to hyperoxia interrupts alveolar The probe was slightly adjusted upwards to obtain a minor axial view of the aorta, and the probe was slightly tilted cephalically to obtain a major axial view of the pulmonary artery. The left atrial pulmonary vein junction was located, and the January 2021 \| Volume 8 \| Article 619853 3 Mitochondrial Fission-Mediated BPD With PH Dai et al. FIGURE 1 \| Morphological changes in rat lungs after hyperoxia. Newborn pups (P0) were exposed to 21% O2 (control) or 80–85% O2 (hyperoxia) for 14 days. (A) Hematoxylin and eosin staining of rat lungs exposed to hyperoxia or control group on day 3, 7, 14, 21, and 28. Scale bar: 100 µm. (B) Compared with the controls, the radial alveolar counts (RACs) of hyperoxia group were signiﬁcantly reduced on ﬁve periods. (C) The Mean alveolar diameter (MAD) of hyperoxia group were signiﬁcantly increased on ﬁve periods. n = 6 per group. Data are shown as mean ± SD; P < 0.01, P < 0.001. FIGURE 1 \| Morphological changes in rat lungs after hyperoxia. Newborn pups (P0) were exposed to 21% O2 (control) or 80–85% O2 (hyperoxia) for 14 days. (A) Hematoxylin and eosin staining of rat lungs exposed to hyperoxia or control group on day 3, 7, 14, 21, and 28. Scale bar: 100 µm. (B) Compared with the controls, the radial alveolar counts (RACs) of hyperoxia group were signiﬁcantly reduced on ﬁve periods. (C) The Mean alveolar diameter (MAD) of hyperoxia group were signiﬁcantly increased on ﬁve periods. n = 6 per group. Data are shown as mean ± SD; P < 0.01, *P < 0.001. with Drp1 levels (Figure 2D). After 14 days of hyperoxia, Drp1 levels in the hyperoxia group were still high (P < 0.01). Furthermore, the ratio of p-Drp1 (Ser616) to Drp1 (P < 0.001) was signiﬁcantly increased compared to the controls, indicating that the phosphorylation of Drp1 at Ser616 (Figure 2C) was actively upregulated (P < 0.001). Pharmacological Inhibition of Drp1 With Mdivi-1 During Hyperoxia Mitigates Pulmonary Vascular Complications Pulmonary Vascular Complications Mdivi-1 is a speciﬁc inhibitor of Drp1. Based on the results that DRP1 started to increase in the hyperoxia group on P7 and the high level persisted until P14, the Drp1 inhibitor, mdivi-1, was injected intraperitoneally daily from days 7 to 14 to investigate whether mdivi-1 had protective eﬀects on chronic hyperoxia-induced lung injury. To evaluate vascular development, vWF-positive small blood vessels were visualized using immunoﬂuorescence staining and counted (Figure 3). We found that long-term hyperoxia signiﬁcantly decreased the number of pulmonary small blood vessels at day 14 (P < 0.001), whereas mdivi-1 treatment in the hyperoxia group (Figure 3B) partially rescued this decrease at day 14 (P < 0.001), indicating that mdivi- 1 can alleviate hyperoxia-induced obstruction of pulmonary vascular development. To evaluate the long-term eﬀects on Hyperoxia Stunts Alveolar Development, Which Is Restored After Recovery in Room Air formation, as evidenced by a decrease in alveolar numbers and increase in alveolar diameter. On day 7 (Figure 1B), the RAC number of control group was higher than hyperoxia group (P < 0.01), and on day 14 this number in control group was much higher than in hyperoxia group (P < 0.001). However, these developmental abnormalities in the hyperoxia group were reversed after recovery in room air for 14 days. As for the MAD (Figure 1C), the MAD value of control group was signiﬁcantly lower than hyperoxia group on day 7 (P < 0.001), on day 14 (P < 0.001), and on day 21 (P < 0.01). Frontiers in Pediatrics \| www.frontiersin.org Hyperoxia Causes Drp1 Overexpression in Newborn Rat Lungs To investigate whether hyperoxia altered the expression of Drp1 in rat lung tissues, we examined the levels of Drp1 and p-Drp1 over time by western blotting (Figure 2A). On the third day of hyperoxia (Figure 2B), the expression of Drp1 in the hyperoxia group was higher than that in the control group (P < 0.05). On the seventh day of hyperoxia, the levels of Drp1 (P < 0.05) and p-Drp1 (Ser616) (P < 0.001) in the hyperoxia group were both signiﬁcantly elevated (Figures 2B,C), although the ratios of p-Drp1 (Ser616)/Drp1 were not diﬀerent between the two groups, suggesting that the elevated p-Drp1 (Ser616) levels increased concomitantly January 2021 \| Volume 8 \| Article 619853 Frontiers in Pediatrics \| www.frontiersin.org 4 Dai et al. Dai et al. Mitochondrial Fission-Mediated BPD With PH FIGURE 2 \| The levels of DRP1 and p-DRP1 (Ser616) in total lung tissues are detected by Western blotting. (A) Representative Western blot images of DRP1 and p-DRP1 (Ser616) in lung tissues from controls or from hyperoxia group on day 3, 7, or 14. (B,C) Protein levels of DRP1 (B) or p-DRP1 (Ser616) (C) in arbitrary units (AU) normalized to β-actin levels. (D) The ratios of p-DRP1 (Ser616)/DRP1 was calculated based on Western blot results. β-actin was the loading control. n = 6 animals/group; Values are expressed as means ± SD; P < 0.05, P < 0.01, P < 0.001. FIGURE 2 \| The levels of DRP1 and p-DRP1 (Ser616) in total lung tissues are detected by Western blotting. (A) Representative Western blot images of DRP1 and p-DRP1 (Ser616) in lung tissues from controls or from hyperoxia group on day 3, 7, or 14. (B,C) Protein levels of DRP1 (B) or p-DRP1 (Ser616) (C) in arbitrary units (AU) normalized to β-actin levels. (D) The ratios of p-DRP1 (Ser616)/DRP1 was calculated based on Western blot results. β-actin was the loading control. n = 6 animals/group; Values are expressed as means ± SD; P < 0.05, P < 0.01, P < 0.001. between the control and hyperoxia groups after 7 days of normal oxygen recovery, the pulmonary vessels showed signiﬁcant abnormalities in the hyperoxia group during the recovery period. We performed continuous pulmonary vascular-related cardiac ultrasonography on rats released from the hypertoxic environment on post-natal day 14, P21, P28, and P42 (Figure 4). Hyperoxia Causes Drp1 Overexpression in Newborn Rat Lungs After measuring indexes from the Doppler ultrasound trace of the pulmonary artery (Figures 4A,B), the results showed that the PAT (Figure 4C) reﬂecting the pulmonary circulation resistance was markedly shortened on day 14 (P < 0.05), day 21 (P < 0.01), and day 28 (P < 0.001). The other two indicators: PVRi and peak pulmonary ﬂow velocity were signiﬁcantly increased (Figures 4D,E). PVRi were signiﬁcantly increased on day 21 (P < 0.001) and day 28 (P < 0.001) in the hyperoxia group compared to the control group. and the peak pulmonary ﬂow velocity were increased on day 21 (P < 0.001), day 28 (P < 0.001), and day 42 (P < 0.05). In addition, cardiac ultrasound- related features of the pulmonary veins were also detected. After hyperoxia, the diameter of the pulmonary vein (Figure 4F) decreased on day 21 (P < 0.05), whereas the diameter of the pulmonary artery (Figure 4G) was not signiﬁcantly changed at day 21. In order to observe the eﬀect of this pulmonary hemodynamic abnormality on the heart, particularly on the right ventricular load, we acquired a short-axis image of the blood vessels, ultrasonic echocardiogram monitoring revealed that the pulmonary vascular resistance index (Figure 3C) of the hyperoxia + mdivi-1 group was signiﬁcantly lower than that of the hyperoxia + vehicle group on P21 (P < 0.05). The peak pulmonary ﬂow velocity was measured on P28 (Figure 3D). The results showed that the pulmonary artery peak ﬂow velocity decreased after administration of mdivi- 1, indicating an improvement in pulmonary artery pressure. In addition, the heart tissue was weighed and Fulton index was calculated on P28. The Fulton index of the hyperoxia + vehicle group was signiﬁcantly higher than that of the control + vehicle group (Figure 3E), indicating that the right ventricle was hypertrophic. Compared to the hyperoxia + vehicle group, the Fulton index of the hyperoxia + mdivi-1 group was lower, suggesting that right ventricular hypertrophy had improved (Figure 3E). Recovery in Room Air Leaves Hyperoxia-Exposed Rats With Abnormal Pulmonary Hemodynamics Until Adolescence Although the alveolar developmental obstruction caused by hyperoxia had no signiﬁcant diﬀerence in lung morphology January 2021 \| Volume 8 \| Article 619853 Frontiers in Pediatrics \| www.frontiersin.org 5 Dai et al. Dai et al. Mitochondrial Fission-Mediated BPD With PH E 3 \| The beneﬁcial effect of Mdivi-1 on pulmonary vasculature after chronic hyperoxia-induced lung injury. DRP1 inhibitor Mdivi-1 was injected intraperitoneally he rats from day 7 to 14. (A) Representative images of immunoﬂuorescence staining. Green ﬂuorescence represented vWF expression. Scale bar = 50 µm. -positive vessels whose diameters were < 50 µm were calculated accordingly. (C) The pulmonary vascular resistance index (PVRi) measured on day 21. (D) k pulmonary ﬂow velocity was measured on P28 (E) the Fulton’s index was measured on day 28. n = 6 animals/group; Values are presented as mean ± SD. 01, P < 0.001, hyperoxia vs. control; #P < 0.05, ###P < 0.001, hyperoxia+Mdivi-1 vs. hyperoxia. stolic ventricle at the mitral valve level, and outlined the right ventricular dilation in the hyperoxia group compared to the FIGURE 3 \| The beneﬁcial effect of Mdivi-1 on pulmonary vasculature after chronic hyperoxia-induced lung injury. DRP1 inhibitor Mdivi-1 was injected intraperitoneally daily to the rats from day 7 to 14. (A) Representative images of immunoﬂuorescence staining. Green ﬂuorescence represented vWF expression. Scale bar = 50 µm. (B) vWF-positive vessels whose diameters were < 50 µm were calculated accordingly. (C) The pulmonary vascular resistance index (PVRi) measured on day 21. (D) The peak pulmonary ﬂow velocity was measured on P28 (E) the Fulton’s index was measured on day 28. n = 6 animals/group; Values are presented as mean ± SD. P < 0.01, **P < 0.001, hyperoxia vs. control; #P < 0.05, ###P < 0.001, hyperoxia+Mdivi-1 vs. hyperoxia. end-diastolic ventricle at the mitral valve level, and outlined the cross-sections of the right and left ventricular cavities. The area ratio (RVEDA/LVEDA) is a measurement of the right ventricular load. We found that the area ratio was higher in the hyperoxia group on day 14 (P < 0.05) and on day 21 (P < 0.01), suggesting that the right ventricle was dilated, and this trend continued for 7 days (up to day 21) in the hyperoxia group (Figure 4H). Recovery in Room Air Leaves Hyperoxia-Exposed Rats With Abnormal Pulmonary Hemodynamics Until Adolescence A representative two-dimensional echocardiography image of the left and right ventricular dimensions on day 21 demonstrated right ventricular dilation in the hyperoxia group compared to the control group, indicative of diastolic right ventricle dysfunction (Figures 4I,J). DISCUSSION In this study, we attempted to explore the relationship between BPD and mitochondrial ﬁssion induced by hyperoxia. To simulate severe BPD in rodent models, we January 2021 \| Volume 8 \| Article 619853 Frontiers in Pediatrics \| www.frontiersin.org 6 Dai et al. Mitochondrial Fission-Mediated BPD With PH Dai et al. FIGURE 4 \| Abnormal pulmonary hemodynamics after hyperoxia. Newborn (P0) pups were exposed to 21% O2 (control) or 80–85% O2 (hyperoxia) for 14 days and returned to air to receive echocardiography detection on post-natal day 14, 21, 28, and 42. (A) Representative Doppler ultrasound images of the pulmonary artery (PA) on day 21 after exposure to either normoxia or hyperoxia for 14 days from birth. (B) Representative 2-dimensional echocardiography images of left and right ventricular dimensions on day 21 was shown by comparing the difference between exposure to normoxia and hyperoxia for 14 days after birth. (C–H) Echocardiography and pulse-wave Doppler-derived indexes of pulmonary acceleration time (PAT) (C), pulmonary vascular resistance (PVR) index (D), peak ﬂow velocity of pulmonary artery (E), pulmonary venous diameter (F), pulmonary artery diameter (G), and right ventricular end-diastolic area (RVEDA)-to-left ventricular end-diastolic area ratio (LVEDA) (H) were analyzed and compared between hyperoxia group and control group on P14, P21, P28, and P42. (I,J) Representative two-dimensional echocardiography images of the left and right ventricular dimensions on control and hyperoxia group on day 21. n = 6 animals/group; Values are expressed as means ± SD; P < 0.05, P < 0.01, P < 0.001, compared with hyperoxia and control group. FIGURE 4 \| Abnormal pulmonary hemodynamics after hyperoxia. Newborn (P0) pups were exposed to 21% O2 (control) or 80–85% O2 (hyperoxia) for 14 days and returned to air to receive echocardiography detection on post-natal day 14, 21, 28, and 42. (A) Representative Doppler ultrasound images of the pulmonary artery (PA) on day 21 after exposure to either normoxia or hyperoxia for 14 days from birth. (B) Representative 2-dimensional echocardiography images of left and right ventricular dimensions on day 21 was shown by comparing the difference between exposure to normoxia and hyperoxia for 14 days after birth. DISCUSSION (C–H) Echocardiography and pulse-wave Doppler-derived indexes of pulmonary acceleration time (PAT) (C), pulmonary vascular resistance (PVR) index (D), peak ﬂow velocity of pulmonary artery (E), pulmonary venous diameter (F), pulmonary artery diameter (G), and right ventricular end-diastolic area (RVEDA)-to-left ventricular end-diastolic area ratio (LVEDA) (H) were analyzed and compared between hyperoxia group and control group on P14, P21, P28, and P42. (I,J) Representative two-dimensional echocardiography images of the left and right ventricular dimensions on control and hyperoxia group on day 21. n = 6 animals/group; Values are expressed as means ± SD; P < 0.05, P < 0.01, P < 0.001, compared with hyperoxia and control group. FIGURE 4 \| Abnormal pulmonary hemodynamics after hyperoxia. Newborn (P0) pups were exposed to 21% O2 (control) or 80–85% O2 (hyperoxia) for 14 days and returned to air to receive echocardiography detection on post-natal day 14, 21, 28, and 42. (A) Representative Doppler ultrasound images of the pulmonary artery (PA) on day 21 after exposure to either normoxia or hyperoxia for 14 days from birth. (B) Representative 2-dimensional echocardiography images of left and right ventricular dimensions on day 21 was shown by comparing the difference between exposure to normoxia and hyperoxia for 14 days after birth. (C–H) Echocardiography and pulse-wave Doppler-derived indexes of pulmonary acceleration time (PAT) (C), pulmonary vascular resistance (PVR) index (D), peak ﬂow velocity of pulmonary artery (E), pulmonary venous diameter (F), pulmonary artery diameter (G), and right ventricular end-diastolic area (RVEDA)-to-left ventricular end-diastolic area ratio (LVEDA) (H) were analyzed and compared between hyperoxia group and control group on P14, P21, P28, and P42. (I,J) Representative two-dimensional echocardiography images of the left and right ventricular dimensions on control and hyperoxia group on day 21. n = 6 animals/group; Values are expressed as means ± SD; P < 0.05, P < 0.01, *P < 0.001, compared with hyperoxia and control group. January 2021 \| Volume 8 \| Article 619853 Frontiers in Pediatrics \| www.frontiersin.org 7 Mitochondrial Fission-Mediated BPD With PH Dai et al. in Drp1 after hyper oxygen in this study, the rats were injected intraperitoneally with Mdivi-1 (25 mg/kg) from days 7 to 14 to explore whether inhibition of Drp1 has protective eﬀects on hyperoxia-induced lung injury. It was found that long-term hyperoxia severely hindered the development of small pulmonary vessels, and after mdivi- 1 administration, the number of small pulmonary vessels signiﬁcantly increased. DISCUSSION These results indicate that Mdivi-1 can relieve hyperoxia-induced obstruction of pulmonary microvascular development. From the perspective of the long-term eﬀects on blood vessels, ultrasonic monitoring results showed that the PVR and PFVP measured during the recovery period were lower after the administration of Mdivi-1, suggesting improvements in pulmonary artery pressure. In addition, the heart tissue was weighed on 28 days after birth to calculate the Fulton index the results showed that the right ventricular hypertrophy index in the hyperoxia group was signiﬁcantly higher than that in the control group, suggesting right ventricular hypertrophy. ﬁrst exposed rats to 80–85% oxygen for a long period (14 days). The results showed that the lung morphology was seriously damaged and alveolar structure was simpliﬁed, which was consistent with the pathology of BPD. Protein expression of Drp1/p-Drp1 in the hyperoxia group was signiﬁcantly higher compared to in the control group. We then applied the Drp1 inhibitor, Mdivi-1, and found an improvement in the reduction of pulmonary microvasculature under hyperoxia. Finally, we discussed whether hyperoxia-induced BPD would have adverse eﬀects on pulmonary circulation function and followed up with echocardiography. In recent years, many studies have shown that mitochondrial dysfunction plays an important role in BPD and PH (21). Mitochondrial dynamics are essential for maintaining mitochondrial integrity and regulating apoptosis (22). Drp1 is a mitochondrial outer membrane protein that mediates ﬁssion of mitochondria and controls mitochondrial morphology (23). The latest research shows that by inducing overexpression of hypoxia inducible factor-1, hypoxia stimuli can promote the expression of Drp1 to regulate mitochondrial dynamics in pulmonary vascular remodeling (24). In this study Drp1 changes were investigated after hyperoxia exposure in newborn rats to further our understanding of the relationship between hyperoxia and Drp1. We found that Drp1 reached its peak on day 7 in the hyperoxia group and maintained this level until day 14. These results showed that Drp1 protein expression could be enhanced by hyperoxia. Many studies have suggested that abnormally high expression of Drp1 in the lungs is an indicator of poor prognosis, particularly in chronic malignant diseases (25, 26). Drp1 has been regarded as an attractive therapeutic target. To investigate whether this BPD model would have an adverse eﬀect on pulmonary circulation function, follow-up detection was carried out by echocardiography in rats at 14, 21, 28, and 42 days after birth. DISCUSSION It was demonstrated that PAT in the hyperoxia group was shortened, while the PVRi and PFVP increased signiﬁcantly compared to the control group. These parameters all reﬂected higher pulmonary pressure after exposure to hyperoxia for 2 weeks. In addition, by measuring the area ratio and Fulton index, we found that over-circulation inﬂuenced right ventricular structure and function. It is worth mentioning that in this study, in addition to focusing on the pulmonary artery, cardiac echocardiography indicators related to the pulmonary vein were also detected, and it was found that the pulmonary vein diameter showed signs of narrowing after exposure to hyperoxia, while there was no signiﬁcant diﬀerence in pulmonary artery diameter between the two groups. Pulmonary vein stenosis is a rare problem that is often neglected (36); however, it is a severe and increasingly common complication of preterm infants with BPD (37). Although this study identiﬁed the manifestations of pulmonary vein stenosis, it did not elucidate the underlying mechanisms, which require further investigation. Mitochondrial oxidative stress is a component of general oxidative stress, and excessive reactive oxygen species would lead to increased mitochondrial ﬁssion (27). Studies have found that particulate matter (PM2.5) can lead to oxidative stress in lung epithelial cells, increasing mitochondrial ﬁssion, resulting in cell apoptosis (28), and Drp1 and oxidative stress are essential mediators in cigarette smoke-induced pulmonary endothelial Injury (29). Combined with our ﬁndings, we speculated that lung injury caused by hyperoxia in newborn rats would increase mitochondrial ﬁssion, namely the expression of Drp1, due to oxidative stress. In addition, experiments have conﬁrmed the relationship between hypoxia and Drp1, studies were not only involved in animal models about Lung Ischemia-reperfusion Injury (30) and lung vascular ischemic/hypoxic injury (31), but also in others like hepatocellular carcinoma cells in hypoxia (32), and Hypoxia-Reoxygenation Injury of Cardiomyocytes (33). Although hypoxia or hyperoxia can trigger similar pathological responses, such as oxidative stress and inﬂammation, these underlying mechanisms need to be further studied at the cellular level. Lastly, there are some limitations present in this study. First, we did not further explore the mechanism between hyperoxia and Drp1. Secondly, we did not carry out in vitro experiments, such as on pulmonary epithelial cells and microvascular endothelial cells. Thirdly, this experiment only discussed the development of pulmonary vessels after hyperoxia stimulation, but future studies will focus on the eﬀects of Drp1 and Mdivi-1 on alveolar development. Frontiers in Pediatrics \| www.frontiersin.org REFERENCES 15. Liu X, Tan H, Liu X, Wu Q. Correlation between the expression of Drp1 in vascular endothelial cells and inﬂammatory factors in hypertension rats. Exp Ther Med. (2018) 15:3892–98. doi: 10.3892/etm.2018.5899 1. Jobe AH, Bancalari E. Bronchopulmonary dysplasia. Am J Respir Crit Care Med. 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(2018) 50:209–15. doi: 10.1093/abbs/gmx137 28. Liu X, Zhao X, Li X, Lv S, Ma R, Qi Y, et al. PM2.5 triggered apoptosis in lung epithelial cells through the mitochondrial apoptotic way mediated by a ROS-DRP1-mitochondrial ﬁssion axis. J Hazard Mater. (2020) 397:122608. doi: 10.1016/j.jhazmat.2020.122608 13. Chiang YY, Chen SL, Hsiao YT, Huang CH, Lin TY, Chiang IP, et al. Nuclear expression of dynamin-related protein 1 in lung adenocarcinomas. Mod Pathol. (2009) 22:1139–50. doi: 10.1038/modpathol.2009.83 29. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. FUNDING The animal study was reviewed and approved by Wenzhou Medicial University. This work was supported by Wenzhou science and technology project (Y20180092 and Y20180006). This work was supported by Wenzhou science and technology project (Y20180092 and Y20180006). The animal study was reviewed and approved by Wenzhou Medicial University. AUTHOR CONTRIBUTIONS development and function. However, further studies are required to determine the precise mechanism of Drp1 in BPD and BPD-PH. YD conceived and designed the experiments. YD and BY performed the experiments and wrote the paper. YD, DA, and LY analyzed the data. XW, RH, and XF contributed materials and analysis tools. CC and SC edited and approved ﬁnal draft. All authors contributed to the article and approved the submitted version. YD conceived and designed the experiments. YD and BY performed the experiments and wrote the paper. YD, DA, and LY analyzed the data. XW, RH, and XF contributed materials and analysis tools. CC and SC edited and approved ﬁnal draft. All authors contributed to the article and approved the submitted version. DISCUSSION In conclusion, the present study identiﬁed the echocardiographic features of hyperoxia-induced BPD-PH models and conﬁrmed that the expression of Drp1 is increased in hyperoxia-induced lung injury. Treatment with Mdivi-1 during hyperoxia was protective against pulmonary vasculature Mdivi-1, a speciﬁc inhibitor of DRP1 (34) is reported to be eﬀective in suppressing the pulmonary artery smooth muscle cells in lungs with PH (35). Because of the changes January 2021 \| Volume 8 \| Article 619853 Frontiers in Pediatrics \| www.frontiersin.org 8 Mitochondrial Fission-Mediated BPD With PH Dai et al. REFERENCES Wang Z, White A, Wang X, Ko J, Choudhary G, Lange T, et al. Mitochondrial ﬁssion mediated cigarette smoke-induced pulmonary endothelial injury. Am J Respir Cell Mol Biol. (2020) 63:637–51. doi: 10.1165/rcmb.2020-0008OC 14. Kim YM, Youn SW, Sudhahar V, Das A, Chandhri R, Cuervo Grajal H, et al. Redox regulation of mitochondrial ﬁssion protein Drp1 by protein disulﬁde isomerase limits endothelial senescence. Cell Rep. (2018) 23:3565– 78. doi: 10.1016/j.celrep.2018.05.054 30. Lin KC, Yeh JN, Chen YL, Chiang JY, Sung PH, Lee FY, et al. Xenogeneic and allogeneic mesenchymal stem cells eﬀectively protect the lung against Frontiers in Pediatrics \| www.frontiersin.org January 2021 \| Volume 8 \| Article 619853 9 Dai et al. Mitochondrial Fission-Mediated BPD With PH therapeutic target in pulmonary hypertension. Circ Res. (2012) 110:1484– 97. doi: 10.1161/CIRCRESAHA.111.263848 therapeutic target in pulmonary hypertension. Circ Res. (2012) 110:1484– 97. doi: 10.1161/CIRCRESAHA.111.263848 therapeutic target in pulmonary hypertension. Circ Res. (2012) 110:1484– 97. doi: 10.1161/CIRCRESAHA.111.263848 ischemia-reperfusion injury through downregulating the inﬂammatory, oxidative stress, and autophagic signaling pathways in rat. Cell Transplant. (2020) 29:963689720954140. doi: 10.1177/0963689720954140 ischemia-reperfusion injury through downregulating the inﬂammatory, oxidative stress, and autophagic signaling pathways in rat. Cell Transplant. (2020) 29:963689720954140. doi: 10.1177/0963689720954140 36. Mahgoub L, Kaddoura T, Kameny AR, Lopez Ortego P, Vanderlaan RD, Kakadekar A, et al. Pulmonary vein stenosis of ex-premature infants with pulmonary hypertension and bronchopulmonary dysplasia, epidemiology, and survival from a multicenter cohort. Pediatr Pulmonol. (2017) 52:1063– 70. doi: 10.1002/ppul.23679 31. Duan C, Wang L, Zhang J, Xiang X, Wu Y, Zhang Z, et al. Mdivi-1 attenuates oxidative stress and exerts vascular protection in ischemic/hypoxic injury by a mechanism independent of Drp1 GTPase activity. Redox Biol. (2020) 37:101706. doi: 10.1016/j.redox.2020.101706 37. Nasr VG, Callahan R, Wichner Z, Odegard KC, DiNardo JA. Intraluminal pulmonary vein stenosis in children: a “New” lesion. Anesth Analg. (2019) 129:27–40. doi: 10.1213/ANE.0000000000003924 32. Lin XH, Qiu BQ, Ma M, Zhang R, Hsu SJ, Liu HH, et al. Suppressing DRP1- mediated mitochondrial ﬁssion and mitophagy increases mitochondrial apoptosis of hepatocellular carcinoma cells in the setting of hypoxia. Oncogenesis. (2020) 9:67. doi: 10.1038/s41389-020-00251-5 33. Luo H, Song S, Chen Y, Xu M, Sun L, Meng G, et al. Inhibitor 1 of protein phosphatase 1 regulates Ca2+/calmodulin-dependent protein kinase II to alleviate oxidative stress in hypoxia-reoxygenation injury of cardiomyocytes. Oxid Med Cell Longev. (2019) 2019:2193019. Frontiers in Pediatrics \| www.frontiersin.org January 2021 \| Volume 8 \| Article 619853 REFERENCES doi: 10.1155/2019/2193019 Conﬂict of Interest: 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. 34. Bordt EA, Clerc P, Roelofs BA, Saladino AJ, Tretter L, Adam-Vizi V, et al. The putative Drp1 inhibitor mdivi-1 is a reversible mitochondrial complex I inhibitor that modulates reactive oxygen species. Dev Cell. (2017) 40:583– 94.e6. doi: 10.1016/j.devcel.2017.02.020 Copyright © 2021 Dai, Yu, Ai, Yuan, Wang, Huo, Fu, Chen and Chen. 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) 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. 35. Marsboom G, Toth PT, Ryan JJ, Hong Z, Wu X, Fang YH, et al. Dynamin-related protein 1-mediated mitochondrial mitotic ﬁssion permits hyperproliferation of vascular smooth muscle cells and oﬀers a novel January 2021 \| Volume 8 \| Article 619853 Frontiers in Pediatrics \| www.frontiersin.org 10
https://openalex.org/W1532061548	https://europepmc.org/articles/pmc4511234?pdf=render	English	null	Genetic variation of pfhrp2 in Plasmodium falciparum isolates from Yemen and the performance of HRP2-based malaria rapid diagnostic test	Parasites & vectors	2,015	cc-by	8,237	* Correspondence: halmekhlafi@yahoo.com 1Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia 3Azal National Research Centre, Azal University for Human Development, 447 Sana’a, Yemen Full list of author information is available at the end of the article © 2015 Atroosh 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 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. Abstract Background: The genetic variation in the Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene that may compromise the use of pfhrp2-based rapid diagnostic tests (RDTs) for the diagnosis of malaria was assessed in P. falciparum isolates from Yemen. Methods: This study was conducted in Hodeidah and Al-Mahwit governorates, Yemen. A total of 622 individuals with fever were examined for malaria by CareStart™malaria HRP2-RDT and Giemsa-stained thin and thick blood films. The Pfhrp2 gene was amplified and sequenced from 180 isolates, and subjected to amino acid repeat types analysis. Results: A total of 188 (30.2 %) participants were found positive for P. falciparum by the RDT. Overall, 12 different amino acid repeat types were identified in Yemeni isolates. Six repeat types were detected in all the isolates (100 %) namely types 1, 2, 6, 7, 10 and 12 while types 9 and 11 were not detected in any of the isolates. Moreover, the sensitivity and specificity of the used PfHRP2-based RDTs were high (90.5 % and 96.1 %, respectively). Conclusion: The present study provides data on the genetic variation within the pfhrp2 gene, and its potential impact on the PfHRP2-based RDTs commonly used in Yemen. CareStart™Malaria HRP2-based RDT showed high sensitivity and specificity in endemic areas of Yemen. Keywords: Malaria, Plasmodium falciparum, Rapid diagnostic test, Plasmodium falciparum histidine-rich protein 2, Yemen Keywords: Malaria, Plasmodium falciparum, Rapid diagnostic test, Plasmodium falciparum histidine-rich protein 2, Yemen alaria, Plasmodium falciparum, Rapid diagnostic test, Plasmodium falciparum histidine-rich protein Genetic variation of pfhrp2 in Plasmodium falciparum isolates from Yemen and the performance of HRP2-based malaria rapid diagnostic test Wahib M. Atroosh1,2, Hesham M. Al-Mekhlafi1,3,4, Adel Al-Jasari5, Hany Sady1, Ahmed K. Al-Delaimy1, Nabil A. Nasr1, Salwa Dawaki1, Awatif M. Abdulsalam1, Init Ithoi1, Yee Ling Lau1, Mun Yik Fong1 and Johari Surin1 Atroosh et al. Parasites & Vectors (2015) 8:388 DOI 10.1186/s13071-015-1008-x Atroosh et al. Parasites & Vectors (2015) 8:388 DOI 10.1186/s13071-015-1008-x Background region; Pakistan, Afghanistan and Yemen account for more than 99 % of the 56,000 regional deaths due to malaria [4]. Malaria is still a major public health problem in Yemen, with almost 66 % of the population living in areas that suffer from stable malaria transmission [1]. Plasmodium falciparum is the predominant species and was respon- sible for almost 99 % of malaria cases in Yemen during 2012, a large number of which consisted of drug-resistant P. falciparum parasites [2, 3]. Among 17 countries with malaria-endemic areas in the Middle East and Eurasia The national malaria control programme in Yemen (NMCP) has achieved substantial success in controlling local cases of malaria, achieving a significant reduction in the number of malaria cases, dropping from 900,000 cases in the early 2000s to around 150,000 cases by 2013 [1]. However, Yemen is still classified among areas of high malaria transmission, making it the only country in the Arabian Peninsula and greater Middle Eastern region that is still plagued with malaria to the extent that resi- dents still suffer from considerably high mortality and morbidity rates [2]. Imported malaria cases are still Correspondence: halmekhlafi@yahoo.com 1Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia 3Azal National Research Centre, Azal University for Human Development, 447 Sana’a, Yemen Full list of author information is available at the end of the article Page 2 of 8 Atroosh et al. Parasites & Vectors (2015) 8:388 on the national malaria records of 2010–2013 provided by NMCP. reported in neighbouring countries, threatening the mal- aria control and elimination programmes in the region. For instance, 2788 malaria cases were diagnosed in southern Saudi Arabia between 2011 and 2012, with about 97 % of the cases having been identified as origin- ating outside the country, particularly from the Tehama region, a Yemen bordered area [5]. The Hodeidah governorate (14.46° North, 43.15° East) is located in the Tehama region in the western part of Yemen, about 226 km from Sana’a, the capital of Yemen. It is a coastal area located along the Red Sea, covering a total area of 117,145 km2 with a total population of 2.16 million [13]. Al-Mahwit governorate (16.25° N, 44.717° E) is located between Hodeidah and Sana’a (about 111 km west of Sana’a), covering a total area of 2858 km2 with a total population of 597,000 people [13]. Plasmodium falciparum isolates p A total of 622 individuals with fever were recruited to this study and examined for malaria. Finger prick blood samples were collected from participants and tested using the RDT (CareStart™Malaria HRP2, Cat no. G0141, Access Bio, Inc, USA), and for preparing both thick and thin blood films. Filter paper blood spots were also collected from each participant on 3MM Whatman® filter paper (Whatman International Ltd., Maidstone, England), and kept in clean, dry, well-sealed aluminum pouches with desiccated silicon bags for molecular ana- lysis. Blood films were stained with 5 % of buffer-diluted Giemsa stain for 30 min and were examined microscop- ically for the presence of malaria parasites. All RDT tests were performed and interpreted by trained and skilled laboratory personnel from the NMCP following the manufacturer’s instructions. Briefly, one purple line in the control line position interpreted negative for falcip- arum malaria and two purple lines at the test line position along with the control line were to be defined as positive. In case the control line did not appear, the result was considered invalid and the test was repeated. In Yemen, PfHRP2-based RDTs have been imple- mented by the NMCP in 2009, and are being used exclu- sively for malaria active case detection (ACD) targeting only falciparum malaria infections [12]. However, data on the genetic variation of the pfhrp2 are not available. Hence, the present study aims to investigate the genetic variations of the pfhrp2 gene in malaria isolates from the Hodeidah and Al-Mahwit governorates, Yemen (areas with high malaria endemicity) and the possible impact of this variation on the efficacy of the currently used pfhrp2-based RDTs. The present study is the first to pro- vide data on the genetic variation of PfHRP2 in Yemen, and therefore has the potential to impact on control strategies and efforts to eradicate malaria from Yemen and the Arabian Peninsula. Background The climate of the selected districts is a combination of tropical monsoon with occasional rains in the summer and dry weather in winter, with a mean rainfall of 200 mm/year. The mean temperature is 37.5 °C in summer and 24 °C in winter, with humidity ranging between 70 and 90 %. Malaria is highly prevalent in Tehama region with high transmission peaking between January and March each year. Figure 1 shows the study area and the distribution of malaria bur- den in Yemen in 2012. Early and accurate diagnosis of malaria, along with prompt treatment, are essential to reduce the burden of the disease worldwide. Rapid diagnostic tests (RDTs) have been widely used for the diagnosis of malaria and be- come an indispensable tool for malaria case-management, control and elimination worldwide, especially in rural en- demic areas without laboratory access [6, 7]. Besides the affordability and shorter turnover time of RDT-based diagnosis, significant reductions in the over-prescription of antimalarials have been reported when RDTs are in- troduced in presumptive treatment settings, especially with the new policy of using the expensive artemisinin- combination therapy (ACT) as the first line treatment for uncomplicated falciparum malaria infection [8]. However, recent studies revealed that the sensitivity of RDTs could be compromised due to genetic poly- morphism of the parasite PfHRP2 antigens, particularly with regards to certain amino acid repeat types, causing false-negative results when using the HRP2-based RDT to diagnose P. falciparum malaria [9–11]. Methods For positive slides, parasite species and stages were reported and parasitaemia (parasite density) was deter- mined by counting only the asexual stages against 300 white blood cells (WBC) and then multiplied by 25; assuming the average of total WBC count of individuals equal to 7500 cells per μl of blood. The level of parasit- aemia was graded as low (<1000 parasites/μl of blood), moderate (1000–9999 parasites/μl of blood) and severe (≥10,000 parasites/μl of blood). A double check for malaria microscopy was performed by two senior mal- aria microscopists; slides were examined twice and the Methods Study area An active case detection survey targeting individuals with fever suspected of having a malaria infection was carried out in some malaria endemic districts of the Tehama region in both the Hodeidah and Al-Mahwit governorates, Yemen. The survey was carried out from March to May 2014, during the malaria transmission season. Districts with high malaria endemicity; namely AdDahi, Al-Marawiah, and Bajil from Hodeidah, and Khamis Bani Saad from Al-Mahwit, were selected based Atroosh et al. Parasites & Vectors (2015) 8:388 Page 3 of 8 Fig. 1 A geographic map showing study area (Hodeidah and Al-Mahwit governorates) and the distribution of malaria in Yemen according to incidence in 2012 Fig. 1 A geographic map showing study area (Hodeidah and Al-Mahwit governorates) and the distribution of malaria in Yemen according to incidence in 2012 Evaluation of HRP2-RDT performance average parasitemia per microlitre of blood was re- corded for every microscopy positive slide. Genomic DNA was extracted from the filter paper blood spots and subjected to pfhrp2 amplification using conven- tional single-run PCR. p A malaria-positive blood sample, with a known parasite density (8500 asexual stage/μl) of P. falciparum mono- infection, was chosen to evaluate the CareStart™Malaria HRP2-RDT. The blood sample was also checked not to have any sexual stage (gametocytes). Duplicate thick and thin blood films were prepared and stained with Giemsa stain as previously mentioned, and then examined micro- scopically by two senior malaria microscopists; the average of parasitaemia was recorded. The sample was then diluted in a ratio of 2:3 serial dilutions with healthy human O positive blood group. A total of 12 serial dilutions (la- belled 1–12) were prepared in duplicates and the results of asexual-stage parasites density were recorded for each tube [14]. Each tube dilution was then tested for PfHRP2- RDT according to manufacturer instructions. The protocol used in this study was approved by the Ethics Committee of the University of Malaya Medical Centre, Malaysia (Ref. 974.19). The protocol was also ap- proved by the Ministry of Health and Population, in con- junction with the National Malaria Control Programme in Yemen. Written and signed or thumb-printed informed consents were taken from adult participants and parents or guardians on behalf of their children before starting the sample collection; these procedures were also approved by the ethics committees. Methods Study area RDT-positive participants were treated with artemisinin combination therapy (artemisinin + sulfadoxine/pyrimethamine) according to the national malaria treatment policy, Ministry of Health and Popula- tion, Yemen. Molecular identification and pfhrp2 sequencing All DNA from malaria positive samples were confirmed by PCR [15], with P. falciparum samples then being considered for pfhrp2 molecular characterization. Amp- lification of hrp2 was carried out in a single-run PCR using a specifically designed oligonucleotide primer pair flanking the region of exon-2 of pfhrp2 gene [16]. A 50 μL reaction mixture was made up containing 10 μM of each forward (PfHRP2-F 5′-TGTGTAGCAA AAATGCAAAAGG-3′) and reverse primer (PfHRP2-R 5′ TTAATGGCGTAGGCAATGTG-3′), along with 20 μL ExPrime Taq Premix ready-mix PCR reagent (Genet Bio, DNA extraction d One or two discs (6 mm diameter) of 3MM Whatman’s filter paper blood spot (cut by flamed-sterile punch) were used for DNA extraction using a Qiagen blood and tissue kit (QIAGEN, DNeasy® Blood & Tissue Kit, Cat. no. 69506, Germany) according to the manufacturer’s in- struction. DNA was eluted using 50 μl AE (10 mM Tris- Cl; 0.5 mM EDTA; pH 9.0) elution buffer (included in the kit) and kept at −20 °C until used. Page 4 of 8 Page 4 of 8 Atroosh et al. Parasites & Vectors (2015) 8:388 Korea) and 2 μL of the DNA extract. The amplification thermal conditions were initiated with DNA denaturation at 95 °C for 5 min, followed by 40 cycles of (95 °C/ 30 s, 57 °C/ 40 s and 72 °C/ 90 s) and a single extension step at 72 °C for 10 min. All PCR amplification reactions were amplified using thermal cycler (MyCycler- BioRad, Hercules, USA). Genomic DNA of P. falciparum lines 3D7 (MRA-102G), Dd2 (MRA-150G) and HB3 (MRA- 155G) provided by Malaria Research and Reference Re- agent Resource Centre (MR4), ATCC®, Manassas, VA, USA were used as positive and negative controls for PCR amplification of HRP2 and HRP3. 3D7 was used as a posi- tive control for both HRP2 and HRP3, and Dd2 (a labora- tory line known to lack pfhrp2) and HB3 (a laboratory line known to lack pfhrp3) were used as negative controls for HRP2 and HRP3 respectively [17, 18]. on microscopy, the detection rate was the highest in Khamis Bani Saad (39.4 %) followed by Bajil (23.5 %), while Al-Marawiah has the lowest (5.3 %). The level of asexual P. falciparum parasitaemia in positive cases ranged from 40 to 55,555 parasites/μl of blood with a geometric mean of 5261 parasites/μl. High parasitaemic individuals (parasite count ≥10,000 parasites/μl of blood) represented 17.9 %, while 41.9 % and 40.2 % of the malaria-positive individuals had moderate and low parasit- aemia respectively. The overall sensitivity and specificity of the used CareStart™malaria HRP2-RDT was rated as 90.5 % (95 % CI = 85.4, 94.3) and 96.1 % (95 % CI = 93.8, 97.7), respectively (Table 1). The PPV and NPV were 91.0 % (95 % CI = 85.9, 94.6) and 95.9 % (95 % CI = 93.5, 97.5), respectively. The agreement between the microscopy and HRP2-RDT was statistically significant by Kappa (K = 0.867; P < 0.001). DNA extraction d In the same vein, HRP2-RDT was examined against serial dilutions of a blood sample with known P. falciparum parasite density. The test re- vealed a high detection rate of CareStart™malaria HRP2-RDT for falciparum malaria isolates; 10 out of the 12 tubes with parasitaemia levels ranged from 8500 to 147 asexual stage/μl were found positive (visible clear band), with a faint band appearing for tube no.11 (98 parasites/μl). The last tube, no. 12, with parasit- aemia of 65 parasites/μl was found negative by HRP2- RDT (Table 2 and Additional file 1). With our Yemeni isolates, it was found that performance of RDT in- creased with parasitaemia level, with the lowest detec- tion rate (44.4 %) reported with parasitaemia levels of 1–100 parasites/μl while it was ≥97.0 % with parasit- aemia levels of ≥1000 parasites/μl. Although most of the negative RDT results were found in samples with low parasitaemia, but negative RDT results were also reported with two samples with moderate and high parasitaemia levels. The PCR products were then analysed using agarose- gel electrophoresis. Ten microliters of each amplicon was loaded into a 1.5 % agarose gel and run in a TAE buffer (Tris acetate EDTA), stained with SYBR® safe DNA gel stain (Invitrogen, USA). The fragments size was visualized under UV compared to 100 bp DNA ladder. The amplicons were sent for purification and sequen- cing, each amplicon was subjected to sequencing using the same forward and reverse primers as were used dur- ing PCR amplification. Both forward and reverse se- quences were aligned using BioEdit Sequence Alignment Editor Software (version 7.1.9) and then translated into corresponding amino acids. Each sequence of amino acid repeats was identified and given a code from Type 1 to Type 14 based on the motif being repeated [19]. Data analysis h f The performance of RDT was calculated based on the following indicators: sensitivity, specificity, positive pre- dictive value (PPV) and negative predictive values (NPV) which were calculated with their corresponding 95 % confidence intervals (CI) using Medcalc® online calcula- tor. Moreover, Kappa statistics were used to assess the agreement between RDT and microscopy. This was calculated in IBM SPSS Statistics, version 18.0 (IBM Corporation, NY, USA) by creating a 2 × 2 contingency table. A P-value of < 0.05 was considered significant. Overall, 180/189 cases were successfully amplified for pfhrp2 and yielded good and quality sequences, and were therefore subjected to amino acid repeat type ana- lysis. The sequence lengths of the isolates were found to Table 1 The sensitivity and specificity of PfHRP2-based RDT against the reference technique (microscopy) using samples collected from Hodeidah and Al-Mahwit, Yemen (n = 622) against the reference technique (microscopy) using samples collected from Hodeidah and Al-Mahwit, Yemen (n = 622) RDT Microscopy Negative Positive Total Negative 416 18 434 Positive 17 171 188 Total 433 189 622 Sensitivity: true positive = 171/189 = 90.5 %; false negative error rate = 18/189 = 9.5 %. Specificity: true negative = 416/433 = 96.1 %; false positive error rate = 17/ 433 = 3.9 %. PPV: 171/188 = 91.0 %; NPV: 416/434 = 95.9 % Results falciparum isolates from Hodeidah and Al-Mahwit, Yemen Type Amino acids sequence No. of repeats Meana Frequency minimum maximum n % 1 AHHAHHVAD 1 5 3.01 180 100 2 AHHAHHAAD 6 18 9.85 180 100 3 AHHAHHAAY 0 2 1.23 177 98.3 4 AHH 0 2 0.19 28 15.6 5 AHHAHHASD 0 2 0.49 85 47.2 6 AHHATD 2 8 4.11 180 100 7 AHHAAD 3 10 4.97 180 100 8 AHHAAY 0 2 0.52 89 49.4 9 AAY 0 0 0.00 0 0 10 AHHAAAHHATD 1 2 1.25 180 100 11 AHN 0 0 0.00 0 0 12 AHHAAAHHEAATH 1 1 1.00 180 100 13 AHHASD 0 1 0.05 10 5.6 14 AHHAHHATD 0 1 0.03 5 2.8 aAverage number of amino acid repeat types in the relevant isolates (n) Table 2 Evaluation of CareStart™PfHRP2-based RDT against serial dilutions of P. falciparum parasitaemia Tube No. Blood Volume Diluent (O+ Blood) Parasite Density RDT Result 0 Whole Blood 0 8500 Positive 1 200 μl of Tube No. 0 100 μl 5666 Positive 2 200 μl of Tube No. 1 100 μl 3777 Positive 3 200 μl of Tube No. 2 100 μl 2518 Positive 4 200 μl of Tube No. 3 100 μl 1679 Positive 5 200 μl of Tube No. 4 100 μl 1119 Positive 6 200 μl of Tube No. 5 100 μl 746 Positive 7 200 μl of Tube No. 6 100 μl 497 Positive 8 200 μl of Tube No. 7 100 μl 331 Positive 9 200 μl of Tube No. 8 100 μl 221 Positive 10 200 μl of Tube No. 9 100 μl 147 Positive 11 200 μl of Tube No. 10 100 μl 98 Positive 12 200 μl of Tube No. 11 100 μl 65 Negative Table 3 Frequency of PfHRP-2 repeat types from P. falciparum isolates from Hodeidah and Al-Mahwit, Yemen Type Amino acids sequence No. 18SrRNA [15], pfhrp2 and pfhrp3 genes [16]. The DNA was successfully amplified for 18SrRNA and pfhrp3 genes while it was confirmed negative for pfhrp2 gene. 18SrRNA [15], pfhrp2 and pfhrp3 genes [16]. The DNA was successfully amplified for 18SrRNA and pfhrp3 genes while it was confirmed negative for pfhrp2 gene. Results 5 100 μl 746 Positive 7 200 μl of Tube No. 6 100 μl 497 Positive 8 200 μl of Tube No. 7 100 μl 331 Positive 9 200 μl of Tube No. 8 100 μl 221 Positive 10 200 μl of Tube No. 9 100 μl 147 Positive 11 200 μl of Tube No. 10 100 μl 98 Positive 12 200 μl of Tube No. 11 100 μl 65 Negative Table 3 Frequency of PfHRP-2 repeat types from P. falciparum isolates from Hodeidah and Al-Mahwit, Yemen Type Amino acids sequence No. of repeats Meana Frequency minimum maximum n % 1 AHHAHHVAD 1 5 3.01 180 100 2 AHHAHHAAD 6 18 9.85 180 100 3 AHHAHHAAY 0 2 1.23 177 98.3 4 AHH 0 2 0.19 28 15.6 5 AHHAHHASD 0 2 0.49 85 47.2 6 AHHATD 2 8 4.11 180 100 7 AHHAAD 3 10 4.97 180 100 8 AHHAAY 0 2 0.52 89 49.4 9 AAY 0 0 0.00 0 0 10 AHHAAAHHATD 1 2 1.25 180 100 11 AHN 0 0 0.00 0 0 12 AHHAAAHHEAATH 1 1 1.00 180 100 13 AHHASD 0 1 0.05 10 5.6 14 AHHAHHATD 0 1 0.03 5 2.8 aAverage number of amino acid repeat types in the relevant isolates (n) vary, ranging from 477 to 879 bp (giving proteins of Table 2 Evaluation of CareStart™PfHRP2-based RDT against serial dilutions of P. falciparum parasitaemia Tube No. Blood Volume Diluent (O+ Blood) Parasite Density RDT Result 0 Whole Blood 0 8500 Positive 1 200 μl of Tube No. 0 100 μl 5666 Positive 2 200 μl of Tube No. 1 100 μl 3777 Positive 3 200 μl of Tube No. 2 100 μl 2518 Positive 4 200 μl of Tube No. 3 100 μl 1679 Positive 5 200 μl of Tube No. 4 100 μl 1119 Positive 6 200 μl of Tube No. 5 100 μl 746 Positive 7 200 μl of Tube No. 6 100 μl 497 Positive 8 200 μl of Tube No. 7 100 μl 331 Positive 9 200 μl of Tube No. 8 100 μl 221 Positive 10 200 μl of Tube No. 9 100 μl 147 Positive 11 200 μl of Tube No. 10 100 μl 98 Positive 12 200 μl of Tube No. 11 100 μl 65 Negative Table 3 Frequency of PfHRP-2 repeat types from P. Results Of the 622 screened individuals, 188 (30.2 %) were positive for malaria by the CareStart™Malaria HRP2- RDT. Of these 188 RDT-positive, only 171 (91.0 %) were confirmed microscopically by detecting either asexual or sexual stages or both. On the other hand, 18 (2.9 %) microscopy-positive individuals were found to be negative with RDT. Similarly, 17 (2.7 %) RDT-positive cases were found to be negative with microscopy. Based Sensitivity: true positive = 171/189 = 90.5 %; false negative error rate = 18/189 = 9.5 %. Specificity: true negative = 416/433 = 96.1 %; false positive error rate = 17/ 433 = 3.9 %. PPV: 171/188 = 91.0 %; NPV: 416/434 = 95.9 % Page 5 of 8 Atroosh et al. Parasites & Vectors (2015) 8:388 Page 5 of 8 vary, ranging from 477 to 879 bp (giving proteins of 159–293 amino acids), with a sequence of 540 bp (180 amino acids) being the most frequent genotype (43.9 %). All isolates sequences were found to be similar in that they started with either a single or multiple Type 1 (AHHAHHVAD), and ended with a single Type 12 repeat (AHHAAAHHEAATH). Overall, Type 1 (AHHAHH- VAD), Type 2 (AHHAHHAAD), Type 6 (AHHATD), Type 7 (AHHAAD), Type 10 (AHHAAAHHATD) and Type 12 (AHHAAAHHEAATH) were found in all se- quenced isolates. On the other hand, type 9 (AAY) and T 11 (AHN) t t ll b t i ll i l t ll t d 18SrRNA [15], pfhrp2 and pfhrp3 genes [16]. The DNA was successfully amplified for 18SrRNA and pfhrp3 genes while it was confirmed negative for pfhrp2 gene. Discussion Many rapid diagnostic tests (RDTs) are available to detect different malaria-specific antigens. Most RDTs detect P. falciparum specific proteins; either histidine- rich protein 2 (PfHRP2) or P. falciparum lactate de- h d (PfLDH) hil th i b th Table 2 Evaluation of CareStart™PfHRP2-based RDT against serial dilutions of P. falciparum parasitaemia Tube No. Blood Volume Diluent (O+ Blood) Parasite Density RDT Result 0 Whole Blood 0 8500 Positive 1 200 μl of Tube No. 0 100 μl 5666 Positive 2 200 μl of Tube No. 1 100 μl 3777 Positive 3 200 μl of Tube No. 2 100 μl 2518 Positive 4 200 μl of Tube No. 3 100 μl 1679 Positive 5 200 μl of Tube No. 4 100 μl 1119 Positive 6 200 μl of Tube No. Results of repeats Meana Frequency minimum maximum n % 1 AHHAHHVAD 1 5 3.01 180 100 2 AHHAHHAAD 6 18 9.85 180 100 3 AHHAHHAAY 0 2 1.23 177 98.3 4 AHH 0 2 0.19 28 15.6 5 AHHAHHASD 0 2 0.49 85 47.2 6 AHHATD 2 8 4.11 180 100 7 AHHAAD 3 10 4.97 180 100 8 AHHAAY 0 2 0.52 89 49.4 9 AAY 0 0 0.00 0 0 10 AHHAAAHHATD 1 2 1.25 180 100 11 AHN 0 0 0.00 0 0 12 AHHAAAHHEAATH 1 1 1.00 180 100 13 AHHASD 0 1 0.05 10 5.6 14 AHHAHHATD 0 1 0.03 5 2.8 aAverage number of amino acid repeat types in the relevant isolates (n) Table 3 Frequency of PfHRP-2 repeat types from P. falciparum isolates from Hodeidah and Al-Mahwit, Yemen vary, ranging from 477 to 879 bp (giving proteins of 159–293 amino acids), with a sequence of 540 bp (180 amino acids) being the most frequent genotype (43.9 %). All isolates sequences were found to be similar in that they started with either a single or multiple Type 1 (AHHAHHVAD), and ended with a single Type 12 repeat (AHHAAAHHEAATH). Overall, Type 1 (AHHAHH- VAD), Type 2 (AHHAHHAAD), Type 6 (AHHATD), Type 7 (AHHAAD), Type 10 (AHHAAAHHATD) and Type 12 (AHHAAAHHEAATH) were found in all se- quenced isolates. On the other hand, type 9 (AAY) and Type 11 (AHN) were totally absent in all isolates collected from both governorates. 18SrRNA [15], pfhrp2 and pfhrp3 genes [16]. The DNA was successfully amplified for 18SrRNA and pfhrp3 genes while it was confirmed negative for pfhrp2 gene. Discussion Overall, 12 different amino acid repeat types were identified in Yemeni isolates examined by the present study. Of these, six repeat types (types 1, 2, 6, 7, 10 and 12) were found to be present in all isolates, while type 3 was found in 98.3 % of the isolates. These findings are in agreement with isolates from different countries in Africa, Asia and America [21]. In India, six (types 1, 2, 3, 6, 7 and 11) out of 13 identified types of amino acid re- peats were detected in all the examined P. falciparum isolates [25]. On the other hand, only four types were found in over 98 % of the Senegalese isolates from Dakar [10, 11]. Our study further showed that repeat types 9 (AAY) and 11 (AHN) were totally absent, a result which has been previously reported in isolates obtained from Senegal, Mali, Uganda and Madagascar [10, 11, 16]. We found that all of the amplified isolates (100 %) started with a type 1 repeat (AHHAHHVAD), with ei- ther a single or multiple of 2 to 5 copies, before ending with a single Type 12 repeat (AHHAAAHHEAATH). Many previous studies in Africa and Asia showed that all pfhrp2 sequences begin with a type 1 repeat and concluded with a type 12 repeat [11, 16, 19, 25]. How- ever, a previous study from Dakar, Senegal on 122 sequenced P. falciparum isolates had detected three isolates for which pfhrp2 protein sequences did not begin with a type 1 repeat, and only one isolate found in that study possessed a sequence ending in a type 12 repeat [10]. Overall, the present study showed that the sensitivity and specificity of the used HRP2-based RDT were high (90.5 % and 96.1 %, respectively). This was supported by a very good level of agreement between the results of microscopy and RDT tests (Kappa = 0.867). The sensitiv- ity of PfHRP2-based RDT from other reports varied worldwide, ranging from > 90 % (high) to 43.7 % (low) compared to the gold standard microscopy and/or PCR [30–34]. Within the same context, a previous study conducted at the eastern part of Yemen during a malaria outbreak aimed to evaluate the accuracy of PfHRP2- based RDT among 25 falciparum malaria patients, revealed a sensitivity, specificity and positive predictive value of 74 %, 94 % and 68 % respectively when com- pared to the microscopy [35]. Discussion Parasites & Vectors (2015) 8:388 the same country [16, 19]. Yemeni isolates showed high to moderate numbers of the repeat types 2, 7, 6 and 1 within pfhrp2 (means: 9.85, 4.97, 4.11 and 3.01, respect- ively) which is higher than what it has been reported in Thailand, the Philippines, Madagascar, Papua New Guinea and South America [16, 19, 26]. were successfully amplified and sequenced for PfHRP2. The sequence lengths of the sequenced isolates varied from 477 to 879 bp (giving proteins of 159 to 293 amino acids). The variations in the number of total amino acids for pfhrp2 for the Yemeni isolates were found to be lower than the global variation (187 to 306 amino acids), as reported for isolates from 19 countries in Africa, South America, the Pacific region and Southeast Asia [19]. On the other hand, similar variations were reported in Madagascar (145 to 309 amino acids), while a higher number of variations (157 to 333 amino acids) were reported in isolates collected from six different geo- graphical areas in India [16, 25). Our findings revealed that the HRP2-based RDT pos- sessed good sensitivity; a low parasitaemia level of 139 asexual stages/μl was strongly detectable by RDT. On the other hand, parasitaemia levels below 100 parasites/ μl were found either weakly detectable (98 parasites/μl) or totally not detected (65 parasites/μl). These results correspond to RDT results on the Yemeni isolates as we found that detection rate increased with parasitaemia level. However, low detection rate (44.4 %) was reported with samples of very low parasitaemia (i.e., < 100 para- sites/μl), as well as two samples (positive with microscopy and pfhrp2-PCR) with moderate and high parasitaemia were found negative. These are in agreement with previ- ous studies which revealed that most of the RDT show ex- cellent detection rates for P. falciparum at a parasitaemia greater than 500 parasites/μl, with most of the variation reported at relatively low-level parasitaemia [7, 10, 21]. Moreover, RDT negative results were reported with sam- ples of high-level parasitaemia [27, 28]. This variation could be attributed to either RDT device-related factors such as poor manufacture and deterioration of the device or parasite-related factors such as the level of parasitemia, variability in the target epitopes of the parasite antigen, or quantity of parasite antigen produced by the parasite or present in the peripheral blood, or malaria transmission season [7, 19, 29]. Discussion Many rapid diagnostic tests (RDTs) are available to detect different malaria-specific antigens. Most RDTs detect P. falciparum specific proteins; either histidine- rich protein 2 (PfHRP2) or P. falciparum lactate de- hydrogenase (PfLDH), while others can recognize both P. falciparum-specific and pan-specific antigens (aldol- ase (pALD) and pan-species (pLDH) [20]. Previous studies have reported genetic variation in PfHRP2 (gen- etic deletions, frame shift mutations or alterations in protein expression), which can affect the sensitivity of HRP2-based RDTs, while no variability was observed for pAldolase or pLDH [21, 22]. The HRP2 of P. falcip- arum is a 2-exons gene connected with an intron, located on chromosome 7 (98671–99734 bp) [23]. The amino acid repeats of pfhrp2 have been characterized into 14 types, based on amino acid motifs being re- peated, with Type 2 and Type 7 having been described as possible epitopes targeted by the monoclonal anti- bodies used to detect hrp2 [10, 19, 21, 24] Other amino acid repeat types were varied in their frequencies. Type 8 (AHHAAY), Type 5 (AHHAHHASD) and Type 4 (AHH) ranged between moderate and low, with percentages of 49.4 %, 47.2 % and 15.6 % respectively. Moreover, Type 13 (AHHASD) and Type 14 (AHHAH- HATD) were rarely reported in the present study (5.6 % and 2.8 %, respectively). Further, the mean numbers for 2 and 7 repeat types in pfhrp2 were the highest among the 12 types detected by this study (mean = 9.85 and 4.97, respectively). No association was found between HRP2 repeats and age, sex and districts of participants as well as parasitaemia level. The frequency and mean number of PfHRP2 repeat types from P. falciparum isolates from Hodeidah and Al-Mahwit, Yemen are shown in Table 3. The present study investigated the genetic variation of PfHRP2 among isolates from Yemen, with a possible predicting effect on the performance of the PfHRP2- based RDTs (CareStart® one-step HRP2 RDT; Access Bio Inc., New Jersey, USA) that are used solely for active case detection (ACD) by the national malaria control programme in Yemen (NMCP). A total of 180 isolates Interestingly, one isolate with low parasitaemia (184 parasites/μl) was found to be positive by both CareStart™ malaria HRP2-RDT and microscopy but it was PCR- negative for pfhrp2 gene. DNA of this isolate was processed for further PCR confirmation using three PCR protocols that aimed at amplifying P. falciparum Page 6 of 8 Atroosh et al. Conclusions The findings of this study provide insights into the gen- etic diversity of pfhrp2 in P. falciparum isolates from Yemen, and reveal that pfhrp2 is highly polymorphic in these isolates. CareStart™one-step HRP2-based RDT showed high performance especially for those cases with parasitaemia of >100 parasites/μl. The isolates used by this study were from endemic areas of the Tehama re- gion only. Therefore, population-based studies from other endemic areas throughout Yemen are recom- mended in order to genetically analyze the P. falciparum isolates based on PfHRP2, and to accurately determine the prevalence of parasites that cannot be detected using the PfHRP2 RDT. 4. Partnership RBM. The Global Malaria Action Plan for a Malaria Free World. Geneva: RBMP; 2008. 5. Malaria in Saudi Arabia. Middle East Health. 2013. http:// www.middleeasthealthmag.com/jul2013/meupdate.htm. Accessed 13 Oct 2014. 6. Murray CK, Bennett JW. Rapid diagnosis of malaria. Interdiscip Perspect Infect Dis. 2009;415953. doi:10.1155/2009/415953. 7. World Health Organization. Malaria rapid diagnostic test performance results of WHO product testing of malaria RDTs: round 3 (2010–2011). Geneva: WHO; 2011. 8. Bastiaens GJH, Bousema T, Leslie T. Scale-up of malaria rapid diagnostic tests and artemisinin- based combination therapy: challenges and perspectives in sub-Saharan Africa. PLoS Med. 2014;11(1):e1001590. doi:10.1371/journal.pmed.1001590. 9. Koita O, Doumbo OK, Ouattara A, Tall LK, Konaré A, Diakité M, et al. False- negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene. Am J Trop Med Hyg. 2012;86(2):194–8. doi:10.4269/ajtmh.2012.10-0665. 9. Koita O, Doumbo OK, Ouattara A, Tall LK, Konaré A, Diakité M, et al. False- negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene. Am J Trop Med Hyg. 2012;86(2):194–8. doi:10.4269/ajtmh.2012.10-0665. Author details 1 1Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2Department of Microbiology and Parasitology, Faculty of Medicine and Health Sciences University of Aden, Aden, Yemen. 3Azal National Research Centre, Azal University for Human Development, 447 Sana’a, Yemen. 4Department of Parasitology, Faculty of Medicine and Health Sciences, Sana’a University, 1247 Sana’a, Yemen. 5National Malaria Control programme, Ministry of Public Health and Population, Sana’a, Yemen. Received: 13 May 2015 Accepted: 13 July 2015 Received: 13 May 2015 Accepted: 13 July 2015 Discussion Moreover, the HRP2 PCR protocol was tested against falciparum malaria DNA samples with the same and lower parasite densities which all yielded successful amplification for HRP2 gene suggesting PfHRP2 gene deletion. Due to pfhrp3 and pfhrp2 structural homology, pfhrp3 can cross-react with HRP2-coated antibodies in the RDT [36], and this may explain the false positive PfHRP2-RDT result by our study. On the other hand, pfhrp2 gene deletion was reported worldwide and more extensively from South America. It was first reported among Peruvian isolates; 41 % of the malaria-microscopically positive isolates have been found negative by RDT, and failed to amplify the PfHRP2 gene by PCR [17]. Later, studies from Brazil and Peru [22, 37, 38], Mali in Africa [9], and from India in Asia have reported false negative RDT results due to a pfhrp2 gene deletion [25]. management. We thank Malaria Research and Reference Reagent Resource Centre (MR4), ATCC®, Manassas, VA, USA for providing us with Plasmodium falciparum parasite lines 3D7 (MRA-102G), HB3 (MRA-155G) and Dd2 (MRA- 150G) deposited by MR4. The work presented in this paper was funded by University of Malaya High Impact Research Grant UM-MOHE (UM.C/625/1/ HIR/MOHE/MED/18) from the Ministry of Higher Education Malaysia. Competing interests 11. Deme A, Park DJ, Bei AK, Sarr O, Badiane AS, Gueye Pel H, et al. Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests. Malar J. 2014;13:34. doi:10.1186/1475-2875-13-34. 11. Deme A, Park DJ, Bei AK, Sarr O, Badiane AS, Gueye Pel H, et al. Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests. Malar J. 2014;13:34. doi:10.1186/1475-2875-13-34. The authors declare that they have no competing interests. Additional file Additional file 1: Performance of CareStart™malaria HRP2-RDT against serial dilutions of parasite densities. 10. Wurtz N, Fall B, Bui K, Pascual A, Fall M, Camara C, et al. Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests. Malar J. 2013;12:34. doi:10.1186/ 1475-2875-12-34. 10. Wurtz N, Fall B, Bui K, Pascual A, Fall M, Camara C, et al. Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests. Malar J. 2013;12:34. doi:10.1186/ 1475-2875-12-34. Discussion However, the small sample size used in the above study, as well as the potential genetic variation in isolates from the eastern part of Yemen collected during an outbreak when compared to the isolates in this study from malaria endemic areas in western Yemen should be taken into consideration. The present study also showed that the existence of other repeat types in Yemeni isolates varied from mod- erate (types 8; 49.4 % and 5; 47.2 %) to low (type 4; 15.6 %). Moreover, other types were found only rarely (types 13; 5.6 % and 14; 2.8 %). Our findings are consist- ent with a previous report on Senegalese P. falciparum isolates [10]. By contrast, repeat types 11 and 14 were not detected in isolates from Senegal, Mali and Uganda, while type 4 repeats were detected in all of the isolates [11]. Furthermore, Ugandan isolates were found to be different in that they all contained type 8 (100 %), and lacked type 5 repeats [11]. Moreover, the number of each motif and the total number of repeats within pfhrp2 vary considerably between countries and within In the present study, one sample was found positive for falciparum malaria by microscopy and HRP2-RDT Page 7 of 8 Page 7 of 8 Atroosh et al. Parasites & Vectors (2015) 8:388 while it was negative by PfHRP2-PCR. The DNA quality was confirmed by successful PCR amplification of both falciparum 18S rRNA and pfhrp3 genes. Moreover, the HRP2 PCR protocol was tested against falciparum malaria DNA samples with the same and lower parasite densities which all yielded successful amplification for HRP2 gene suggesting PfHRP2 gene deletion. Due to pfhrp3 and pfhrp2 structural homology, pfhrp3 can cross-react with HRP2-coated antibodies in the RDT [36], and this may explain the false positive PfHRP2-RDT result by our study. On the other hand, pfhrp2 gene deletion was reported worldwide and more extensively from South America. It was first reported among Peruvian isolates; 41 % of the malaria-microscopically positive isolates have been found negative by RDT, and failed to amplify the PfHRP2 gene by PCR [17]. Later, studies from Brazil and Peru [22, 37, 38], Mali in Africa [9], and from India in Asia have reported false negative RDT results due to a pfhrp2 gene deletion [25]. while it was negative by PfHRP2-PCR. The DNA quality was confirmed by successful PCR amplification of both falciparum 18S rRNA and pfhrp3 genes. Authors’ contributions 12. World Health Organization. Report on the regional workshop on strengthening quality management systems for parasitological diagnosis of malaria. Geneva: WHO; 2011. 12. World Health Organization. Report on the regional workshop on strengthening quality management systems for parasitological diagnosis of malaria. Geneva: WHO; 2011. Conceived and designed the experiments: WMA HMA JS LYL. Sampling and perform the lab. experiments: WMA HS AKA NAN SD AMA. Analyzed the data and wrote the paper: WMA HMA. Provided logistic support for data collection and field work: AA. Revised the manuscript critically for important intellectual content: HMA JS II LYL FMY. All authors read and approved the final manuscript. Conceived and designed the experiments: WMA HMA JS LYL. Sampling and perform the lab. experiments: WMA HS AKA NAN SD AMA. Analyzed the data and wrote the paper: WMA HMA. Provided logistic support for data collection and field work: AA. Revised the manuscript critically for 13. National Information Centre, Yemen. http://www.yemen-nic.info/sectors/ popul. Accessed 18 Mar 2015. important intellectual content: HMA JS II LYL FMY. All authors read and approved the final manuscript. 14. Avila PE, Kirchgatter K, Brunialti KC, Oliveira AM, Siciliano RF, Di Santi SM. Evaluation of a rapid dipstick test, Malar-CheckÔ, for the diagnosis of Plasmodium falciparum malaria in Brazil. Rev Inst Med Trop Sao Paulo. 2002;44(5):293–6. References 1 W ld H 1. World Health Organization. World Malaria Report 2014. Geneva: WHO; 2014. 2 Al M khl fi AM M hd MA Al M khl fi HM A AA F MY Hi h 1. World Health Organization. World Malaria Report 2014. Geneva: WHO; 2014. 2 Al M khl fi AM M hd MA Al M khl fi HM A AA F MY Hi h 1. World Health Organization. World Malaria Report 2014. Geneva: WHO; 2014. 2 Al Mekhlafi AM Mahdy MA Al Mekhlafi HM Azazy AA Fong MY High 2. Al-Mekhlafi AM, Mahdy MA, Al-Mekhlafi HM, Azazy AA, Fong MY. High frequency of Plasmodium falciparum chloroquine resistance marker (pfcrt T76 mutation) in Yemen: an urgent need to re-examine malaria drug policy. Parasit Vectors. 2011;4:94. doi:10.1186/1756-3305-4-94. 3. Abdul-Ghani R, Farag HF, Allam AF, Shawky SM. Prevailing Plasmodium falciparum dihydrofolate reductase 108-asparagine in Hodeidah, Yemen: A questionable sulfadoxine-pyrimethamine partner within the artemisinin- based combination therapy. Acta Trop. 2014;132:39–44. doi:10.1016/ j.actatropica.2013.12.022. Acknowledgments 15. Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiological studies. Am J Trop Med Hyg. 1999;60(4):687–92. We gratefully acknowledge the National Malaria Control Programme (NMCP) staff for their generous cooperation during this study. The authors are very grateful to Mr. Khaled Al-Mansab of the NMCP for providing the map of the malaria distribution in Yemen in 2012. The authors are also very thankful to Bajil malaria team represented by Dr. Yehea A. Aamer, Fathya S. Bukair and Fatima Y. Hasan for their fruitful help in samples collection and malaria cases 16. Mariette N, Barnadas C, Bouchier C, Tichit M, Menard D. Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and 16. Mariette N, Barnadas C, Bouchier C, Tichit M, Menard D. Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Page 8 of 8 Page 8 of 8 Atroosh et al. Parasites & Vectors (2015) 8:388 field treatment in an outbreak of falciparum malaria. Trop Parasitol. 2012;2(1):35–7. doi:10.4103/2229-5070.97237. Plasmodium vivax antigens detected with rapid diagnostic tests for malaria. 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Acknowledgments 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: Submit your next manuscript to BioMed Central and take full advantage of: 32. 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https://openalex.org/W3120468023	https://ora.ox.ac.uk/objects/uuid:05d0b1d0-61e8-45b1-8969-23c0611420bd/files/rb8515n68w	English	null	Detection and attribution of precipitation trends associated with the poleward shift of the South Atlantic Convergence Zone using CMIP5 simulations	International journal of climatology	2,021	cc-by	14,723	K E Y W O R D S anthropogenic forcing, CMIP5 models, natural variability, precipitation trend, South Atlantic Convergence Zone 1 \| INTRODUCTION The austral summer (December–February, DJF) precipi- tation over tropical South America (SA) is strongly linked to the South American Monsoon System (SAMS) and the South Atlantic Convergence Zone (SACZ; Zhou and Lau, 1998; Barros et al., 2000; Zhou and Lau, 2001; Carvalho et al., 2004; 2011; Vera et al., 2006a; Muza et al., 2009). This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. International Journal of Climatology published by John Wiley & Sons Ltd on behalf of the Royal Meteorological Society. Detection and attribution of precipitation trends associated with the poleward shift of the South Atlantic Convergence Zone using CMIP5 simulations Marcia T. Zilli1,2 \| Leila M. V. Carvalho1,3 1Department of Geography, University of California, Santa Barbara, California 2School of Geography and the Environment, University of Oxford, Oxford, UK Abstract Abstract Observational studies demonstrate a poleward shift in the South Atlantic Conver- gence Zone (SACZ) in recent decades with regional impacts on extreme precipita- tion. However, the relative contribution of anthropogenic forcing and natural variability to the observed trends is presently unknown. To evaluate the main forcings associated with the recent trends in precipitation rate consistent with the poleward shift of the SACZ, this study examines the last 40 years of the historical scenario of 20 global climate models participating in the fifth phase of the Coupled Model Intercomparison Project (CMIP5). The relative contribution of natural variability and anthropogenic-related forcings to precipitation in the 20th century is assessed based on historical, natural, anthropogenic, and pre-industrial simulations from CMIP5 models. Over the study area, precipitation trends simu- lated by the CMIP5 historical scenario are small, with large disagreement among models and members. Despite the small magnitude of the trends, the spatial pat- tern emerging is coherent with a poleward displacement of the SACZ, as indi- cated by a reduction (increase) in the precipitation rate over Southeastern Brazil (Southeastern South America). Even with large discrepancies among models, the attribution analysis suggests that precipitation trends could partially result from land-use change, followed by changes in ozone concentrations, with more signifi- cant influence over Southeastern Brazil. The large uncertainty in the simulated precipitation suggests that not all mechanisms related to the position and inten- sity of the SACZ events are well captured by the CMIP5 models considered here. 3Earth Research Institute, University of California, Santa Barbara, California Correspondence Marcia T. Zilli, School of Geography and the Environment, University of Oxford, Oxford, OX1 3QY, UK. Email: marcia.zilli@ouce.ox.ac.uk Correspondence Correspondence Marcia T. Zilli, School of Geography and the Environment, University of Oxford, Oxford, OX1 3QY, UK. Email: marcia.zilli@ouce.ox.ac.uk Correspondence Marcia T. Zilli, School of Geography and the Environment, University of Oxford, Oxford, OX1 3QY, UK. Email: marcia.zilli@ouce.ox.ac.uk wileyonlinelibrary.com/journal/joc Received: 1 December 2019 Revised: 20 November 2020 Accepted: 8 January 2021 Received: 1 December 2019 Revised: 20 November 2020 Accepted: 8 January 2021 Received: 1 December 2019 Revised: 20 November 2020 Accepted: 8 January 2021 DOI: 10.1002/joc.7007 DOI: 10.1002/joc.7007 R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Int J Climatol. 2021;1–22. 1 \| INTRODUCTION The austral summer (December–February, DJF) precipi- tation over tropical South America (SA) is strongly linked wileyonlinelibrary.com/journal/joc Int J Climatol. 2021;1–22. 2 ZILLI AND CARVALHO 2 Coupled Model Intercomparison Project (CMIP5). Models and observations agree that trends are larger over central-eastern Brazil (Carvalho and Jones, 2013; de Bar- ros Soares et al., 2017). The rapid rate of warming cannot be explained by natural variability alone, suggesting that the contribution of anthropogenic-related forcings is already detectable (de Barros Soares et al., 2017). As expected for a warmer troposphere, the water vapour content is also increasing, particularly over the regions with larger warming (Carvalho and Jones, 2013) with implications for duration and amplitude of the SAMS (Jones and Carvalho, 2013). The SACZ is a diagonal convergence zone extending from the Amazon toward west South Atlantic Ocean and is responsible for advecting moisture to the subtropical SA (Kodama, 1992; 1993; Zhou and Lau, 1998; Liebmann et al., 2001; Carvalho et al., 2002; 2004; Marengo et al., 2012). It is characterized by the convergence of low-level northerly inflow originating along the western periphery of the South Atlantic Subtropical High (SASH) and west- erly flows along the convergence zone, increasing in intensity with height and forming part of the subtropical jet in upper levels (Kodama, 1992; 1993). The spatial variability of the SACZ is dominated by a dipole-like pattern of opposing precipitation anomalies over Eastern Brazil (EBr) and Southeastern South America (SESA), recurrent on timescales ranging from intra- seasonal to decadal (Nogues-Paegle and Mo, 1997; Zhou and Lau, 1998; Nogués-Paegle et al., 2000; Carvalho et al., 2002; 2011; Cunningham and Cavalcanti, 2006; Grimm and Zilli, 2009; Grimm and Saboia, 2015). During the SACZ active phase, precipitation is enhanced (inhibited) over EBr (SESA); the opposite is observed during its inac- tive phase (Zhou and Lau, 1998; Nogués-Paegle et al., 2000; Carvalho et al., 2002; Jones and Carvalho, 2002; Grimm and Zilli, 2009). On intraseasonal timescales, the SACZ cir- culation and convective activity are modulated by the Mad- den-Julian Oscillation (MJO) and by the Pacific South America (PSA) mode (Mo and Nogues-Paegle, 2001; Car- valho et al., 2004; 2011; Cunningham and Cavalcanti, 2006). Observational studies (Zilli et al., 2017; 2019) and his- torical and future scenarios of CMIP3 and CMIP5 models simulations (Seth et al., 2010; Junquas et al., 2012; Jones and Carvalho, 2013; Díaz and Vera, 2017) identified evi- dence of changes in the SACZ-related dipole. 1 \| INTRODUCTION Using future changes in sea surface temperature (SST) derived from the CMIP3 model ensemble to force a Regional Climate Model (RCM) interactively coupled (i.e., using a two-way nesting technique) to an Atmospheric Global Climate Model (AGCM), Junquas et al. (2013) demonstrated a prevalence of SACZ inactive phase as a response to an asymmetric increase in SST over the Indian and equato- rial western Pacific. The asymmetric equatorial warming generates a Rossby wave-train pattern that propagates toward South America, favouring cyclonic (anticyclonic) anomalies and consequent increase (decrease) in precipi- tation over SESA (SACZ). On decadal timescales, the dipole is partially influenced by the Interdecadal Pacific Oscillation (IPO) and the Atlan- tic Multidecadal Oscillation (AMO; Grimm and Saboia, 2015; Jones and Carvalho, 2018). Previous studies suggest that during AMO positive (negative) phase, cold (warm) sea surface temperature anomalies over South Atlantic Ocean reduce (increase) the intensity of cyclones over the region (Kayano et al., 2019), which in turn decrease (increase) the SACZ activity, reducing precipitation over EBr (Chiessi et al., 2009; Bombardi et al., 2013). Moreover, Jones and Carvalho (2018) have shown that the AMO mod- ulates the decadal-to-multidecadal activity of the South American Low-Level Jet (LLJ), with the negative AMO phase related to enhanced precipitation over Paraguay, southern Brazil, and northern Argentina, favouring the inactive phase of the SACZ-dipole. However, this same study showed that the differences in precipitation between AMO phases are exacerbated in area and intensity when trends are not removed, suggesting that the AMO cannot explain the complete variability in rainfall in the SACZ domain. The increase in total precipitation over SESA, the sub- tropical portion of the SACZ dipole, has been identified in observational studies and is mostly related to an increase in the number of intense events and a reduction in the number of consecutive dry days over the region (Haylock et al., 2006; Barros et al., 2008; Skansi et al., 2013; Zilli et al., 2017; 2019). These trends, as well as the precipita- tion climatology over the region, are correctly reproduced by historical scenarios of coupled GCMs and RCM models (Vera et al., 2006b; Chou et al., 2014; Díaz and Vera, 2017) and have been attributed to the anthropogenic-related increase in the concentration of well-mixed greenhouse gases (Zhang et al., 2016) and stratospheric ozone deple- tion (Polvani et al., 2011; Gonzalez et al., 2014). 1 \| INTRODUCTION Previous observational studies also detected negative trends in precipitation over EBr, the tropical portion of the SACZ dipole (Zilli et al., 2017; 2019). Zilli et al. (2017) detected changes in the observed precipitation distribu- tion over Southeastern Brazil, with fewer rainy days and more intense events observed in recent decades along the northern margin of the SACZ during the rainy season (October to March). Zilli et al. (2019) related the reduc- tion in precipitation rate over EBr to the weakening of the northerly winds and decrease in the available The increase in low tropospheric temperatures over the SA during the last decades of the 20th century has been consistently shown in reanalyses and global climate models (GCM) contributing to the fifth phase of the ZILLI AND CARVALHO 3 moisture at the mid-to-lower troposphere (700 hPa) co- located with the climatological position of the SASH. These changes in circulation and low-level moisture have reduced convergence and moisture advected to EBr. Using an Atmospheric Global Climate Model (AGCM), Talento and Barreiro (2017) identified negative precipita- tion trends and related circulation changes over tropical Brazil as a response to extratropical warming over the northern hemisphere, similar to the atmospheric warming observed over the last decades. The hemispheric warming has been associated with changes in the Hadley circulation, resulting in weakened trade winds along the equator and enhanced SASH, increasing subsidence over the northern margin of the SACZ. Together with the well-documented positive precipitation trends over SESA, these results suggest a poleward shift in the SACZ posi- tion over recent decades (Zilli et al., 2019). However, there are significant discrepancies among the CMIP5 his- torical and future projections over the region, typically because the models do not accurately reproduce the pre- cipitation variability related to the SACZ position, inten- sity and seasonal variability (Carvalho and Jones, 2013; de Barros Soares et al., 2017; Díaz and Vera, 2017). merges directly observed precipitation from rain gauge over land with various satellite-based estimates (Adler et al., 2018). Its previous version (GPCP V2.1) accurately reproduces the spatial and temporal precipitation vari- ability related to the SAMS (Carvalho et al., 2012). The simulated precipitation is from various GCMs selected from the CMIP5 archive (Taylor et al., 2012) avail- able through the Program for Climate Model Diagnosis and Intercomparison (PCMDI). 1 \| INTRODUCTION Precipitation trends are detected in the historical scenario (HIST) of 20 CMIP5 GCM, considering all available ensemble members (Table 1) and resulting in 76 simulations. The attribution analysis is based on the external forcing scenarios, divided into nat- ural and anthropogenic-related. The external natural forc- ing influence is accessed through the natural scenario (NAT), in which the atmospheric composition is kept con- stant at pre-industrial levels and external forcings are only due to changes in incoming solar radiation and strato- spheric aerosol from volcanic sources. The anthropogenic influence can be estimated using four single-forcing scenar- ios: anthropogenic-related changes in greenhouse gas con- centration (GHG); anthropogenic aerosol emissions (AA), which may include direct and indirect effects; anthropo- genic-related changes in tropospheric and stratospheric ozone concentration (OZ), and the influence of land-use changes (LU). Table 2 describes these scenarios and lists the models utilized in each case. A more in-depth discus- sion of these scenarios can be found in Taylor et al. (2012). The objective of this study is to evaluate the contribu- tion of natural variability and anthropogenic-related forc- ings to precipitation trends in the SACZ-related dipole, associated with a poleward shift of the convergence zone in the 20th century. The attribution analysis is conducted using single forcing scenarios from the CMIP5 archive. Challenges due to large uncertainties related to the cli- mate system internal variability and models' representa- tion of the climate system are accounted for by evaluating multiple CMIP5 ensemble members. This study is orga- nized as follows: dataset and methodology are described in Section 2. The observed and simulated DJF precipitation climatology are presented in Section 3. Section 4 discusses the trends in recent decades, while Section 5 focuses on the attribution analysis, emphasizing the influence of nat- ural internal variability and external forcings on the simu- lated trends. The main results are discussed in Section 6 and conclusions are presented in Section 7. In addition to external forcings, simulated trends can ensue from natural internal variability intrinsic to the cli- mate system, represented by climatic modes such as ENSO, AMO, IPO, among others. Here, we use the pre- industrial control run (PICONTROL) as a proxy for this variability. In the PICONTROL experiment, all external forcings are kept constant at pre-industrial levels, with the simulated precipitation variability and its trends related to interactions between the different components of the climate system (Taylor et al., 2012). 1 \| INTRODUCTION The results described are based on the analysis of individual ensemble members of each model and scenario, their averages, which are referred to as Multi-Member Mean (MMeM), and the average of all MMeM, forming the Multi-Model Mean (MMoM) for each scenario. With this approach, each MMeM have the same weight in the MMoM, independent of their number of members. 2.2 \| Precipitation trend detection This study compares precipitation trends simulated by CMIP5 climate models with those identified in observa- tions. The observational dataset is from the Global Pre- cipitation Climatology Project Version 2.3 (GPCP; Adler et al., 2016; 2018), a monthly dataset with 2.5 lon/lat horizontal resolution and spanning 1979–2019. GPCP To analyse how CMIP5 models simulate the location and intensity of the SACZ, we compare the austral summer (December to February—DJF) average of daily precipita- tion rate (PP) and its trend (τ) from GPCP with respective ZILLI AND CARVALHO TABLE 1 CMIP5 models, institutes, resolution, and references Model Institute and country Resolution Reference A ACCESS1-3 Centre for Australian Weather and Climate Research (CAWCR), Australia 1.875×1.25 (N96) Bi et al. (2013) B BCC-CSM1-1 Beijing Climate Center (BCC), China Meteorological Administration, China 1.89×1.89 (T42L26) Wu et al. (2013) C CanESM2 Canadian Centre for Climate Modelling and Analysis (CCCMA), Canada 2.8×2.8 (T63L35) Arora et al. (2011) D CCSM4 National Center for Atmospheric Research (NCAR), USA 1.25×0.9 (F09G16) Gent et al. (2011) E CMCC-CESM Centro Euro-Mediterraneo per i Cambiamenti Climatici, Bologna, Italy 3.75×3.75 Vichi et al. (2011) F CNRM-CM5 Centre National de Recherches Météorologiques (CNRM) and Centre Européen de Recherche et de Formation Avancée) (CERFACS), France 1.4×1.4 (TL127L31) Voldoire et al. (2013) G CSIRO_Mk3-6- 0 Centre for Australian Weather and Climate Research (CSIRO) and Queensland Climate Change Centre of Excellence, Australia 1.875×1.875 (T63) Jeffrey et al. (2013) H FGOALS-g2 Institute of Atmospheric Physics, Chinese Academy of Sciences, Beijing, China (IAP) and Tsinghua University (THU) 2.1825×3.0 (L62) Li et al. (2013) I GFDL-ESM2M Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric Administration (GFDL/ NOAA), USA 2.5×2 (M45L24) Dunne et al. (2012) J GISS-E2H Goddard Institute for Space Studies, National Aeronautics and Space Administration (GISS/NASA), USA 2.5×2.0 Schmidt et al. (2014) K HadGEM2-CC Met Office Hadley Centre, UK 1.875×1.25 (N96L60) Collins et al. (2011) Martin et al. (2011) L HadGEM2-ES Met Office Hadley Centre, UK 1.875×1.25 (N96L38) Collins et al. (2011) Jones et al. (2011) M INMCM4 Institute for Numerical Mathematics (INM), Russia 2.0×1.5 Volodin et al. (2010) N IPSL-CM5A-LR Institut Pierre Simon Laplace (IPSL), France 3.75×1.875 Dufresne et al. (2013) O MIROC4h Atmosphere and Ocean Research Institute (AORI) of the University of Tokyo, National Institute for Environmental Studies (NIES), and Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Japan 0.5625×0.5625 (T213L56) Sakamoto et al. 2.2 \| Precipitation trend detection (2012) P MIROC5 Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba, Japan (AORI), National Institute for Environmental Studies, Ibaraki, Japan (NIES), Japan Agency for Marine-Earth Science and Technology, Kanagawa, Japan (JAMSTEC) 1.40625×1.40625 Watanabe et al. (2010) Q MIROC-ESM Atmosphere and Ocean Research Institute (The University of Tokyo), National Institute for Environmental Research, and Japan Agency for Marine-Earth Science and Technology, Japan 2.8125×2.8125 (T42L80) Watanabe et al. (2011) R MPI-ESM-LR Max Planck Institute for Meteorology (MPI-M), Germany 1.875×1.875 (T63L47) Giorgetta et al. (2013) S MRI-CGCM3 Meteorological Research Institute, Japan 1.125×1.125 (TL159L48) Yukimoto et al. (2012) T NorESM1-M Norwegian Climate Centre (NorClim), Norway 2.5×1.875 (F19L26) Bentsen et al. (2013) TABLE 1 CMIP5 models, institutes, resolution, and references ZILLI AND CARVALHO TABLE 2 Description of forcings related to and a list of models considered in each scenario Scenario Description Models PI control (PICONTROL) Pre-industrial conditions fixed at pre-industrial levels. May include prescribed atmospheric concentrations or non-evolving emissions, solar radiation, and unperturbed land use All 20 models listed in Table 1, 76 members Historical (HIST) Simulation of the recent past (1850–2005), imposing observed natural and anthropogenic changes in atmospheric composition (such as GHG, as in GHG scenario, tropospheric and stratospheric ozone (Oz), direct and indirect effects of anthropogenic sulfate aerosol (SA), black carbon (BC), organic carbon (OC), solar radiation (Sl), volcanic aerosol (Vl), mineral dust (MD), sea salt (SS)) and land use All 20 models listed in Table 1, 76 members Natural (NAT) Recent past (1850–2005) observed variations in solar radiation (Sl) and volcanic aerosol (Vl). Other forcings are fixed at pre-industrial levels 12 models, 42 members: ACCESS1-3; BCC- CSM1-1; CanESM2; CCSM4; CNRM-CM5; CSIRO_Mk3-6-0; GFDL-ESM2M; HadGEM2-ES; IPSL-CM5A-LR; MIROC- ESM; MRI-CGM3; NorESM1-M GHG (GHG) Recent past (1850–2005) observed GHG concentration: CO2, N2O, CH4, CFC11, CFC12, CFC113, HCFC22, HFC125, and HFC134a 12 models, 41 members: ACCESS1-3; BCC- CSM1-1; CanESM2; CCSM4; CNRM-CM5; CSIRO_Mk3-6-0; GFDL-ESM2M; HadGEM2-ES; IPSL-CM5A-LR; MIROC- ESM; MRI-CGM3; NorESM1-M Anthropogenic aerosols (AA) Recent past (1850–2005) observed changes in anthropogenic aerosol concentration, which may include: Direct (SD) and indirect (SA) effects of anthropogenic sulfate aerosol, SS, dust (Ds), SD, BC, MD, OC. Other forcings are fixed at pre-industrial levels 5 models, 20 members: CanESM2; CCSM4; CSIRO_Mk3-6-0; GFDL-ESM2M; IPSL- CM5A-LR Ozone (OZ) Recent past (1850–2,205) changes in tropospheric and stratospheric ozone. Other forcings are fixed at pre-industrial levels 1 model, 2 members: CCSM4 Land use (LU) Recent past (1850–2005) observed land use change. 2.2 \| Precipitation trend detection ρ is the spatial correlation between the observed and simulated datasets and is cal- culated as: ρ= P NP i=1 Wi xi,o−μo ð Þ xi,s −μs ð Þ P NP i=1 Wi xi,o −μo ð Þ2 P NP i=1 Wi xi,s−μs ð Þ2 ð2Þ ð2Þ where xi is either PP (in mmday−1) or τ (in mmday−1 10 yr−1) at each i = 1, …, NP grid points in the considered study region, and μ is the spatial average over the area. Wi is the spatial weight, calculated as 1=cosφi ð Þ=P NP i=1 1=cosφi ð Þ, where φi is the latitude of each grid point (in radians). The spatial standard deviation is calculated as: where xi is either PP (in mmday−1) or τ (in mmday−1 10 yr−1) at each i = 1, …, NP grid points in the considered study region, and μ is the spatial average over the area. Wi where xi is either PP (in mmday−1) or τ (in mmday−1 10 yr−1) at each i = 1, …, NP grid points in the considered study region, and μ is the spatial average over the area. Wi is the spatial weight, calculated as 1=cosφi ð Þ=P NP i=1 1=cosφi ð Þ, where φi is the latitude of each grid point (in radians). The spatial standard deviation is calculated as: is the spatial weight, calculated as 1=cosφi ð Þ=P NP i=1 1=cosφi ð Þ, where φi is the latitude of each grid point (in radians). The spatial standard deviation is calculated as: σ= ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ NP NP−1 X NP i=1 Wi xi −μ ð Þ2 v u u t ð3Þ ð3Þ and the areal average μ of each variable is: and the areal average μ of each variable is: FIGURE 1 Legend on next coloumn μ= X NP i=1 Wixi ð4Þ ð4Þ FIGURE 1 PP (contours, each 2 mmday−1) and Sen's slope τ (shades, in mmday−110 yr−1) for GPCP DJF (a) 40-year period (1979–2019) and (b) 26-year period (1979–2005). (c) As in (a), for anomalies in PP for MMoM HIST scenario (1966–2005). Note reduced τ range in (c). Red cross-hatching indicates areas where the trends are distinguishable from internal natural variability while stippling indicates where the trends are statistically significant (p < 0.1) according to the Mann–Kendall trend test. Note that none of the grid points in (c) passed the PICONTROL test. 2.2 \| Precipitation trend detection Other forcings are fixed at pre- industrial levels 3 models, 8 members: CanESM2; CCSM4; GFDL-ESM2M PP and τ from all 20 CMIP5 models. Note that the DJF PP of a specific year is calculated using December of that year and January and February of the next year. For the observational dataset (GPCP), the statistics are calculated between 1979 and 2019 and between 1979 and 2005, cov- ering the overlapping period of historical CMIP5 data; for CMIP5 models we considered the last 40 years of the his- torical scenario (1966–2005) so both series have the same length. Despite covering different periods, having obser- vational and simulated time series with the same length result in more reliable statistics when comparing trends using non-parametric tests such as the Mann–Kendall (Wilks, 2011). The caveat of this approach is that it excludes the period after 2005 from the simulated dataset, which could result in underestimated trends. Other options, such as using CMIP5 future projections after 2005 would increase the uncertainties of the results. All CMIP5 data (including PICONTROL) are interpo- lated to GPCP 2.5 resolution using linear interpolation. Trends in all datasets and scenarios are estimated based on Mann–Kendall (Wilks, 2011) and Sen's Slope (Sen, 1968) tests, as described in Zilli et al. (2017). The Sen's slope measures the magnitude of the trend (τ) in all datasets and the Mann Kendall test indicates the areas where trends are statistically significant (p < 0.1). As the main goal of this study is to investigate changes in precip- itation resulting from changes in the SACZ position, the evaluation criteria are based on the spatial representation of PP and τ over Southeastern Brazil, (SEBr; brown box in Figure 1) and SESA (green box in Figure 1). These two 6 ZILLI AND CARVALHO 6 regions represent the main centres of action of the SACZ- related dipole. The pattern analysis metrics considered are the Root Mean Square Error (RMSE) and uncentered regression coefficient (R) obtained with respect to GPCP. The RMSE is calculated according to Taylor (2001): FIGURE 1 Legend on next coloumn. RMSE= ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ σ2 o +σ2 s −2σoσsρ q ð1Þ ð1Þ ð1Þ where σo and σs are the spatial standard deviation of the GPCP (observed—“o” index) and CMIP5 (simulated—“s” index) statistics, respectively. 3 \| One of the advantages of using CMIP5 simulations to identify trends in climatic variables is the availability of the PICONTROL, with simulations undisturbed by exter- nal forcings. Data from the PICONTROL of all 20 CMIP5 models are averaged over austral summer (DJF) and interpolated to the GPCP 2.5 resolution. For each model, we remove the model's drift (Gupta et al., 2013) by adjusting a linear regression to the entire time series before dividing them into non-overlapping 40-year periods. For each period, we estimate PP and τ, resulting in 251 independent realizations used to calculate the probability distribution and confidence intervals of the variables. The GPCP climatology for DJF PP over SA (contours in Figure 1a, b) is characterized by the Intertropical Conver- gence Zone (ITCZ) over equatorial Atlantic and Pacific oceans and by the SAMS, with maximum seasonal aver- ages over the central Amazon extending toward subtropi- cal South Atlantic, forming the SACZ. These features are correctly reproduced by the MMoM of the HIST scenario, with the exception to the double ITCZ over equatorial Atlantic (contours in Figure 1c). Note that in Figure 1c, contours represent the MMoM's bias, that is, the differ- ence between MMoM's and GPCP's PP. In the MMoM HIST simulation, the maximum pre- cipitation associated with the SACZ is located northward of its climatological position (Figure 1c), resulting in a positive (negative) bias in PP over the north (south) part of the SEBr study area (brown box in Figure 1c). Despite that, the spatial average (grey bars in Figure 2a) and stan- dard deviation (red whiskers in Figure 2a) of PP over SEBr are well reproduced by the MMoM (see also values in Table 3). The spatial pattern of PP is similar to the GPCP's, with spatial correlation ρ = 0.89 (Table 3), which is larger than the 75th percentile of the probability distri- bution derived from the 251 non-overlapping 40-year periods from the PICONTROL simulations. As a result, the RMSE between HIST and GPCP is small (=0.45, The analysis is divided into two parts: detection of trends in observed and historical simulation precipita- tion, and the attribution of the simulated trends to external forcings, represented by natural- and anthropogenic- forcing scenarios. In both cases, the PICONTROL distribu- tion is used to assess the similarity between observed and simulated trends and the influence of internal natural variability. 2.2 \| Precipitation trend detection Brown (green) box represents the location of the study area over the SACZ (SESA) [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 1 Legend on next coloumn. ZILLI AND CARVALHO 7 The uncentered spatial regression coefficient R (Hegerl and Zwiers, 2011; Barkhordarian et al., 2012; 2013; Polson and Hegerl, 2017) is used to evaluate the similarity between the observed and simulated spatial patterns over the study areas. It can be defined as: the observed when they are within the confidence inter- val defined by the PICONTROL. In the same way, the PICONTROL distribution can also be considered as a confidence interval for the RMSE and R similarity metrics (as defined by Equations (1) and (5), respectively). In this case, the probability distribution is estimated after calcu- lating the RMSE and R between PP from GPCP and each of the non-overlapping 40-year PICONTROL intervals. Additionally, both observed and simulated τ are consid- ered significantly different from those caused by natural internal variability when their statistics are outside the confidence intervals based on the PICONTROL τ distribution. R= P NP i=1 xi,oxi,s P NP i=1 xi,s ð Þ2 ð5Þ R= P NP i=1 xi,oxi,s P NP i=1 xi,s ð Þ2 ð5Þ ð5Þ The R index indicates whether the co-variability between observations (xi,o) and simulations (xi,s), repre- sented by the numerator in Equation (5), can be distin- guished from the simulation variability, represented by the denominator in Equation (5). It measures the relative magnitude between observed and simulated trends (Barkhordarian et al., 2012), with positive (negative) values of R indicating that they are similar (dissimilar). Values of R greater (smaller) than 1 indicate an underes- timation (overestimation) of the trend by the simulations while R = 1 indicating a perfect simulation of the observed spatial patterns (Polson and Hegerl, 2017). The attribution analysis is based on the evaluation of the simulated τ in each scenario. In this part of the analy- sis, the objective is to identify the contribution of each forcing to the simulated τ, independently on its sign. The spatial pattern of the trends in a given scenario is consid- ered significantly different from those caused by natural internal variability when the R between the scenario's and GPCP's τ is outside the confidence interval of the R distribution, obtained when regressing the simulated τ with each of the non-overlapping 40-year PICONTROL intervals. 3 \| In the first part of the analysis, our objective is to identify whether the CMIP5 HIST simulations can cor- rectly reproduce the observed precipitation rate and its trends. Simulated values of PP are considered similar to FIGURE 2 (a,b) Spatially averaged PP for GPCP, MMoM, each MMeM (bars), and individual members (dots; in MMoM bar dots represent each MMeM). Red bars with whiskers: Spatially averaged standard deviation for GPCP, MMoM, and each MMeM. Dotted line: 5th–95th confidence interval defined by the PICONTROL distribution. (c,d) Taylor diagram for spatially averaged PP, comparing GPCP values to HIST scenario of MMoM, each MMeM (dots), each member of MMeM (“x”). Dashed lines represent the interquartile range interval of the RMSE for the PICONTROL distribution. See Table 1 for the correspondence between letters and CMIP5 models. Values are averaged over (a) and (c) SEBr and (b) and (d) SESA [Colour figure can be viewed at wileyonlinelibrary.com] 8 ZILLI AND CARVALHO 8 ZILLI AND CARVALHO FIGURE 2 (a,b) Spatially averaged PP for GPCP, MMoM, each MMeM (bars), and individual members (dots; in MMoM bar dots represent each MMeM). Red bars with whiskers: Spatially averaged standard deviation for GPCP, MMoM, and each MMeM. Dotted line: 5th–95th confidence interval defined by the PICONTROL distribution. (c,d) Taylor diagram for spatially averaged PP, comparing GPCP values to HIST scenario of MMoM, each MMeM (dots), each member of MMeM (“x”). Dashed lines represent the interquartile range interval of the RMSE for the PICONTROL distribution. See Table 1 for the correspondence between letters and CMIP5 models. Values are averaged over (a) and (c) SEBr and (b) and (d) SESA [Colour figure can be viewed at wileyonlinelibrary.com] Table 3 and Figure 2c) and below the 25th percentile of the PICONTROL distribution. that, the MMoM correctly simulates the PP spatial pat- tern, with ρ (RMSE) equal 0.74 (0.58) and in the upper (lower) quartile of the PICONTROL distribution (Table 3 and Figure 2d). It is interesting to notice that the observed PP is outside the interquartile range of PP simu- lated by the PICONTROL (represented by the dotted lines in Figure 2b). Over SESA (green box in Figure 1c), the MMoM HIST simulation is not as accurate, underestimating the spa- tially averaged PP and its standard deviation (Figure 2b and Table 3). 3 \| Similar underestimation of the DJF precipi- tation by CMIP5 models has been identified in previous studies (Gulizia and Camilloni, 2015) and was related to underestimation in the southerly flow east of the Andes, responsible for advecting moisture from the Amazon region to the SESA (Barros and Doyle, 2018). Despite Considering each MMeM individually, not all HIST simulations accurately reproduce the DJF average precip- itation over SA, with the largest bias occurring over the Andes and the Amazon basin (contours in Figure 3). ZILLI AND CARVALHO 9 TABLE 3 Spatial average (μ) of GPCP and each MMoM scenario, normalized standard deviation (σ/σGPCP),spatial correlation (ρ), and normalized RMSE (Equations (1)–(4)) with respect to GPCP for PP (in mmday−1) and τ (in mmday−110 yr−1) calculated over the SEBr and SESA (brown and green boxes in Figure 1, respectively) SCENARIO PP (mmday−1) τ (mmday−110 yr−1) SEBr SEBr μ σ/σGPCP ρ RMSE μ σ/σGPCP ρ RMSE GPCP 6.39 1.00 1.00 0.00 −0.32 1.00 1.00 0.00 HIST 6.49 0.95 0.89 0.45 −0.01 0.15 −0.04 1.02 NAT 6.59 1.00 0.92 0.40 −0.06 0.15 −0.04 1.02 GHG 6.72 1.04 0.92 0.42 0.05 0.21 0.12 1.00 AA 6.02 1.15 0.95 0.37 −0.05 0.25 0.01 1.03 OZ 6.04 1.06 0.93 0.40 −0.10 0.62 0.31 1.00 LU 6.19 1.11 0.95 0.34 −0.18 0.40 −0.09 1.11 SESA SESA GPCP 4.14 1.00 1.00 1.00 0.13 1.00 1.00 0.00 HIST 2.78 0.80 0.74 0.58 0.03 0.11 0.06 1.00 NAT 2.77 0.79 0.75 0.58 0.02 0.16 0.33 0.96 GHG 2.92 0.82 0.78 0.55 0.01 0.11 −0.08 1.01 AA 2.81 0.86 0.71 0.62 0.02 0.20 0.08 1.00 OZ 3.70 0.85 0.75 0.58 0.02 0.32 −0.04 1.06 LU 3.26 1.01 0.73 0.65 0.06 0.35 0.27 0.97 (CCSM4, GFDL-ESM2M, INMCM4, IPSL-CM5A_LR, and NorESM1-M) suggesting a reliable representation of the precipitation rate in these MMeM. On the other hand, in 5 MMeM (ACCESS1-3, BCC-CSM1-1, CSIRO- Mk3-6-0, GISS-E2-H, and MIROC5), the RMSE was above the upper quartile of the PICONTROL distribution (i.e., below 0.87; outermost dashed black circle in Figure 2c), suggesting large discrepancies between observations and simulations. Most of the selected models displace the maximum pre- cipitation from the Amazon eastward/southeastward of the observed climatological position, overestimating pre- cipitation over semi-arid northeastern Brazil. 3 \| In previous studies, the dry bias over the Amazon has been related to overestimated precipitation over the ITCZ, changing the moisture flux to the Amazon and affecting the precipita- tion annual cycle (Bombardi and Carvalho, 2009; Yin et al., 2013). This displacement is manifested in the MMoM HIST scenario as negative (positive) bias over the Amazon (Northeastern Brazil; Figure 1c). Five models fail in reproducing the spatial characteristics of SAMS precipitation, placing the maximum precipitation as a zonal band over the equator (CMCC-CESM, GISS-E2-H, INMCM4, and MPI-ESM-LR) or the subtropics (CSIRO- Mk3-6-0). Over SESA, all except two MMeM have RMSE below the 75th percentile of the PICONTROL distribution (Can- ESM2 and MPI-ESM-LR; Figure 2d) and in 8 MMeM, the RMSE is below the 25th percentile of the PICONTROL dis- tribution (ACCESS1-3, CCSM4, GFDL-ESM2M, HadGEM2-CC, HadGEM2-ES, INMCM4, MRI-CGCM3, and NorESM1-M). In Figure 2 it is also evident the small spread of simulated PP (Figure 2a,b, dots) and respective RMSE (Figure 2c,d, “x”) among members of each MMeM member. Over both study areas, most of the selected MMeM correctly simulated the average PP and its spatial pattern, with the spatial average of PP within the interquartile range of PP simulated by the PICONTROL (Figure 2a,b) and their RMSE below the 75th percentile of the probabil- ity distribution from the PICONTROL (Figure 2c,d). Nonetheless, most MMeM displaces the maximum pre- cipitation associated with the SACZ to the north part of SEBr (brown box in Figure 3), resulting in the bias observed in the MMoM. Over SEBr, the RMSE between the observed and simulated spatial pattern of PP is below the 25th percentile of the PICONTROL distribution (i.e., below 0.51, with this value represented as the inner- most dashed black circle in Figure 2c) in 5 MMeM Thus, the MMoM of the 20 selected models accurately simulates the magnitude of the precipitation rates aver- aged over the study regions (SEBr and SESA). However, the MMoM is not able to correctly reproduce the spatial location of the SACZ, with the maximum precipitation located to the north of its climatological location, affect- ing the SACZ-related dipole. Consequently, the MMeMs that better reproduce the precipitation rate over one study area may not be as accurate over the other (see Table 4). Nonetheless, the results obtained here are in agreement with previous studies, among them Cavalcanti ZILLI AND CARVALHO 10 FIGURE 3 Anomalies of PP (in mmday−1; contours each 2 mmday−1) and τ (in mmday−110 yr−1, shades) for MMeM models in the HIST scenario. Red cross-hatchings indicate areas where the trends are distinguishable from internal natural variability while stipplings indicate where τ is statistically significant (p < 0.1) according to the Mann–Kendall trend test. Brown (green) box represents the location of the study area over the SEBr (SESA) [Colour figure can be viewed at wileyonlinelibrary.com] 10 ZILLI AND CARVALHO FIGURE 3 Anomalies of PP (in mmday−1; contours each 2 mmday−1) and τ (in mmday−110 yr−1, shades) for MMeM models in the HIST scenario. Red cross-hatchings indicate areas where the trends are distinguishable from internal natural variability while stipplings indicate where τ is statistically significant (p < 0.1) according to the Mann–Kendall trend test. Brown (green) box represents the location of the study area over the SEBr (SESA) [Colour figure can be viewed at wileyonlinelibrary.com] 11 ZILLI AND CARVALHO 11 TABLE 4 PP columns: Simulations with RMSE below (above) the 75th percentile of the PICONTROL distribution are shaded blue (red); rossed cells indicating RMSE below the 25th percentile of the PICONTROL distribution ote: τHIST column: simulations with τ of the same (opposite) sign as (to) GPCP's are shaded blue (red), with those distinguishable from natural internal ariability (5th to 95th confidence interval of R distribution against PICONTROL) crossed. Other columns: simulations with τ not distinguishable from natural nternal variability are shaded grey; those with τ distinguishable from internal natural variability and in agreement (opposite) with (to) GPCP's trend sign are haded blue (red). Unshaded cells do not have simulations for the scenario. TABLE 4 PP columns: Simulations with RMSE below (above) the 75th percentile of the PICONTROL distribution are shaded blue (red); crossed cells indicating RMSE below the 25th percentile of the PICONTROL distribution Note: τHIST column: simulations with τ of the same (opposite) sign as (to) GPCP's are shaded blue (red), with those distinguishable from natural internal variability (5th to 95th confidence interval of R distribution against PICONTROL) crossed. Other columns: simulations with τ not distinguishable from natural internal variability are shaded grey; those with τ distinguishable from internal natural variability and in agreement (opposite) with (to) GPCP's trend sign are shaded blue (red). Unshaded cells do not have simulations for the scenario. (shades in Figure 1a). In addition to being significant against the Mann–Kendall trend test (stippling in Figure 1a), the trends observed over SEBr and SESA are not within the 5th–95th percentile range defined by the PICONTROL distribution and thus are distinguishable from internal natural variability (red cross-hatching in Figure 1a). There is also significant decrease (increase) in PP over southern (western) Amazon and the northern coast of SA since 1979, which has also been identified in previous observational studies (Marengo, 2004; Espinoza et al., 2019). and Shimizu (2012), who identified an adequate perfor- mance of HadGEM2-ES in simulating the SACZ dipole, and Jones and Carvalho (2013), who identified GFDL- ESM2M and MIROC4h as the models with more realistic simulations of the SAMS characteristics. MIROC4h, in addition to MIROC-ESM and INMCM4, were also identi- fied as agreeing with the observed precipitation over sub- tropical South America by Barros and Doyle (2018). The models mentioned in those studies are among those cor- rectly simulating the precipitation rate over SEBr and SESA (Table 4). We also tested GPCP τ considering only the period in common with CMIP5 (1979–2005; Figure 1b). The resulting trends are similar, with a reduction in precipita- tion over the continental SACZ extending toward south- ern Amazon and over the northern coast of South America. However, the spatial pattern of wetting trends over SESA is not evident yet. The magnitude of the trends 4 \| PRECIPITATION TREND IN RECENT DECADES Consistently with Zilli et al. (2017; 2019), GPCP τ (1979– 2019) is negative over the SACZ and positive to its south FIGURE 4 (a,b) Spatially averaged τ for GPCP, MMoM, each MMeM (bars) and individual members (dots; in MMoM dots represent each MMeM) in the HIST scenario. Dashed (dotted) lines: 5th–95th (10th–90th) confidence interval defined by the PICONTROL scenario. (c,d) R (from Equation (5)) between GPCP and MMoM, each MMeM, and individual members (dots). Red bars with whiskers: Confidence interval (5th–95th percentile) defined by the regression of MMoM and each MMeM onto all 40-year PICONTROL periods. Values are averaged over (a) and (c) SEBr and (b) and (d) SESA [Colour figure can be viewed at wileyonlinelibrary.com] 12 ZILLI AND CARVALHO 12 ZILLI AND CARVALHO FIGURE 4 (a,b) Spatially averaged τ for GPCP, MMoM, each MMeM (bars) and individual members (dots; in MMoM dots represent each MMeM) in the HIST scenario. Dashed (dotted) lines: 5th–95th (10th–90th) confidence interval defined by the PICONTROL scenario. (c,d) R (from Equation (5)) between GPCP and MMoM, each MMeM, and individual members (dots). Red bars with whiskers: Confidence interval (5th–95th percentile) defined by the regression of MMoM and each MMeM onto all 40-year PICONTROL periods. Values are averaged over (a) and (c) SEBr and (b) and (d) SESA [Colour figure can be viewed at wileyonlinelibrary.com] Despite being able to correctly reproduce the DJF pre- cipitation rate, CMIP5 HIST simulations (considering the 40-years period between 1966–2005) are less accurate in reproducing τ (Figure 1c). The MMoM simulation cor- rectly places positive trends over western Amazon and the southern portion of SA. However, over both study areas, τHIST has the same sign as in GPCP, but its spatial average is close to zero (Figure 4a,b and Table 3). Conse- quently, the value of R is large (=4.51 and 3.15 over SACZ and SESA, respectively; Figure 4c,d), indicating the underestimation of the precipitation trend by the MMoM. The large spread between the 5th and the 95th percentiles (MMoM error bar in Figure 4c,d) is also a consequence of the small values of τHIST. Note that the error bar is estimated by Equation (5), with τHIST in each grid point as xi,s and the τ estimated in each PICONTROL realization as over both study areas is smaller and not significant. 4 \| PRECIPITATION TREND IN RECENT DECADES The lack of significant trends over this reduced period rein- forces the need of extending the analysis until 2019 and include important variabilities observed after 2005. Thus, we will focus on the 40-year period (1979–2019) for the remainder of the analysis. over both study areas is smaller and not significant. The lack of significant trends over this reduced period rein- forces the need of extending the analysis until 2019 and include important variabilities observed after 2005. Thus, we will focus on the 40-year period (1979–2019) for the remainder of the analysis. Over SEBr (SESA), the spatial average of GPCP τ (consid- ering the 40-year period) indicates a reduction (increase) of −0.32 mmday−1 per decade (+0.13 mmday−1 per decade) in the precipitation rate since 1979 (Table 3 and Figure 4a,b). Both values are outside the 5th–95th percentile confidence interval defined by the PICONTROL distribution (dotted lines in Figure 4a,b) and thus cannot be attributed to natural internal variability. Gonzalez et al. (2013) found a similar magnitude of trends over SESA when considering precipita- tion rates from gridded datasets and individual stations. ZILLI AND CARVALHO 13 xi,o. Despite the small magnitude of τHIST, the lower limit of the confidence interval of MMoM's R is above zero, suggesting that the spatial patter of τHIST cannot be consid- ered similar to those obtained in the PICONTROL simula- tions and cannot be explained by internal natural variability alone. observed precipitation changes. It is also important to recall that the CMIP5 simulations end in 2005 and does not capture important changes in precipitation in the last decade. Nonetheless, the spatial analysis suggests that the simulated spatial pattern of the precipitation trends is similar to the observed pattern and is already detectable by some of the individual members. The small magnitude of the trends simulated by the MMoM and the large spread of its confidence interval is an outcome of the large discrepancies among MMeMs (dots in Figure 4a,b). Over SEBr, the spatially averaged τHIST is negative in 11 of the 20 MMeM (Figure 4a and Table 4) and 44 of the 76 members. However, only one MMeM and 11 members simulated negative and signifi- cant τHIST (considering a 10th to 90th percentile confi- dence interval; dotted line in Figure 4a). 5 \| ATTRIBUTION ANALYSIS To further investigate whether the observed trends are related to natural external variability (such as changes in solar radiation or volcanic emissions) or are a response to anthropogenic forcings, we repeat the previous analysis considering CMIP5 individual forcing scenarios. The nat- ural external forcing is represented by the NAT scenario while anthropogenic forcings are divided into GHG emis- sions (GHG scenario), anthropogenic aerosols (AA), changes in ozone (OZ), and land-use changes (LU). The climatology of each MMoM scenario is similar to the HIST MMoM and GPCP (Table 3) and will not be further discussed. In this part of the analysis, we focus on ana- lysing whether the simulated spatial pattern of the trends is distinguishable from natural internal variability. g y Natural external forcings (NAT scenario) result in weak negative τNAT over most of SA (Figure 5b). Small areas with positive trends can be found over the ITCZ and the SESA (including the study area). The magnitude of the simulated values of τNAT spatially averaged over SEBr (SESA) are negative (positive) but not significant when compared to the PICONTROL (Figure 6a,b). The small magnitude of τNAT in MMoM and MMeM results from the lack of agreement among members (Figure 6) and is an indication of the large uncertainties regarding the effects of natural external forcings in the precipitation rate over the study regions. Despite their small magni- tude, the spatial pattern of τNAT are significantly different from natural internal variability (5th–95th confidence interval for R does not include zero) in the MMoM and about half of the MMeM (Figure 6c,d, and Table 4). Among the MMeM with trends significantly different from natural internal variability, 6 of them simulate τNAT with a spatial pattern similar to the observed τ, with neg- ative trends over the SEBr and positive ones in SESA (Figure 6a,b and Table 4). However, the small magnitude of the forcings (volcanic activity and very minor changes in insolation over the period considered) suggest that part of the trends in this scenario could be resulting from internal variability. Over SESA, the spatially averaged value of τHIST is positive in 15 of the 20 MMeM (Figure 4b and Table 4) and 50 of the 76 members. However, all except two MMeMs (CanESM2 and GFDL-ESM2-M) underestimate the magnitude of τHIST (Figure 4b), resulting in non-sig- nificant trends. 4 \| PRECIPITATION TREND IN RECENT DECADES The MMeMs that better simulate the intensity of τHIST are those with good agreement among their members: CanESM2M, GFDL-ESM2M, GISS-E2-H (despite its poor representa- tion of PP), INMCM4, IPSL-CM5A-LR, and MRI-CGCM3 (Figure 4a). In these models, τHIST is more intense over land (Figure 3), resulting in values of R close to unity (Figure 4c). Furthermore, the confidence interval of R in these MMeMs is above 0, indicating that the spatial pat- tern of these trends cannot result solely from natural internal variability. 5 \| ATTRIBUTION ANALYSIS Similar underestimation of trends by CMIP5 historical scenarios over the region has been noticed in previous studies (Gonzalez et al., 2013; Vera and Díaz, 2015; Zhang et al., 2016). In addition to Can- ESM2 and GFDL-ESM2M, 6 other MMeMs correctly sim- ulate the spatial pattern of τHIST (R values close to 1 in Figure 4d), although with underestimated magnitude (HadGEM2-CC, INMCM4, MIROC4h, MIROC5, MPI- ESM-LR, and MRI-CGCM3). In these eight MMeMs, the spatial pattern of the trends over SESA cannot be attrib- uted to natural internal variability (Figure 4d). Note, how- ever, that CanESM2 and MPI-ESM-LR MMeMs have the worst performance in simulating PP over this study area. Hence, the HIST scenarios of MMoM and a part of the MMeMs simulate spatial patterns of precipitation trends that are similar to those observed in GPCP and with a spatial pattern that cannot be attributed to internal natural variability alone. However, the large var- iability among MMeM and their members results in an underestimation of the precipitation trends' magnitude over both study areas. The large variability among indi- vidual members suggests that the simulations may fail to correctly reproduce the mechanisms associated with the Among the anthropogenic forcings, changes in anthropogenic greenhouse gases (GHG) result in small and positive precipitation trends (τGHG) over most of (a) GPCP (b) MMoM – NAT (c) MMoM – GHG (d) MMoM – AA (e) MMoM – OZ (f) MMoM – LU FIGURE 5 As in Figure 1 for (a) GPCP and MMoM of the (b) NAT, (c) GHG, (d) AA, (e) OZ, and (f) LU scenarios. Note reduced Sen's slope range in (b)–(f) [Colour figure can be viewed at wileyonlinelibrary.com] 14 ZILLI AND CARVALHO ZILLI AND CARVALHO 14 (a) GPCP (a) GPCP (b) MMoM – NAT (c) MMoM – GHG (b) MMoM – NAT (c) MMoM – GHG (b) MMoM – NAT (c) MMoM – GHG (d) MMoM – AA (e) MMoM – OZ (f) MMoM – LU FIGURE 5 As in Figure 1 for (a) GPCP and MMoM of the (b) NAT, (c) GHG, (d) AA, (e) OZ, and (f) LU scenarios. 5 \| ATTRIBUTION ANALYSIS Note reduced Sen's slope range in (b)–(f) [Colour figure can be viewed at wileyonlinelibrary.com] (e) MMoM – OZ (e) MMoM – OZ (f) MMoM – LU (d) MMoM – AA (d) MMoM – AA (d) MMoM – AA FIGURE 5 As in Figure 1 for (a) GPCP and MMoM of the (b) NAT, (c) GHG, (d) AA, (e) OZ, and (f) LU scenarios. Note reduced Sen's slope range in (b)–(f) [Colour figure can be viewed at wileyonlinelibrary.com] the scenarios considered here, changes in land use (LU) result in the largest values of τLU (Figure 6a,b), with drying (wetting) trends over central-eastern Brazil (SESA) and extending along the northern (southern) margin of the MMoM SACZ (Figure 5f), similar to the observed τ. The influence of LU is stronger over SEBr where the spatial pat- tern of τLU is distinguishable from natural internal variabil- ity in the MMoM and all MMeMs (Figure 6c). In 2 of the 3 MMeMs and 4 of the 8 members, the simulated τLU is similar to GPCP's (R close to 1 in Figure 6c and Table 4). Over SESA, the spatial pattern of τNAT is not attributable to internal natural variability in the MMoM, 2 of the 3 MMeMs and 2 of the 8 members (Figure 6d and Table 4). the scenarios considered here, changes in land use (LU) result in the largest values of τLU (Figure 6a,b), with drying (wetting) trends over central-eastern Brazil (SESA) and extending along the northern (southern) margin of the MMoM SACZ (Figure 5f), similar to the observed τ. The influence of LU is stronger over SEBr where the spatial pat- tern of τLU is distinguishable from natural internal variabil- ity in the MMoM and all MMeMs (Figure 6c). In 2 of the 3 MMeMs and 4 of the 8 members, the simulated τLU is similar to GPCP's (R close to 1 in Figure 6c and Table 4). Over SESA, the spatial pattern of τNAT is not attributable to internal natural variability in the MMoM, 2 of the 3 MMeMs and 2 of the 8 members (Figure 6d and Table 4). Changes in tropospheric and stratospheric ozone (OZ) and anthropogenic aerosol (AA) also result in similar wetting/drying trends over SA, more intense over the SEBr (Figure 5d,e, respectively), where the spatial pattern of both τOZ and τAA in the MMoM are distinguishable from natural internal variability (Figure 6c). 5 \| ATTRIBUTION ANALYSIS Over this region, τOZ has the second-largest value (−0.1 mmday−1 per decade; Table 3 and Figure 6a), but this scenario has only one MMeM with 2 members (CCSM4). The influence of changes in AA over SEBr is not as pronounced, resulting in a spatial central and subtropical SA in the MMoM (Figure 5c). Over SEBr, τGHG simulated by the MMoM is positive but indis- tinguishable from natural internal variability (Figure 6a). Since MMoM τGHG is opposite to the GPCP's trend, R is negative and the spatial pattern of τGHG is distinguishable from natural internal variability over the area (Figure 6c). However, the disagreement among individual MMeMs is large, with 5 (3) of the 12 MMeMs (Figure 6c and Table 4) and 13 (10) of the 41 members simulating positive (nega- tive) values of τGHG with spatial pattern that cannot be attributed to natural internal variability. Over SESA, τGHG is close to zero and its spatial pattern is not distinguishable from natural internal variability in the MMoM (Figure 6b, d). Furthermore, τGHG is not significant in any of the MMeM (Figure 6b), despite having spatial patterns that are not attributable to natural internal variability in some cases (Figure 6d and Table 4). Thus, due to the large uncer- tainties in simulating the responses of precipitation to changes in GHG concentration, their influence on the observed precipitation rate over SEBr and SESA cannot be attributed to GHG in the current set of models. Changes in tropospheric and stratospheric ozone (OZ) and anthropogenic aerosol (AA) also result in similar wetting/drying trends over SA, more intense over the SEBr (Figure 5d,e, respectively), where the spatial pattern of both τOZ and τAA in the MMoM are distinguishable from natural internal variability (Figure 6c). Over this region, τOZ has the second-largest value (−0.1 mmday−1 per decade; Table 3 and Figure 6a), but this scenario has only one MMeM with 2 members (CCSM4). The influence of changes in AA over SEBr is not as pronounced, resulting in a spatial The small magnitude of τ in the GHG scenario suggests the importance of other anthropogenic forcings. Among 15 FIGURE 6 (a,b) Spatially averaged τ for GPCP, MMoM (bars) and each MMeM (dots) in each scenario. Dashed (dot) lines represent the 5th–95th (10th–90th) confidence interval defined by the PICONTROL scenario. (c,d) R (from Equation (5)) between GPCP and MMoM (diamonds), and each MMeM (dots) for each scenario. 5 \| ATTRIBUTION ANALYSIS Black bars with whiskers (colour bars): 5th–95th confidence interval defined by the regression of MMoM (each MMeM) onto all 40-year PICONTROL periods. Values are averaged over (a) and (c) SEBr and (b) and (d) SESA [Colour figure can be viewed at wileyonlinelibrary.com] ZILLI AND CARVALHO 15 ZILLI AND CARVALHO FIGURE 6 (a,b) Spatially averaged τ for GPCP, MMoM (bars) and each MMeM (dots) in each scenario. Dashed (dot) lines represent the 5th–95th (10th–90th) confidence interval defined by the PICONTROL scenario. (c,d) R (from Equation (5)) between GPCP and MMoM (diamonds), and each MMeM (dots) for each scenario. Black bars with whiskers (colour bars): 5th–95th confidence interval defined by the regression of MMoM (each MMeM) onto all 40-year PICONTROL periods. Values are averaged over (a) and (c) SEBr and (b) and (d) SESA [Colour figure can be viewed at wileyonlinelibrary.com] decades (Zilli et al., 2017; 2019). Similar changes have also been identified in future projections of climate change (Seth et al., 2010; Junquas et al., 2013; Talento and Barreiro, 2017). In the present analysis, the historical simulations from MMoM and part of the 20 MMeM cor- rectly simulate the DJF the precipitation rate over the SACZ, even though with maximum precipitation located to the north of its observed position. This affects the SACZ-related precipitation dipole, resulting in an under- estimation of the precipitation rate and trend over SESA, as already described in previous studies (Gulizia and Camilloni, 2015; Vera and Díaz, 2015; Barros and Doyle, 2018). Over SEBr, the large uncertainties among the MMeMs and individual members of each ensemble result in the lack of significant trend in the MMoM. However, it is important to emphasize the different period of analysis, with MMoM not capturing important precipitation trends observed after 2005. The bias observed in precipitation rates over both areas could suggest that the models are not able to correctly reproduce the mechanisms related to the location and intensity of the SACZ (and related SACZ-dipole). average of MMoM τAA of −0.05 mmday−1 per decade (Table 3), which is similar in magnitude (but opposite sign) to the trends resulting from the NAT scenario (Figure 6a). Despite the small magnitude of τAA, this is the scenario with the largest agreement among individual members, with all MMeM (Figure 6a) and 15 of the 20 members simulating negative trends over the area. 5 \| ATTRIBUTION ANALYSIS The spatial pattern of τAA can be considered significantly different from natural internal variability in 2 of the 5 MMeMs (Figure 6c and Table 4) and 9 of 20 members. Over SESA, the spatial pattern of τOZ and τAA simulated by MMoM are distinguishable from natural internal variability only when considering the 10th–90th con- fidence interval from PICONTROL distribution. MMoM τAA is small due to the lack of agreement among the MMeMs and individual members (Figure 6d). In OZ scenario, the spatial pattern of the τOZ is distinguishable from natural vari- ability in only one member. 6.2 \| Attribution analysis While large uncertainties among individual members reduce our ability to conclusively attribute the observed trends to a set of forcings, the emerging patterns from each individual scenario allows a qualitative assessment of the influence of anthropogenic forcings in the local precipita- tion trends. Land-use change is the main forcing associated with the reduction (increase) in precipitation rates over SEBr (SESA), followed by anthropogenic-related changes in stratospheric and tropospheric ozone contributing to the reduction in precipitation rate over SEBr. In addition to changes in land use, anthropogenic forcings related to changes in the concentration of ozone also contribute to precipitation trends, albeit with small intensity. Previous studies suggest that ozone depletion is associated with a poleward expansion of the Hadley cell in the Southern Hemisphere and a consequent shift in the jet position, which would result in increased precipi- tation over SESA (Polvani et al., 2011; Gonzalez et al., 2014). The position of the subtropical jet is one of the ingredients necessary for the development of the SACZ over eastern Brazil, advecting negative vorticity and favouring upper-level divergence over the region (Kodama, 1992; 1993). Thus, a poleward shift of the jet could also affect the location and intensity of the SACZ, resulting in the drying (wetting) trends in the SACZ north (southern) flank, consistent with Zilli et al. (2019). According to the present analysis, changes in land use result in the largest contribution to the simulated changes in precipitation over both study areas. Even though the present analysis does not indicate the location of this forcing, whether it is local (e.g., in the La Plata Basin [LPB], where the largest part of both study regions are located) or remote (in the Amazon Basin), changes in land use could affect the SACZ-related precipitation. The Amazon region is one of the main sources of moisture to the SACZ and SESA (Arraut et al., 2012). The transport of moisture to the SACZ depends strongly on the charac- teristics of the Low-Level Jet (LLJ) east of the Andes (Arraut and Satyamurty, 2009; Nascimento et al., 2016; Montini et al., 2019). Changes in land use in the Amazon, mainly associated with deforestation, may reduce the amount of moisture recycling, affecting precipitation in the region (Zemp et al., 2017a) and reducing the amount of moisture transported by the LLJ toward the SACZ and the SESA (Zemp et al., 2017b). 6.2 \| Attribution analysis Finally, the response of the precipitation to changes in GHG cannot be distinguishable from natural internal vari- ability. The lack of consensus regarding the sign of the trend suggests that internal variability dominates trends in this scenario. Over the LPB, changes in land use since 1950s have reduced the area of native vegetation (mostly savanna and forest) to less than 20% (Tucci and Clarke, 1998). The conversion of native vegetation to crop land can result in drier surface conditions that can modify the thermal structure of the atmosphere, affecting the low level circu- lation, consequently, the moisture transport toward SACZ and SESA (Lee and Berbery, 2012; Yang and Dominguez, 2019). Using simulations from Weather 6.1 \| Detection of precipitation trends Despite the small magnitude, the spatial pattern of the simulated HIST precipitation trend indicates a drying (wetting) trend over SEBr (SESA) coherent with trends in Observational studies have indicated a poleward shift in the SACZ-related summer precipitation rate in recent ZILLI AND CARVALHO 16 16 Research and Forecast Model (WRF) experiments, Lee and Berbery (2012) demonstrated that land-use changes within the LPB are associated with a reduction (increase) in precipitation over the northern (southern) part of the basin. According to them, land-use changes from native vegetation to dry crops increases the albedo and decreases the surface friction over the northern part of the basin, reducing the sensible heat flux and near-sur- face temperature, which in turn reduces convective insta- bility over the region. Additionally, the reduction in surface roughness accelerates the surface winds favouring moisture divergence over the area and reduc- ing precipitation. Further south, land-use changes from native grassland to dry crops have the opposite effect, mostly due to a reduction in albedo, resulting in increased precipitation. The spatial pattern of changes in precipitation described by these authors is similar to those observed in GPCP and simulated by the LU MMoM, suggesting that changes in land use over central and southeastern SA could be affecting the surface pro- cesses associated with the strength and location of the SACZ-related precipitation dipole. the SACZ dipole observed in Zilli et al. (2017; 2019). Pre- vious studies, based on filtered precipitation analysis (such as EOF), have identified similar historical and future changes in the SACZ-related dipole (Junquas et al., 2013; Díaz and Vera, 2017). Our results indicate a simulated spatial pattern of precipitation trends that can- not be attributed to internal natural variability alone and are consistent with the future projections. However, the large variability among MMeM and individual members of each MMeM increases the uncertainties regarding the magnitude of the trends. 6.3 \| Other factors associated with the observed trends Another source of discrepancies between observed and simulated trends is related to the position of the SACZ in CMIP5 models. The present analysis is based on fixed study regions located over areas with large observed pre- cipitation trends. However, some CMIP5 models simulate 17 ZILLI AND CARVALHO 17 the SACZ, where the uncertainty in simulations is large (Carvalho and Jones, 2013; Jones and Carvalho, 2013; Sanchez et al., 2015; de Barros Soares et al., 2017; Díaz and Vera, 2017). Over the SACZ, the number of rainy days is decreasing (Zilli et al., 2017) likely due to weaker northerly winds along the eastern Brazilian coast and reduced avail- able moisture in the lower-to-mid troposphere (Zilli et al., 2019). On the other hand, the intensity of extreme events is increasing over the same region (Zilli et al., 2017), suggesting the importance of the combined influence of dynamic and thermodynamic forcings on the observed changes (Chen et al., 2019; Norris et al., 2019a, 2019b). Thus, besides cor- rectly reproducing the observed average precipitation trends, it is necessary to access the accuracy of the CMIP5 models in reproducing the observed trends in extreme precipitation, as well as dynamic and thermodynamic forcings associated with the intensification of these events. the SACZ displaced to the north of its climatological posi- tion, resulting in trends outside the study regions. The utilization of approaches based on the location of the SACZ in each model could improve the identification of the forcings and mechanisms associated with the observed changes, without being restricted to models that accurately reproduce the SACZ's location. Despite demonstrating that the detected precipitation trends cannot be caused by internal variability alone, trends resulting from NAT forcings suggest that at least part of the changes in the position and intensity of the SACZ could be associated with changes in climatic modes. The analysis period, 1979–2019, encompasses a transition from cold to warm AMO phase, which possibly contributes to the precipitation trends over the study region (Chiessi et al., 2009; Grimm and Saboia, 2015; Jones and Carvalho, 2018; Kayano et al., 2019). A previ- ous study (Zhang and Wang, 2013) demonstrated that the AMO transition from cold to warm phase occurred between 1975 and 2005 in all CMIP5 models analysed, even though most of them are not able to correctly repre- sent the spatial structure and intensity of the mode. ACKNOWLEDGEMENTS GPCP Precipitation data was provided by the NOAA/ OAR/ESRL PSL, Boulder, Colorado, USA, from their website at https://psl.noaa.gov/. All CMIP5 data was downloaded from the PCMDI website at http://pcmdi9. llnl.gov/. The authors acknowledge the World Climate Research Program's Working Group on Coupled Model- ling, which is responsible for CMIP, and we thank the cli- mate modelling groups (listed in Table 1 of this paper) for producing and making available their model output. M. Zilli acknowledges the Brazilian National Council for Scientific and Technological Development (CNPq) for the financial support through the Science without Borders Program (202691/2011-0). L. Carvalho acknowledges the support of NSF-AGS-1505198. ORCID Marcia T. Zilli https://orcid.org/0000-0003-1670-8567 7 \| CONCLUSIONS The present analysis indicates that precipitation trends observed over the SACZ and SESA regions over the last 40 years consistent with Zilli et al. (2019) are detectable in some of the historical simulations of the CMIP5 cli- mate models. The results indicate large disagreement among individual models, suggesting that the mechanism related to the observed precipitation trends are not well represented in the selected models. The large uncer- tainties prevent a robust attribution analysis of the main forcings. Nonetheless, a qualitative assessment of the results suggests that part of the precipitation trends over SEBr and SESA could result from changes in land-use and anthropogenic-related stratospheric and tropospheric ozone concentration. 6.3 \| Other factors associated with the observed trends Thus, it is possible that the AMO have influenced the observed trends and that the lack of agreement among the CMIP5 simulations arises from a misrepresentation of the influ- ence of this mode on the SACZ-related dipole. A more in- depth evaluation of the topic should also include other variables, such as circulation and sea surface tempera- ture, which will be addressed in future research. 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https://openalex.org/W3155767691	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/24/e3sconf_caes2021_03077.pdf	English	null	Considerations on the Protection of Historic Buildings in Wuhan：Starting from Tanhualin Protection	E3S web of conferences	2,021	cc-by	2,572	2 Tanhualin general situation Tanhualin historic district is located in the northeast of the old town of Wuchang. It mainly starts from Zhongshan Road in the east to Desheng Bridge in the west, including Tanhualin, Gejia ying, Taiping Testuan, Madaomen, Sanyi Village, and Huayuan Hill and a strip of land within the two hills of Pangxiejia. According to the regional division, the historical heritage protection work of Tanhualin area is mainly divided into three sections: the western section is positioned as a historical and cultural district. Starting from No. 81 Tanhualin Street and ending at Deshengqiao Street, the total length is 300m. The middle section is positioned as a cultural and leisure block, covering a total length of 280m from Wuhan No. 14 Middle School to No. 81 Tanhualin Street. The eastern section is positioned as a cultural industry block, with a total length of 620m, starting from the entrance of Zhongshan Road and ending at the 14th Wuhan City [1]. 3 Status of Historic Buildings Wuchang Tanhualin historic district has five cultural elements: ancient city culture, religious culture, education culture, street culture and architectural culture. It is an important section for the exchange, collision and integration of Chinese and Western cultures in modern times. The rich historical buildings will still give the city a profound and long-term influence. Taking this area as an example, this paper discusses the thinking on the protection of historic buildings in Wuhan. Considerations on the Protection of Historic Buildings in Wuhan：Starting from Tanhualin Protection Yulu Zhuang1 1School of Civil Engineering and Architecture, Wuhan University of Technology, Wuhan, Hubei, 430070, China Abstract. This paper takes the historical and cultural area of Tanhualin in Wuchang District as an example, from the historical construction present situation, the value of historical architecture, discusses three aspects of the historical building protection measures, and finally draws the conclusion: historical building protection needs to emphasize on protection, do repair old as before, to protect the culture at the same time also should pay attention to regain its vitality, achieve sustainable development, and carefully reconsider historical buildings in the present age, the function of space positioning. 3.1 Historic buildings are scattered The historical district of Tanhualin covers a wide area. The historical buildings are scattered in the district, with poor accessibility, lack of connection and cohesion between them, which can not heighten the historical atmosphere of the district as a whole. The historic buildings that are not adjacent to the main street, due to poor accessibility, there is no continuous and attractive space to attract people, people rarely come. Garden Hill Catholic Church, for example, is a Romanesque basilica church built by Italian missionaries more than two hundred years ago. It is a magnificent and worthy place to visit. But it is located in a long narrow street, almost isolated from the main street, few people arrive. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: yuluzhuang@whut.edu.cn https://doi.org/10.1051/e3sconf/202124803077 https://doi.org/10.1051/e3sconf/202124803077 E3S Web of Conferences 248, 03077 (2021) CAES 2021 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons org/licenses/by/4 0/) y EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 icenses/by/4.0/). 3.2 Most of the historic buildings are not properly preserved Due to the wide range of distribution of historical buildings, historical buildings lose their original features through the spontaneous reconstruction and construction of local residents and the replacement of homeowners under the changes of history. This leads to the phenomenon of mixed architectural layout and the decline of the whole historical atmosphere of the block. For example, the Swedish Diocese, is also the self-inhabited area of Tanhualin villagers [2]. In the decades after the founding of the People's Republic of China, more and more self-owned houses were built around the parishes and many historic buildings were confined to small alleys or courtyards, which affected the original appearance of the historic districts. On account of the excessive human E3S Web of Conferences 248, 03077 (2021) CAES 2021 https://doi.org/10.1051/e3sconf/202124803077 Fig. 2. Modern residential houses are mixed with art shops intervention in daily life and the lack of systematic protection, several historic buildings in the area have been damaged, even partially collapsed. So the rescue and protection work is imminent. 3.3 The newly restored historic buildings do not continue the historical context The historic buildings on both sides of the main street are mainly used as shops after the facades are refurbished. Although their commercial value has been improved, they still face the dilemma of renovating old buildings like new ones and breaking the context. In addition, apart from a few commercial buildings are otago ancient buildings, most of the shops or archaize like new, or follow a typical town shop decoration form of literature and art. (See Figure 1)There is no different from shops in Gulangyu in Xiamen or Tianzifang in Shanghai. Fig. 2. Modern residential houses are mixed with art shops Fig. 1. The literary shop similar as which in Gulangyu and Tianzifang 3.5 The buildings that remain on the block are no longer adequate for the modern lifestyle of the residents According to the investigation, the majority of people left in Tanhualin block are the elderly and children, as well as renters. Most of the residential houses are old houses that are forbidden to be demolished in the protected areas. By reason of their inability to meet the living environment of modern people, these old houses have suffered varying degrees of damage after artificial reconstruction, expansion and construction. Because of the high density of buildings in Tanhualin, it is difficult to add new social service facilities, which can only be solved through the surrounding communities and the traffic distance is too long. Due to serious material aging, the buildings and spaces in Tanhualin are no longer able to support the urban life today(See Figure 3). Fig. 1. The literary shop similar as which in Gulangyu and Tianzifang 3.4 Historic buildings do not form an organic whole with their surroundings Fig. 3. Houses build by the residents themselves Historic buildings exist independently of their surroundings and are not affected by or influenced by the surrounding buildings. For example, in the main street of the middle section of Tanhualin, except for the historical buildings of Renji Hospital, it mainly presents a mixture of antique buildings (such as the historical and cultural exhibition hall of Tanhualin), modern residential buildings and scattered modern art shops (See Figure 2) [3]. Moving east to Wuhan No. 14 Middle School, the main street gradually becomes deserted, with fewer and fewer buildings open to the street, and the main street becomes a corridor for people. p p While the historical buildings that have been greatly transformed and used as shops, there are also some historical buildings that have only been slightly reformed and strengthened. Most of them are dwellings and studios with high privacy, which seem to be out of place with the bustling main street. The doors are often closed and few people pay attention to them. Fig. 3. Houses build by the residents themselves 5 Conservation measures for historic buildings after the modern history play a leading role in the evolution of the architectural form of Wuhan. Discussing Wuhan's modern historical building cultural heritage protection achievements is also to discuss the value attitude of Wuhan's urban space in the construction process. As a well-preserved historical block in Wuchang, Tanhualin Historical and Cultural Area of Wuchang has witnessed the construction, development and protection process of Wuhan's modern historical buildings. Wuhan is the epitome of modern history, also known as the living Wuhan modern history book. It is of great theoretical value and practical significance for the protection and development of urban historic blocks. Therefore, the protection of Tanhualin is very important for the development of Wuhan. 5.1.1 Repair the old as the old should be achieved Excessive rectification should be avoided to make the historical buildings lose their social value, and the collective memory of Old Wuchang should be avoided to make fault. The influence of events in each period should be respected and retained. 5.1.3 Historic buildings need to weaken their commercial value Avoid the phenomenon that the aboriginal people all move out due to excessive commercialization in the middle and late development of Tianzifang and Lijiang Ancient Town.. 5.2.1 Improve the street space Collinlow wrote in his Collage City : European cities are like a collage, which combines elements of different ages such as the Middle Ages, Baroque and Renaissance together to form a city, so there will be no historical fault. We can walk through a street in Europe and browse the history of the development of the whole Europe. Historic buildings are like a piece of genetic information that remains to this day. If we remove all the historic buildings, then there will be a fault line in our history. We will not know what the land was like in the past. Improve the street space so that it can lead people to the historic building and connect the historic building nodes. 4.1 Historic buildings are the material carriers of history Historic buildings bear witness to history and are the evidence of history. A series of historical events before and 2 E3S Web of Conferences 248, 03077 (2021) CAES 2021 E3S Web of Conferences 248, 03077 (2021) https://doi.org/10.1051/e3sconf/202124803077 5.2.2 The cultural characteristics of blocks should reflect the value characteristics of historical buildings The cultural characteristics of blocks should reflect the value characteristics of historical buildings, and should not be occupied by a variety of monotonous art shops. 5.1.2 Rethink the functional positioning of historical buildings in the current era and space Tanhualin historical area tells the story of the unique old Wuchang.Its existence gives Wuhan a unique charm and temperament.The protection of historical buildings is to protect the historical features of the city, to achieve the historical continuity of urban development and cultural diversity. Historic buildings are the cultural gene pool of a city, which records the information superimposing on space by time and interweaved with the urban living environment. By retaining and inheriting the urban memory, the historical buildings reveal the spirit and characteristics of a city. Put historical buildings into the whole district and even the city for consideration, so as to make them become the nodes with strong accessibility and vitality in the urban image. For example, the Renji Hospital in the main street of the middle section of Tanhualin is now an office building, which is not open to the public, and its value is not well utilized. Therefore, the main street nearby is extremely deserted. If it can be changed into a public tour building on the basis of establishing a complete set of protection rules, its value may be better explored. Nowadays, more and more cities are faced with the problem of Same Imagines of the City. The similarity of the appearance of new buildings causes each city to lose its own characteristics. And the old city texture and historical buildings of each city become the valuable characteristics of each city. Just as people think of Beijing when they think of quadrangles, they think of Suzhou when they think of gardens. 4.2 Historic buildings are the unique cultural symbols of a city 5.1.2 Rethink the functional positioning of historical buildings in the current era and space References 1. Mei Yn. (2013) Reflection on the Transformation of Historical and Cultural Districts Oriented by Cultural and Creative Industries : Taking Wuhan Tanhualin Historical and Cultural Districts as an Example. Huazhong Architecture, 12 : 158-160. Fig. 4. The former residence of Shi Ying, which is off-limits to visitors 2. Hu Youbin, Li Haosheng. (2004) A Study on the Development and Development of Urban Historic Districts . Planners, 12 : 61-64. 3. Wang Xu, Zhang Yuanchun. (2010) Research on the protection and renewal of historic blocks -- a case study of Tanhualin block in Wuchang. Huazhong Architecture, 7 : 134-136. 4. Hu Rongrui, Hu Shaoxue. (2007) A study on the development and utilization of modern architectural heritage in Wuhan. Journal of Architecture, 05 : 15- 18. 5. Li Heping. (2003) Preservation and renovation methods of buildings in historic districts. City Planning Review, 04 : 52-56. Fig. 4. The former residence of Shi Ying, which is off-limits to visitors 5.4 Construction of government, community, unit and residents of the four-in-one protection mechanism With the development and evolution of Wuhan, Tanhualin historical and cultural area is not just a street or a few simple historical building nodes. It is a link of urban design in Wuhan urban renewal. Therefore, the protection of Wuhan's historical architectural and cultural heritage, including Tanhualin area, is not only the work of architects and planners, but also requires the cooperation of the government, investors and residents [5]. First, we need to attract investment and create employment opportunities for local residents. Second, it is necessary to build up the local residents' sense of responsibility and honor for the excellent historical buildings in the area, so that they can participate in the protection of historic buildings. 5.3 Enhance the sustainability of historic buildings Both historic blocks and historic buildings cannot be displayed statically like some cultural relics on display, but should be developed dynamically. Some of the historic buildings in Tanhualin are idle or blocked. As time passes, the buildings are not taken care of, and the buildings will 3 3 E3S Web of Conferences 248, 03077 (2021) CAES 2021 https://doi.org/10.1051/e3sconf/202124803077 fall into disrepair, and the buildings will lose their vitality. For example, Shi Ying's former residence in Sanyi Village was tightly sealed by iron gates (See Figure 4), making it inaccessible to people. Such protection method could only temporarily protect its body, but could not protect its cultural spirit and historical connotation. area, for example, can be realized in the future and even all over the country engaged in historic buildings in Wuhan city protection work, the need to focus on protection, do repair the old as sweet, to avoid too many changes to its social value is lost, also avoided the rupture of the old residents' collective memory. At the same time, attention should be paid to enhancing the overall vitality of Tanhualin historical and cultural area to achieve the goal of sustainable development. Both historic blocks and historic buildings cannot be displayed statically like some cultural relics on display, but should be developed dynamically. Some of the historic buildings in Tanhualin are idle or blocked. As time passes, the buildings are not taken care of, and the buildings will fall into disrepair, and the buildings will lose their vitality. For example, Shi Ying's former residence in Sanyi Village was tightly sealed by iron gates (See Figure 4), making it inaccessible to people. Such protection method could only temporarily protect its body, but could not protect its cultural spirit and historical connotation. 6 Conclusion The value of historical buildings deposited through the baptism of time is not only the physical building itself, but also the social value contained in it. Historic architecture is the material carrier of history, the cultural symbol of a city, the memory inheritance of generations, and the perceptual cognition mixed with many vicissitudes of history. In Wuchang Tanhualin area historical and cultural 4
https://openalex.org/W4318212368	https://journal.unpak.ac.id/index.php/pedagogia/article/download/4894/2887	Indonesian	null	Penerapan Model Pembelajaran Kontekstual Untuk Meningkatkan Keterampilan Menulis Biografi	Pedagogia	2,015	cc-by-sa	3,688	PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL 257 Keywords: writing skill, biography, contextual learning model Keywords: writing skill, biography, contextual learning model PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL UNTUK MENINGKATKAN KETERAMPILAN MENULIS BIOGRAFI Yulia1, Eri Sarimanah2, Suhendra3 ABSTRACT The study is aimed at investigating the implementation of Contextual Learning Model in improving students’ ability and their obstacles to write biography. The study was conducted to the students of SMPN 2, Kabandungan, Kabupaten Sukabumi grade VII. Experimental method was employed. There were a control class and an experimental class. The data was collected through test and questionnaire. The sample was taken by using saturated sampling technique. The first hypothesis which stated that the model of contextual learning could improve the ability of students at SMPN 2, Kabandungan, Kabupaten Sukabumi grade VII to write biography is proven to be true. It could be seen from the students’ score of pretest in the experimental class whose average reached 51,0 while the average of the posttest score was 78,3. The average score of students’ pretest in the control class was 47,4 and the model of students as facilitator and explaining was implemented. At the end, the average score of the post test was 65,6. From the score gained, it is clear that the model of contextual learning was more effective to improve students’ ability in writing biography. Based on the mean calculation using t-test formula, it was gained that to =3,72, t0,95=1,67 and the value of t0,99=2,39. It means that to is higher than tt with the comparison of 1,67<3,72>2,39. It clearly shows that the contextual model implemented in the experimental class could improve the students’ biography writing skill. The second hypothesis which stated that the students encountered problems in writing biography was also proven to be true. Based on the result of questionnaire analysis, there were 71% of the students encountering problems in developing the identity of the character, 54% encountering problems in applying the grammar of writing biography, and 49% students encountering problems when they explained the great attitudes of the character. Therefore, it can be concluded that the contextual learning model could improve students’ ability to write biography even though the students still encountered problems. Keywords: writing skill, biography, contextual learning model Pendahuluan Selain hal-hal di atas, hasil penelitian Sri Wasito (2011) menyebutkan ada tiga penyebab lemahnya keterampilan menulis siswa. Salah satu penyebab tersebut adalah guru hanya menggunakan metode ceramah tanpa tanya jawab dan pemodelan. Penyebab kedua adalah guru hanya menggunakan papan tulis sebagai media pembelajaran. Selain itu siswa kurang aktif bertanya saat ada materi yang tidak dimengerti. Penelitian Sri Wasito tersebut dimuat dalam jurnal yang berjudul “Meningkatkan Keterampilan Menulis Karangan Narasi Melalui Media Gambar Seri pada Siswa Kelas V SDN 2 Wonosobo”. Selain hal-hal di atas, hasil penelitian Sri Wasito (2011) menyebutkan ada tiga penyebab lemahnya keterampilan menulis siswa. Salah satu penyebab tersebut adalah guru hanya menggunakan metode ceramah tanpa tanya jawab dan pemodelan. Ada empat keterampilan berbahasa yang dikaji dalam ilmu bahasa. Empat keterampilan tersebut yaitu keterampilan menyimak, keterampilan berbicara, keterampilan membaca, dan keterampilan menulis. Keterampilan menyimak merupakan keterampilan awal yang dimiliki manusia. Hal ini dimulai sejak manusia lahir ke bumi. Setelah keterampilan menyimak, keterampilan kedua yang dimiliki manusia adalah keterampilan berbicara, lalu diikuti keterampilan membaca yang biasanya dimulai pada masa sekolah. Keterampilan terakhir yang sangat kompleks dan paling sulit adalah keterampilan menulis. Bagi semua orang normal tentu akan bisa menyimak dan berbicara tetapi tidak semua orang bisa menulis. Penyebab kedua adalah guru hanya menggunakan papan tulis sebagai media pembelajaran. Selain itu siswa kurang aktif bertanya saat ada materi yang tidak dimengerti. Penelitian Sri Wasito tersebut dimuat dalam jurnal yang berjudul “Meningkatkan Keterampilan Menulis Karangan Narasi Melalui Media Gambar Seri pada Siswa Kelas V SDN 2 Wonosobo”. Guru memegang peran penting dalam keberhasilan pembelajaran, termasuk dalam pembelajaran menulis. Siswa bisa menghasilkan tulisan yang baik jika guru menggunakan metode dan model pembelajaran yang menarik. Oleh karena itu, guru harus mampu menyajikan pembelajaran menggunakan model yang tepat. Keterampilan menulis adalah keterampilan seseorang untuk menuangkan ide dalam sebuah tulisan. Hal ini selalu dianggap sulit karena orang- orang menganggap ide lebih mudah dituangkan dalam bentuk bahasa lisan. Kenyataan ini terjadi juga di kalangan siswa. Siswa sebagai manusia terpelajar hendaknya tidak berpedoman pada pendapat di atas. Namun, faktanya siswa-siswa zaman sekarang lebih asyik bercengkrama dengan ponsel canggih mereka daripada harus menulis. Jika hal ini terus berlanjut maka akan terjadi kemunduran kualitas generasi muda Indonesia di masa mendatang. Salah satu model pembelajaran yang bisa diterapkan adalah model pembelajaran kontekstual. Model pembelajaran kontekstual merupakan pembelajaran yang membawa materi ke dalam dunia nyata. ABSTRAK Tujuan penelitian ini untuk mengetahui penerapan model pembelajaran kontekstual dalam meningkatkan keterampilan menulis biografi siswa kelas VII SMPN 2, Kabandungan, Kabupaten Sukabumi, serta mengetahui kendala siswa saat menulis biografi. Metode yang digunakan dalam penelitian ini adalah eksperimen dengan meneliti kelas eksperimen dan kelas kontrol. Teknik pengumpulan data dilakukan dengan tes dan angket. Teknik pengambilan sampel yang digunakan adalah sampling jenuh. Hipotesis pertama, yaitu penerapan model pembelajaran kontekstual dapat meningkatkan keterampilan menulis biografi siswa kelas VII SMPN 2 Kabandungan, Kabupaten Sukabumi, teruji kebenarannya. Hal ini terlihat dari hasil tes awal (prates) di kelas eksperimen diperoleh nilai rata- rata 51,0 sedangkan hasil tes akhir (postes) nilai rata-rata siswa meningkat menjadi 78,3. Hasil tes awal (prates) di kelas kontrol diperoleh nilai rata-rata 47,4 sedangkan pada tes akhir (postes) setelah diterapkan model student facilitator and explaining, nilai rata-rata siswa sedikit meningkat menjadi 65,6. Berdasarkan data tersebut dapat diketahui bahwa terdapat peningkatan nilai siswa yang lebih baik dengan menggunakan model pembelajaran kontekstual, yaitu dari nilai rata-rata 51,0 menjadi 78,3. Berdasarkan hasil penghitungan perbandingan mean dengan menggunakan rumus t-tes, diperoleh harga to =3,72, harga t0,95=1,67 dan harga t0,99=2,39 dengan demikian to lebih besar dari tt dengan perbandingan 1,67<3,72>2,39. Hal tersebut berarti model pembelajaran kontekstual pada kelas eksperimen dapat meningkatkan keterampilan menulis biografi. Hipotesis kedua, yaitu siswa kelas VII SMPN 2 Kabandungan, Kabupaten Sukabumi mengalami kendala dalam menulis biografi dengan model pembelajaran kontekstual, dapat diuji kebenarannya. Berdasarkan hasil analisis angket, diperoleh hasil 71% siswa mengalami kendala dalam mengembangkan identitas tokoh, 54% siswa mengalami kendala saat menerapkan kaidah kebahasaan biografi, dan 49% siswa mengalami kendala saat memaparkan sikap teladan tokoh. Dengan demikian, dapat disimpulkan bahwa model pembelajaran kontekstual dapat meningkatkan keterampilan menulis biografi meskipun pada saat menulis biografi, siswa mengalami kendala. Kata kunci: keterampilan menulis, biografi, kontekstual Pedagogia, Volume 7 Nomor 2 Tahun 2015 Pedagogia, Volume 7 Nomor 2 Tahun 2015 PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL 258 motivasi. PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL 259 siswa untuk mencapai tujuan pembelajaran. Prinsip kontekstual adalah membawa siswa pada dunia nyata saat belajar. Banyak ahli yang berpendapat mengenai pengertian pembelajaran kontekstual. Sumiati dan Asra (2011: 14) berpendapat bahwa pembelajaran kontekstual adalah upaya guru untuk membantu siswa memahami relevansi materi pembelajaran yang dipelajarinya dan memberikan kesempatan kepada siswa untuk mengaplikasikan apa yang dipelajarinya di kelas. graphien yang berarti tulisan. Jadi, biografi adalah tulisan tentang kehidupan seseorang atau riwayat hidupnya. Dilihat dari asal kata pembentuknya, jelas bahwa biografi merupakan sebuah tulisan yang di dalamnya memuat mengenai kisah kehidupan seseorang. Berkaitan dengan biografi, banyak ahli yang berpendapat mengenai pengertian biografi. Fu’ad (2008: 5) biografi adalah riwayat hidup seseorang yang ditulis oleh orang lain. Pendapat Fu’ad sejalan dengan pemikiran Keraf (2000: 141) yang mengutarakan bahwa biografi adalah tulisan tentang kisah menarik dalam kehidupan seseorang mengenai pengalaman dan kehidupan pribadinya. Pendapat di atas sejalan dengan pendapat La Iru dan La Ode (2012: 71) yang mengatakan bahwa pembelajaran kontekstual adalah konsep belajar yang membantu guru mengaitkan antara materi yang diajarkan dengan situasi dunia peserta didik dan mendorong peserta didik membuat hubungan antara pengetahuan yang dimilikinya dengan penerapannya dalam kehidupan mereka sehari-hari. Pendapat ini sejalan dengan Taniredja (2011: 49) Pendapat para ahli di atas dilengkapi oleh Isnatun dan Farida (2013: 85) yang berpendapat bahwa biografi merupakan kisah kehidupan seseorang yang bersumber pada kisah nyata (nonfiksi) yang lebih kompleks daripada sekadar data tanggal lahir atau tanggal kematian dan data pekerjaan seseorang. Pendapat di atas dipertegas oleh Nurhadi (2002, dalam Rusman, 2012:189) bahwa pembelajaran kontekstual merupakan konsep belajar yang dapat membantu guru mengaitkan antara materi yang diajarkannya dengan situasi dunia nyata siswa dan mendorong siswa membuat hubungan antara pengetahuan yang dimilikinya dengan penerapannya dalam kehidupan mereka sebagai anggota keluarga dan masyarakat. Pendapat ini sebenarnya melangkapi dua pendapat sebelumnya. Nurhadi lebih memperjelas penerapan materi yang dimiliki siswa dalam kehidupan itu adalah kehidupan siswa sebagai anggota keluarga dan masyarakat. Berdasarkan pendapat para ahli di atas, dapat disimpulkan bahwa biografi adalah tulisan yang berisi mengenai riwayat hidup seseorang, misalnya sosok seorang ayah yang ditulis oleh orang lain, misalnya siswa. Riwayat hidup tidak hanya mencantumkan nama dan tanggal lahir saja tetapi lebih lengkap dari itu terdapat pengalaman menarik orang tersebut berdasarkan kisah nyata hidupnya. Pengalaman- pengalaman tersebut misalnya kisah saat menempuh pendidikan, pengalaman saat mencapai cita-cita, dan lain-lain. Pendahuluan Siswa bisa lebih mudah memahami materi karena mereka bisa menghubungkan pelajaran yang sedang mereka pelajari dengan pengalaman yang telah dialaminya. Siswa sering mengalami kendala saat menulis sehingga nilai keterampilan menulis mereka menjadi rendah. Kendala yang dialami siswa beragam. Berdasarkan hasil pengamatan awal pada siswa kelas VII SMPN 2 Kabandungan Kabupaten Sukabumi, kendala siswa saat menulis dialami ketika menentukan judul dan menyesuaikan isi tulisan dengan judul. Khusus dalam menulis biografi, siswa sering mengalami kendala saat menuliskan kelengkapan identitas tokoh, mencantumkan perjuangan, prestasi, dan hal-hal yang dapat diteladani dari tokoh, serta penggunaan bahasa dalam tulisan mereka. Guru bisa mengaitkan materi dengan kehidupan di keluarga, sekolah dan di masyarakat sekitar siswa. Misalnya saat guru menyajikan materi biografi, bawalah siswa pada dunia nyata mereka. Biografi adalah tulisan tentang kehidupan seseorang atau riwayat hidup seseorang. Berikanlah contoh biografi salah satu dari anggota keluarga, misalnya ayah. Cara ini akan membantu siswa agar lebih mudah memahami materi karena setiap siswa pasti memiliki ayah. Jadi, mereka lebih mudah menafsirkan mengenai biografi. Guru bisa memberikan ilustrasi untuk mempermudah penyampaian materi. Iliustrasi tersebut bisa berupa benda, gambar, atau kejadian yang ada di sekeliling siswa, seperti biografi seorang ayah. Penerapan model pembelajaran kontekstual diharapkan bisa meningkatkan keterampilan menulis siswa. Fenomena di atas tidak terjadi begitu saja, tentu ada pemicunya. Rahardja (2010) mengemukakan penyebab lemahnya keterampilan menulis seseorang. Penyebab itu adalah lemahnya kesadaran pentingnya menulis, keterbatasan mengakses informasi (membaca) sehingga tidak tahu mengenai hal yang akan ditulis, tidak tahu manfaat menulis, tidak menguasai metode menulis, dan kurang Model pembelajaran kontekstual merupakan salah satu pembelajaran yang membantu guru dan Pedagogia, Volume 7 Nomor 2 Tahun 2015 Pedagogia, Volume 7 Nomor 2 Tahun 2015 Rekapitulasi Data Prates Kelas Eksperimen Interval Persentase Tingkat Penguasaan Frekuensi Persentase Kemapuan 85%-100% - Sangat Mampu 75%-84% - Mampu 60%-74% 7 20% Cukup Mampu 40%-59% 25 71,42% Kurang Mampu 0%-39% 3 8,57% Tidak Mampu Jumlah 35 100% Diagram 1 Isnatun dan Farida (2013: 102) mengemukakan ada empat unsur yang dinilai dalam biografi. Unsur- unsur tersebut adalah judul harus sesuai dengan isi, ketepatan dan kelengkapan identitas tokoh, mencantumkan perjuangan, prestasi, dan hal-hal yang dapat diteladani dari tokoh, serta menggunakan kata sifat dan kata kerja dengan tepat. g Data Prates Kelas Eksperimen Data Prates Kelas Eksperimen Tabel 2 Data Prates Kelas Eksperimen T b l 2 Pedagogia, Volume 7 Nomor 2 Tahun 2015 PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL Isnatun dan Farida (2013: 101) mengemukakan bahwa biografi dapat diklasifikasikan berdasarkan sisi penulis, isi, dan persoalan yang dibahas.i Dari beberapa pendapat ahli di atas, dapat disimpulkan bahwa pembelajaran kontekstual adalah pembelajaran yang mendorong siswa untuk memahami materi dan mengaitkannya dengan kehidupan sehari-hari. Lalu, siswa juga didorong untuk menerapkan materi dalam kehidupan pribadi, sosial, dan budaya. Materi yang mereka kuasai juga akan menjadi bekal dalam menyelesaikan masalah. Siswa juga didorong untuk berperan aktif saat proses pembelajaran. i Berdasarkan sisi penulis dikenal istilah biografi dan otobiografi. Biografi adalah riwayat hidup yang ditulis oleh orang lain sedangkan otobiografi adalah riwayat hidup atau biografi yang ditulis oleh tokohnya sendiri. Istilah “ditulis oleh tokohnya sendiri” ini tidak selalu berarti harus benar-benar sang tokoh yang menulisnya. Sang tokoh dapat meminta bantuan orang lain (profesional) untuk menuliskan biografi dirinya. Jadi sang tokoh memaparkan riwayat hidupnya pada penulis, lalu si penulislah yang merangkai kalimat dan menyusun teks biografinya.ii Biografi merupakan bagian dari karangan narasi ekspositoris, yaitu narasi yang hanya bertujuan untuk memberi informasi kepada pembaca agar pengetahuannya bertambah luas. Biografi memberikan informasi mengenai riwayat hidup seseorang kepada pembaca. Biografi berasal dari bahasa Yunani, yaitu bios yang berarti hidup dan Berdasarkan isinya, biografi diklasifikasikan berdasarkan perjalanan hidup dan perjalanan karier. Biografi perjalanan hidup berisi cerita perjalanan Pedagogia, Volume 7 Nomor 2 Tahun 2015 PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL 260 dilaksanakan. Postes adalah tes yang dilakukan setelah pembelajaran dilaksanakan. Tes dilakukan untuk mengetahui mengetahui kemampuan siswa dalam menulis biografi. Angket digunakan untuk mengukur dan mengetahui kendala siswa dalam menulis biografi. Angket yang digunakan adalah angket tertutup. hidup lengkap atau sebagian yang paling berkesan. Biografi perjalanan karier berisi cerita perjalanan karier dari awal karier hingga karier terbaru, atau sebagian perjalanan karier dalam mencapai sukses tertentu. Berdasarkan persoalan yang dibahas, biografi dapat diklasifikasikan menjadi biografi politik, intelektual, dan biografi jurnalistik. Biografi politik memuat riwayat tokoh-tokoh dari sudut pandang politik, bahan penulisan dikumpulkan melalui riset, tetapi tidak lepas dari kepentingan politik penulis ataupun tokoh yang ditulisnya. Biografi intelektual juga disusun berdasarkan riset dan segenap temuannya disajikan dalam gaya penulisan ilmiah. Sementara itu, biografi jurnalistik atau biografi sastra adalah biografi yang materinya disusun berdasarkan hasil wawancara terhadap tokoh yang akan ditulis maupun orang lain yang menjadi rujukan. Hasil Penelitian Tabel 1 Rekapitulasi Data Prates Kelas Eksperimen Simpulan Berdasarkan hasil penelitian dengan judul Penerapan Model Pembelajaran Kontekstual untuk Meningkatkan Keterampilan Menulis Biografi Siswa Kelas VII SMPN 2 Kabandungan Kabupaten Sukabumi, dapat disimpulkan sebagai berikut: Berdasarkan tabel dan diagram tersebut dapat dijelaskan bahwa hasil prates materi biografi yang diperoleh di kelas eksperimen dengan nilai rata-rata 51,0 dapat diketahui siswa kurang mampu dalam menulis biografi. Pada hasil postes materi menulis biografi dengan model kontekstual pada kelas eksperimen terlihat kemampuan siswa meningkat menjadi mampu dengan nilai rata-rata 78,3. Berdasarkan data tersebut dapat diketahui bahwa ada peningkatan nilai siswa sebelum dan sesudah diterapkan model kontekstual dari kemampuan kurang mampu menjadi mampu. Sementara itu, hasil prates pada kelas kontrol diperoleh nilai rata-rata 47,4 dan dapat diketahui kemampuan siswa kelas kontrol dalam menulis biografi kurang mampu. Pada hasil postes materi biografi di kelas kontrol kemampuan siswa sedikit meningkat menjadi cukup mampu dengan nilai rata-rata 65,6. Berdasarkan data tersebut dapat diketahui terdapat sedikit peningkatan sebelum dan sesudah menerapkan model student facilitator and explaining dari kemampuan kurang mampu menjadi cukup mampu. Pertama, penerapan model pembelajaran kontekstual di kelas VII SMPN 2 Kabandungan Kabupaten Sukabumi dapat meningkatkan keterampilan menulis siswa, khususnya menulis biografi. Hal tersebut terbukti berdasarkan hasil analisis prates dan postes siswa. Hasil prates dan postes menunjukkan adanya peningkatan yang signifikan saat pembelajaran menggunakan model kontekstual dibandingkan dengan model lain. Nilai rata-rata hasil prates keterampilan menulis biografi pada kelas ekperimen adalah 51,0. Nilai tersebut menunjukkan bahwa kemampuan siswa berada pada tingkatan kurang mampu. Setelah dilakukan penerapan model kontekstual pada kelas eksperimen, nilai rata-rata postes siswa mengalami peningkatan, yaitu 78,3 dan berada pada tingkat mampu. Sementara itu, hasil prates di kelas kontrol diperoleh nilai rata-rata 47,4. Nilai tersebut menunjukkan bahwa kemampuan siswa menulis biografi berada pada tingkat kurang mampu. Setelah dilakukan penerapan model student facilitator and explaining, kemampuan menulis biografi di kelas kontrol sedikit meningkat menjadi cukup mampu dengan nilai rata-rata 65,6. Hasil perhitungan perbandingan mean dengan menggunakan rumus t-tes diperoleh harga to=3,72, harga t0,95=1,67, dan harga t0,99=2,39. Perbandingan to lebih besar dari tt, artinya model kontekstual dalam kelas eksperimen dapat meningkatkan keterampilan menulis biografi siswa. Berdasarkan perhitungan perbandingan mean dengan menggunakan t-tes diperoleh harga to= 3,72, harga t0,95= 1,67, dan harga t0,99= 2,39. Perbandingan to dengan tt yaitu 1,67 <3,72> 2,39, dengan demikian to lebih besar dari tt artinya model kontekstual dalam kelas eksperimen dapat meningkatkan keterampilan menulis biografi siswa. Metode Penelitian Prates adalah tes yang dilakukan sebelum pembelajaran Pedagogia, Volume 7 Nomor 2 Tahun 2015 PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL 261 Diagram 4 Data Postes Kelas Kontrol Diagram 2 Diagram 2 Data Postes Kelas Eksperimen Data Postes Kelas Eksperimen Tabel 3 Diagram 5 Data Angket Pembahasan Tabel 5 Perbandingan Nilai Prates Dan Postes Kelas Eksperimen Dan Kelas Kontrol Kelas Eksperimen Kelas Kontrol Prates Postes Prates Postes 51,0 78,3 47,4 65,6 Rekapitulasi Data Prates Kelas Kontrol Rekapitulasi Data Prates Kelas Kontrol Diagram 5 Data Angket Diagram 5 Data Angket Rekapitulasi Data Prates Kelas Kontrol Interval Persentase Tingkat Penguasaan Frekuensi Persentase Kemapuan 85%-100% - - Sangat Mampu 75%-84% - - Mampu 60%-74% 2 5,26% Cukup Mampu 40%-59% 27 71,05 Kurang Mampu 0%-39% 9 23,68% Tidak Mampu Jumlah 38 100% Diagram 3 Data Prates Kelas Kontrol Data Prates Kelas Kontrol Tabel 4 Pembahasan Tabel 5 Perbandingan Nilai Prates Dan Postes Kelas Eksperimen Dan Kelas Kontrol Kelas Eksperimen Kelas Kontrol Prates Postes Prates Postes 51,0 78,3 47,4 65,6 Rekapitulasi Data Postes Kelas Kontrol Rekapitulasi Data Postes Kelas Kontrol Rekapitulasi Data Postes Kelas Kontrol Interval Persentase Tingkat Penguasaan Frekuensi Persentase Kemapuan 85%-100% - - Sangat Mampu 75%-84% 7 18,42% Mampu 60%-74% 18 47,36% Cukup Mampu 40%-59% 13 34,21% Kurang Mampu 0%-39% - - Tidak Mampu Jumlah 38 100% Pedagogia, Volume 7 Nomor 2 Tahun 2015 PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL 262 Diagram 6 Perbandingan Nilai Prates Dan Postes Kelas Eksperimen Dan Kelas Kontrol Diagram 6 Perbandingan Nilai Prates Dan Postes Kelas Eksperimen Dan Kelas Kontrol separuh siswa mengalami kendala saat menentukan judul, menyesuaikan isi tulisan dengan judul, menuliskan identitas tokoh, menuliskan prestasi tokoh, menuliskan perjuangan tokoh, memaparkan sikap teladan tokoh, dan tidak menguasai kaidah kebahasaan biografi. Selain itu, sebagian besar siswa mengalami kendala saat mengembangkan identitas tokoh dan menerapkan kaidah kebahasaan dalam tulisan biografi ayah mereka. Metode Penelitian Metode yang digunakan dalam penelitian ini adalah metode penelitian eksperimen. Pada penelitian eksperimen, ada dua aspek yang dibandingkan, yaitu satu kelas eksperimen dan satu kelas kontrol. Pada kelas eksperimen, pembelajaran menulis biografi menggunakan model kontekstual sedangkan pada kelas kontrol pembelajaran menulis biografi menggunakan model student facilitator and explaining. Populasi pada penelitian ini adalah siswa kelas VII SMPN 2 Kabandungan, Kabupaten Sukabumi. Teknik pengambilan sampel dilakukan dengan teknik sampling jenuh karena jumlah populasi sedikit dan seluruhnya menjadi sampel. Pengambilan data dilakukan dengan tes, angket, dan observasi. Rekapitulasi Data Postes Kelas Eksperimen Interval Persentase Tingkat Penguasaan Frekuensi Persentase Kemapuan 85%-100% 6 17,14% Sangat Mampu 75%-84% 21 60% Mampu 60%-74% 8 22,85% Cukup Mampu 40%-59% - - Kurang Mampu 0%-39% - - Tidak Mampu Jumlah 35 100% Teknik pengumpulan data yang digunakan dalam penelitian ini yaitu tes dan angket. Dalam penelitian ini tes digunakan untuk mengukur keterampilan siswa dalam menulis biografi. Tes yang dilakukan pada penelitian ini adalah prates dan postes. Simpulan Berdasarkan data angket dapat diketahui bahwa siswa mengalami kendala dalam menulis biografi dengan menggunakan model pembelajaran kontekstual. Berdasarkan jawaban angket terlihat bahwa sebagian kecil siswa mengalami kendala saat menuliskan sikap teladan tokoh, hampir Kedua, hasil analisis data angket menunjukkan bahwa siswa SMPN 2 Kabandungan Kabupaten Sukabumi mengalami kendala saat menulis biografi. Pedagogia, Volume 7 Nomor 2 Tahun 2015 PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL 263 Berdasarkan jawaban angket terlihat bahwa sebagian besar siswa mengalami kendala saat mengembangkan identitas tokoh dengan persentase 71% atau 25 dari 35 siswa. Selain itu, sebagian besar siswa mengalami kendala saat menerapkan kaidah kebahasaan dalam tulisan biografi ayah mereka. Kendala ini dialami oleh 19 dari 35 siswa dengan persentase 54%. Kendala juga dialami siswa saat memaparkan hal yang dapat diteladani dari tokoh. Kendala ini dialami oleh 17 dari 35 siswa dengan persentase 49%. Finoza, Lamuddin. 2008. Komposisi Bahasa Indonesia Edisi Revisi. Jakarta: Diksi.ii Fuad, Zulfikar. 2008. Menulis Biografi. Yogyakarta: Life Story.i Fuad, Zulfikar. 2012. The Secret of Biography Rahasia Menulis Biografi ala Ramadhan K.H. Jakarta: Akademia Permata. Jakarta: Akademia Permata. Hidayat, Kosadi. 1994. Evaluasi dan Pengajarannya dalam Bahasa Indonesia. Bandung: CV Alfabeta. Iru, La., dan La Ode. 2012. Analisis Penerapan Pendekatan, Metode, Strategi, dan Model- model Pembelajaran. Bantul: Multi Presindo. Saran Iskandar, Alex. 1992. Beberapa Pilihan dalam Penelitian Pendidikan. Bogor: FKIP Unpak Penelitian yang telah dilakukan dalam kegiatan pembelajaran keterampilan menulis biografi dengan menerapkan model pembelajaran kontekstual menunjukkan bahwa kemampuan siswa dalam menulis biografi meningkat. Berdasarkan hasil penelitian tersebut, maka peneliti menyarankan hal- hal sebagai berikut: Isnatun, Siti., dan umi Farida. 2013. Mahir Berbahasa Indonesia 2. Jakarta: Yudhistira. Johnson, Elaine. 2010. Contextual Teaching and Learning. Bandung: Kaifa. Keraf, Gorys. 2000. Argumentasi dan Narasi. Jakarta: Gramedia Pustaka Utama. g Pertama, pada kegiatan pembelajaran, guru diharapkan menjadikan model pembelajaran kontekstual sebagai salah satu pilihan dalam pembelajaran keterampilan menulis, khususnya menulis biografi. Kedua, pada kegiatan pembelajaran, guru hendaknya memberikan penjelasan yang lebih rinci mengenai cara mengembangkan identitas seseorang karena identitas bukan hanya nama dan tempat tanggal lahir saja. Guru juga harus memberikan contoh-contoh penerapan kaidah kebahasaan biografi dalam bentuk kalimat sehingga siswa menjadi lebih mudah mengerti. Selain itu, guru juga harus memberikan arahan kepada siswa mengenai cara mengembangkan kalimat utama terutama dalam memaparkan hal teladan tokoh biografi. Misalnya dengan cara mengenalkan pola pengembangan kalimat deduktif dan induktif supaya siswa bisa mengembangkan tulisannya dengan baik. Ketiga, bagi sekolah, penelitian eksperimen ini diharapkan dapat dijadikan sebagai kegiatan wajib bagi setiap tenaga pengajar untuk meningkatkan kualitas pembelajaran yang lebih baik. Nurgiyantoro, Burhan. 2001. Penilaian dalam Pengajaran Bahasa dan Sastra, Edisi Ketiga, Cetakan Pertama. Yogyakarta: BPFE. Nurgiyantoro, Burhan. 2011. Penilaian Pembelajaran Bahasa Berbasis Kompetensi. Yogyakarta: BPFE. Rusman. 2012. Model-model Pembelajaran. Jakarta: Rajagrafindo Persada. Sanjaya, Wina. 2010. Strategi Pembelajaran Berorientasi Standar Proses Pendidikan. Jakarta: Kencana. Soebachman, Agustina. 2014. 4 Hari Mahir Menulis Artikel, Cerpen, Novel, Skripsi. Yogyakarta: Syura Media Utama. Sugiyono. 2012. Metode Penelitian Pendidikan (Pendekatan Kuantitatif, Kualitatif, dan R&D). Bandung: Alfabeta. Sumardjo, Jakob., dan Saini. 1986. Apresiasi Kesusastraan. Jakarta: Gramedia. Sumiati, dan Asra. 2011. Metode Pembelajaran. Bandung: CV Wacana Prima. Taniredja, Tukiran., dkk. 2011. Model-model Pembelajaran Inovatif. Bandung: Alfabeta. PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL 264 Daftar Pustaka Daftar Pustaka Arikunto, Suharsimi. 2006. Prosedur Penelitian Suatu Pendekatan Praktik. Jakarta: Rineka Cipta. Arikunto, Suharsimi. 2006. Prosedur Penelitian Suatu Pendekatan Praktik. Jakarta: Rineka Cipta. Tarigan, Henry Guntur. 2008. Menulis Sebagai Suatu Keterampilan Berbahasa. Bandung: Angkasa. Pedagogia, Volume 7 Nomor 2 Tahun 2015 PENERAPAN MODEL PEMBELAJARAN KONTEKSTUAL Pedagogia, Volume 7 Nomor 2 Tahun 2015 Riwayat Hidup Penulis 1. Yulia, lahir di Sukabumi pada tanggal 6 Maret 1992, putri pertama dari dua bersaudara. Alamat Pendidikan: SDN Cihamerang lulus tahun 2004, MTs Darul Ahkam lulus pada tahun 2007, dan SMK Nurul Bayan lulus pada tahun 2010, Program Studi Pendidikan 2. Eri Sarimanah, Staf Pengajar Program Studi Pendidikan Bahasa dan Sastra Indonesia, FKIP Unpak. 3. Suhendra, Staf Pengajar Program Studi Pendidikan Bahasa dan Sastra Indonesia, FKIP Unpak.

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